Bell-shaped agonist activation of 5-HT1A receptor-coupled Gαi3 G-proteins: Receptor density-dependent switch in receptor signaling.

A previous study observed bell-shaped concentration-response isotherms for activation of Gαi3 G-protein subunits by high efficacy 5-HT1A receptor agonists in a Chinese hamster ovary (CHO) cell line expressing high levels of these receptors. This suggested that a signaling switch took place in that cell line (from Gαi3 to activation of other G-proteins) but it was unclear if such effects are observed for 5-HT1A receptors in other cellular environments. Here, using an antibody capture-based [35S]GTPγS binding assay for Gαi3 activation, we investigated whether efficacious 5-HT1A receptor agonists (5-HT, F13714, befiradol, NLX-101), prototypical agonists ((+) and (-)8-OH-DPAT), and partial agonist, antagonists, inverse agonists (pindolol, WAY100635, spiperone) produced similar effects on 5 cell lines expressing different levels of human 5-HT1A receptors. In membranes from cell lines (HeLa, C6-glia and CHO-low) expressing moderate receptor levels (between 1 and 4 pmol/mg of protein), 5-HT, F13714, befiradol and NLX-101 elicited classical sigmoid concentration-response isotherms. In contrast, in cell lines (CHO-high, HEK-293F) expressing high receptor levels (>9 pmol/mg) these agonists elicited bell-shaped concentration-response isotherms that peaked at nanomolar-range concentrations and then returned to baseline or below. Spiperone elicited inverse agonist inhibitory sigmoid isotherms in all membrane preparations while WAY100635 was mostly 'silent' for Gαi3 activation. The other compounds elicited diverse responses in the different cell lines suggesting that other factors, in addition to receptor expression levels, could be influencing Gαi3 activation. These data indicate that Gαi3 G-protein activation by 5-HT1A receptor ligands is highly dependent on receptor expression levels and on cellular background. Moreover, the induction of bell-shape concentration-response isotherms by 5-HT and other high-efficacy agonists is consistent with a switch in signaling to other G-protein-mediated signaling cascades, possibly elicited by receptor conformational changes.

Levomilnacipran (F2695), a norepinephrine-preferring SNRI: profile in vitro and in models of depression and anxiety.

Levomilnacipran (LVM; F2695) is the more active enantiomer of the serotonin/norepinephrine (5-HT/NE) reuptake inhibitor (SNRI) milnacipran and is currently under development for the treatment of major depressive disorder. LVM was benchmarked against two other SNRIs, duloxetine and venlafaxine, in biochemical, neurochemical and pharmacological assays. LVM exhibited high affinity for human NE (Ki = 92.2 nM) and 5-HT (11.2 nM) transporters, and potently inhibited NE (IC50 = 10.5 nM) and 5-HT (19.0 nM) reuptake (human transporter) in vitro. LVM had 2-fold greater potency for norepinephrine relative to serotonin reuptake inhibition (i.e. NE/5-HT potency ratio: 0.6) and 17 and 27 times higher selectivity for NE reuptake inhibition compared with venlafaxine and duloxetine, respectively. LVM did not exhibit affinity for 23 off-target receptors. LVM (i.p.) increased cortical extracellular levels of 5-HT, and NE (minimal effective doses: MEDs = 20 and 10 mg/kg, respectively). In anti-depressive/anti-stress models, i.p. LVM diminished immobility time in the mouse forced swim (MED = 20 mg/kg) and tail suspension (MED = 2.5 mg/kg) tests, and reduced shock-induced ultrasonic vocalizations in rats (MED = 5 mg/kg). Duloxetine and venlafaxine were less potent (MEDs ≥ 10 mg/kg). At doses active in these three therapeutically-relevant models, LVM (i.p.) did not significantly affect spontaneous locomotor activity. In summary, LVM is a potent, selective inhibitor of NE and 5-HT transporters with preferential activity at the former. It is efficacious in models of anti-depressive/anti-stress activity, with minimal potential for locomotor side effects.

5-HT1A receptors are involved in the effects of xaliproden on G-protein activation, neurotransmitter release and nociception.

BACKGROUND AND PURPOSE: Xaliproden (SR57746A) is a 5-HT(1A) receptor agonist and neurotrophic agent that reduces oxaliplatin-mediated neuropathy in clinical trials. The present study investigated its profile on in vitro transduction, neurochemical responses and acute nociceptive pain tests in rats. EXPERIMENTAL APPROACH: Xaliproden was tested on models associated with 5-HT(1A) receptor activation including G-protein activation, extracellular dopamine and 5-HT levels measured by microdialysis and formalin-induced pain. Activation of 5-HT(1A) receptors was confirmed by antagonism with WAY100635. KEY RESULTS: Xaliproden exhibited high affinity for rat (r) and human (h) 5-HT(1A) receptors (pK(i)= 8.84 and 9.00). In [(35)S]GTPgammaS (guanosine 5'-O-(3-[(35)S]thio)triphosphate) assays it activated both hippocampal r5-HT(1A)[pEC(50)/E(MAX) of 7.58/61% (%5-HT)] and recombinant h5-HT(1A) receptors (glioma C6-h5-HT(1A): 7.39/62%; HeLa-h5-HT(1A): 7.24/93%). In functional [(35)S]GTPgammaS autoradiography, xaliproden induced labelling in structures enriched with 5-HT(1A) receptors (hippocampus, lateral septum, prefrontal and entorhinal cortices). Xaliproden inhibited in vivo binding of [(3)H]WAY100635 to 5-HT(1A) receptors in mouse frontal cortex and hippocampus (ID(50): 3.5 and 3.3 mg x kg(-1), p.o. respectively). In rat, it increased extracellular dopamine levels in frontal cortex and reduced hippocampal 5-HT levels (ED(50): 1.2 and 0.7 mg x kg(-1), i.p. respectively). In a rat pain model, xaliproden inhibited paw licking and elevation (ED(50): 1 and 3 mg x kg(-1), i.p. respectively) following formalin injection in the paw. All effects were reversed by pretreatment with WAY100635. CONCLUSIONS AND IMPLICATIONS: These results indicate that activation of 5-HT(1A) receptors is the principal mechanism of action of xaliproden and provide further support for the utility of 5-HT(1A) receptor activation as an anti-nociceptive strategy.

Signal transduction and functional selectivity of F15599, a preferential post-synaptic 5-HT1A receptor agonist.

BACKGROUND AND PURPOSE: Activation of post-synaptic 5-HT(1A) receptors may provide enhanced therapy against depression. We describe the signal transduction profile of F15599, a novel 5-HT(1A) receptor agonist. EXPERIMENTAL APPROACH: F15599 was compared with a chemical congener, F13714, and with (+)8-OH-DPAT in models of signal transduction in vitro and ex vivo. KEY RESULTS: F15599 was highly selective for 5-HT(1A) receptors in binding experiments and in [(35)S]-GTPgammaS autoradiography of rat brain, where F15599 increased labelling in regions expressing 5-HT(1A) receptors. In cell lines expressing h5-HT(1A) receptors, F15599 more potently stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, compared with G-protein activation, internalization of h5-HT(1A) receptors or inhibition of cAMP accumulation. F13714, (+)8-OH-DPAT and 5-HT displayed a different rank order of potency for these responses. F15599 stimulated [(35)S]-GTPgammaS binding more potently in frontal cortex than raphe. F15599, unlike 5-HT, more potently and efficaciously stimulated G(alphai) than G(alphao) activation. In rat prefrontal cortex (a region expressing post-synaptic 5-HT(1A) receptors), F15599 potently activated ERK1/2 phosphorylation and strongly induced c-fos mRNA expression. In contrast, in raphe regions (expressing pre-synaptic 5-HT(1A) receptors) F15599 only weakly or did not induce c-fos mRNA expression. Finally, despite its more modest affinity in vitro, F15599 bound to 5-HT(1A) receptors in vivo almost as potently as F13714. CONCLUSIONS AND IMPLICATIONS: F15599 showed a distinctive activation profiles for 5-HT(1A) receptor-mediated signalling pathways, unlike those of reference agonists and consistent with functional selectivity at 5-HT(1A) receptors. In rat, F15599 potently activated signalling in prefrontal cortex, a feature likely to underlie its beneficial effects in models of depression and cognition.

F15063, a potential antipsychotic with D2/D3 antagonist, 5-HT 1A agonist and D4 partial agonist properties. I. In vitro receptor affinity and efficacy profile.

BACKGROUND AND PURPOSE: Combining 5-HT(1A) receptor activation with dopamine D(2)/D(3) receptor blockade should improve negative symptoms and cognitive deficits in schizophrenia. We describe the in vitro profile of F15063 (N-[(2,2-dimethyl-2,3-dihydro-benzofuran-7-yloxy)ethyl]-3-(cyclopent-1-enyl)-benzylamine). EXPERIMENTAL APPROACH: F15063 was characterised in tests of binding affinity and in cellular models of signal transduction at monoamine receptors. KEY RESULTS: Affinities (receptor and pK(i) values) of F15063 were: rD(2) 9.38; hD(2L) 9.44; hD(2S) 9.25; hD(3) 8.95; hD(4) 8.81; h5-HT(1A) 8.37. F15063 had little affinity (40-fold lower than D(2)) at other targets. F15063 antagonised dopamine-activated G-protein activation at hD(2), rD(2) and hD(3) receptors with potency (pK (b) values 9.19, 8.29 and 8.74 in [(35)S]GTP gamma S binding experiments) similar to haloperidol. F15063 did not exhibit any hD(2) receptor agonism, even in tests of ERK1/2 phosphorylation and G-protein activation in cells with high receptor expression. In contrast, like (+/-)8-OH-DPAT, F15063 efficaciously activated h5-HT(1A) (E(max) 70%, pEC(50) 7.57) and r5-HT(1A) receptors (52%, 7.95) in tests of [(35)S]GTP gamma S binding, cAMP accumulation (90%, 7.12) and ERK1/2 phosphorylation (93%, 7.13). F15063 acted as a partial agonist for [(35)S]GTP gamma S binding at hD(4) (29%, 8.15) and h5-HT(1D) receptors (35%, 7.68). In [(35)S]GTP gamma S autoradiography, F15063 activated G-proteins in hippocampus, cortex and septum (regions enriched in 5-HT(1A) receptors), but antagonised quinelorane-induced activation of D(2)/D(3) receptors in striatum. CONCLUSIONS AND IMPLICATIONS: F15063 antagonised dopamine D(2)/D(3) receptors, a property underlying its antipsychotic-like activity, whereas activation of 5-HT(1A) and D(4) receptors mediated its actions in models of negative symptoms and cognitive deficits of schizophrenia (see companion papers).

Effects of the alpha 2-adrenoreceptor antagonist dexefaroxan on neurogenesis in the olfactory bulb of the adult rat in vivo: selective protection against neuronal death.

A dysfunction of noradrenergic mechanisms originating in the locus coeruleus has been hypothesised to be the critical factor underlying the evolution of central neurodegenerative diseases [Colpaert FC (1994) Noradrenergic mechanism Parkinson's disease: a theory. In: Noradrenergic mechanisms in Parkinson's disease (Briley M, Marien M, eds) pp 225-254. Boca Raton, FL, USA: CRC Press Inc.]. alpha(2)-Adrenoceptor antagonists, presumably in part by facilitating central noradrenergic transmission, afford neuroprotection in vivo in models of cerebral ischaemia, excitotoxicity and devascularization-induced neurodegeneration. The present study utilised the rat olfactory bulb as a model system for examining the effects of the selective alpha(2)-adrenoceptor antagonist dexefaroxan upon determinants of neurogenesis (proliferation, survival and death) in the adult brain in vivo. Cell proliferation (5-bromo-2'-deoxyuridine labelling) and cell death associated with DNA fragmentation (terminal dideoxynucleotidyl transferase-catalysed 2'-deoxyuridine-5'-triphosphate nick end-labelling assay) were quantified following a 7-day treatment with either vehicle or dexefaroxan (0.63 mg/kg i.p., three times daily), followed by a 3-day washout period. The number of terminal dideoxynucleotidyl transferase-catalysed 2'-deoxyuridine-5'-triphosphate nick end-labelling-positive nuclei in the olfactory bulb was lower in dexefaroxan-treated rats, this difference being greatest and significant in the subependymal layer (-52%). In contrast, 5-bromo-2'-deoxyuridine-immunoreactive nuclei were more numerous (+68%) in the bulbs of dexefaroxan-treated rats whilst no differences were detected in the proliferating region of the subventricular zone. Terminal dideoxynucleotidyl transferase-catalysed 2'-deoxyuridine-5'-triphosphate nick end-labelling combination with glial fibrillary acidic protein or neuronal-specific antigen immunohistochemistry revealed that terminal dideoxynucleotidyl transferase-catalysed 2'-deoxyuridine-5'-triphosphate nick end-labelling-positive nuclei were associated primarily with a neuronal cell phenotype. These findings suggest that dexefaroxan increases neuron survival in the olfactory bulb of the adult rat in vivo, putatively as a result of reducing the apoptotic fate of telencephalic stem cell progenies.

Protective effects of the alpha 2-adrenoceptor antagonist, dexefaroxan, against degeneration of the basalocortical cholinergic system induced by cortical devascularization in the adult rat.

It has been hypothesized [Colpaert, F.C., 1994. In: Briley, M., Marien, M. (Eds.), Noradrenergic Mechanisms in Parkinson's Disease. CRC Press, Boca Raton, FL, pp. 225-254] that a deficiency in the noradrenergic system originating from the locus coeruleus is a decisive factor in the progression of central neurodegenerative disorders including Alzheimer's disease, and that treatments which boost noradrenergic transmission (e.g. via blockade of alpha(2)-adrenoceptors) could provide both symptomatic and trophic benefits against the disease. Studies in the rat in vivo demonstrating that the selective alpha(2)-adrenoceptor antagonist dexefaroxan increases acetylcholine release in the cortex, improves measures of cognitive performance and protects against excitotoxin lesions, support this concept. As a further test of the hypothesis, we investigated the effect of dexefaroxan in a rat model of unilateral cortical devascularization that induces a loss of the cortical cholinergic terminal network and a retrograde degeneration of the cholinergic projections that originate in the nucleus basalis magnocellularis. Lesioned and sham-operated rats received a 28-day subcutaneous infusion of dexefaroxan (0.63 mg/rat/day) or vehicle, delivered by osmotic minipumps implanted on the day of the cortical devascularization procedure. In lesioned rats, the dexefaroxan treatment was associated with a significantly higher number and size of vesicular acetylcholine transporter-immunoreactive boutons in comparison to the vehicle treatment; this effect was most marked within cortical layer V. Dexefaroxan also significantly reduced the atrophy of cholinergic neurons within the nucleus basalis magnocellularis. Dexefaroxan had no observable effect on any of these parameters in sham-operated cohorts. These results show that systemically administered dexefaroxan mitigates cholinergic neuronal degeneration in vivo, and provide further evidence for a therapeutic potential of the drug in neurodegenerative diseases such as Alzheimer's disease, where central cholinergic function is progressively compromised.

Drug treatments to reduce excitotoxicity in vivo: a potential for alpha2-adrenoceptor antagonists?

It is hypothesized that the locus coeruleus-noradrenergic system controls compensatory and repair mechanisms in the CNS, and that its dysfunction is a critical factor in the progression of central neurodegenerative diseases. Pharmacological activation of locus coeruleus neurons can be achieved with alpha2-adrenoceptor antagonists, and such compounds are protective in vivo in some models of brain injury where excitotoxicity is thought to be a causative factor. To further explore this neuroprotective potential, the effects of a 7-day treatment with the alpha2-antagonists, (+)-efaroxan and (+/-)-idazoxan, were evaluated in rats undergoing a unilateral lesioning of the striatum with the excitotoxin, quinolinic acid. The alpha2-antagonist treatments reduced both the ipsiversive circling response to apomorphine and the deficit of choline acetyltransferase in the lesioned animals. To elucidate the mechanisms underlying this neuroprotective effect, a modulation of the extracellular levels of amino acids within the striatum was investigated using in vivo microdialysis. Intrastriatal injection of quinolinic acid increased taurine and tyrosine levels by 2-2.5 fold, while most other amino acids were not significantly altered; the effect of (+)-efaroxan on these changes is being investigated. Further research is required to identify which of several possible mechanisms is involved in the neuroprotective action of alpha2-antagonists in vivo.

Levodopa or D2 agonist induced dyskinesia in MPTP monkeys: correlation with changes in dopamine and GABAA receptors in the striatopallidal complex.

Dopamine D1 and D2 receptors as well as the GABA/benzodiazepine receptor complex in the striatum and the globus pallidus (internal: GPi and external: GPe) were studied by autoradiography using [3H]SCH 23390, [3H]spiperone, and [3H]flunitrazepam ([3H]FNZ) respectively, in five groups of cynomolgus monkeys. These included (i) untreated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-monkeys; (ii) MPTP monkeys treated chronically with levodopa injections; (iii) MPTP monkeys treated chronically with injections of the novel D2 agonist U91356A; (iv) MPTP monkeys treated chronically with U91356A delivered through an osmotic mini-pump; and (5) naive controls. Animals treated in a pulsatile mode with U91356A or levodopa injections showed progressive sensitization to their respective drug and developed choreic dyskinesia. In contrast, animals treated in a continuous mode with U91356A showed behavioral tolerance but did not develop dyskinesia. A trend for a down-regulation of putaminal D2 receptors was observed following D2 agonist stimulation with U913356A. Striatal [3H]FNZ binding was significantly decreased only in animals treated in a continuous mode with U91356A. The dopamine receptor decrease in the striatum could be implicated with the development of tolerance but cannot explain the appearance of dyskinesia. Denervation by MPTP was associated with a decrease of the GPe/GPi [3H]FNZ binding ratio which reflects an imbalance of striatal output pathways; this ratio was not reversed by any of the treatments although changes were observed in the GPe and GPi. Indeed, pulsatile U91356A treatment restored the decreased [3H]FNZ binding in the GPe near control values and levodopa showed a similar tendency. A significant increase of [3H]FNZ binding in the GPi only of dyskinetic monkeys, namely those treated with pulsatile U91356A or levodopa was seen compared to untreated MPTP or naive controls. This GABAA receptor up-regulation might lead to a supersensitive state of the GPi to gabaergic input which may be involved in the mechanism underlying the development of dopaminomimetic-induced dyskinesia.

Continuous administration decreases and pulsatile administration increases behavioral sensitivity to a novel dopamine D2 agonist (U-91356A) in MPTP-exposed monkeys.

We compared the behavioral effects of a novel and highly selective dopamine D2 receptor agonist, U-91356A, administered to 6 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-exposed parkinsonian monkeys for 27 days following an intermittent (n = 3) or continuous (n = 3) schedule, using subcutaneous osmotic minipumps for the latter group. Each group received equivalent amount of drug daily. Dopamine D1 and D2 receptor binding assays were performed on striatal tissue homogenates with tritiated selective antagonists and were compared with those of 3 healthy control animals and 3 MPTP-exposed monkeys treated in parallel with daily doses of levodopa and 2 additional MPTP-exposed monkeys otherwise untreated. U-91356A quickly relieved all parkinsonian features and greatly stimulated locomotion in all animals. The pulsatile administration group showed progressive sensitization to the drug, and all 3 animals developed chorea during the first week of treatment that subsequently increased in intensity. The same pattern was seen in the levodopa-treated animals. In contrast, an apparent, incomplete tachyphylaxis were observed in 2 of 3 animals in the continuous infusion group during the first 10 days of treatment. Only 1 of these animals developed minimal and transient choreic dyskinesia. An apparent decrease of D2 receptor binding was observed. No upregulation of dopamine receptors occurred in the dyskinetic monkeys of the pulsatile group, but a tendency toward upregulation of putaminal D1 receptors was observed in the levodopa-treated, dyskinetic animals. These results confirm that the mode of administration of dopaminergic agents may result in a markedly different clinical outcome.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain somatostatin receptors in spontaneously hypertensive rats: an autoradiographic study.

The apparent densities of brain somatostatin (SRIF) receptor sites were compared in adult spontaneously hypertensive rats (SH) and their normotensive genetic counterparts (Wistar-Kyoto; WKY) using quantitative receptor autoradiography. Globally, the distribution of brain [125I][Tyr0, D-Trp8]SRIF14 binding sites was very similar in both strains. However, apparent densities of specific labeling were either higher (subfornical organ, 3.2 x; locus coeruleus, 1.9 x; lateroanterior hypothalamic nucleus, 1.3 x) or lower (basolateral amygdaloid nucleus, 0.8 x; spinal trigeminal sensory nucleus, 0.6 x) in SH than WKY rats in areas especially relevant to CNS cardiovascular integration. This provides further evidence for the possible involvement of brain SRIF neurons in cardiovascular regulation.

Comparative structural requirements of brain neuropeptide Y binding sites and vas deferens neuropeptide Y receptors.

A series of fragments and analogues of neuropeptide Y (NPY), both human (hNPY) and porcine (pNPY), were synthesized and tested for their affinities at brain NPY receptor binding sites and their potencies in inhibiting the electrically stimulated twitch response of rat vas deferens. Results with N- and C-terminal fragments suggest that amino acid residues in the N-terminal portion of the molecule are mostly important for recognition of brain and vas deferens NPY receptors, in addition to being relevant for the maintenance of adequate receptor affinity. On the other hand, C-terminal amino acid residues appear to be responsible for triggering receptor activation in the rat vas deferens preparation, because full intrinsic activity is maintained with fragments up to NPY18-36. C-terminal fragment NPY25-36 and N-terminal fragment NPY1-15 were devoid of affinity for [3H]NPY brain receptor sites and showed no activity in the rat vas deferens preparation. Similarly, N-terminal fragment hNPY1-24CONH2 showed no affinity toward [3H]NPY brain receptor sites and no inhibition of the twitch response in the rat vas deferens preparation at concentrations up to 1.0 microM. On the contrary, this fragment appears to selectively increase the amplitude of the twitch response to electrical stimulation at low micromolar concentrations, an effect opposite to that of NPY and all other NPY fragments and analogues studied here. The exact mechanism mediating this contractile action of hNPY1-24CONH2 remains to be established. Modifications of the tyrosine residue in position 20 led to the development of two analogues, [D-Tyr20]hNPY and [D-Trp20]hNPY, which show an apparent preference for the vas deferens NPY receptor. On the other hand, substitutions of the tyrosine residue in position 21 by a phenylalanine ([Phe21]hNPY) or a methylated tyrosine residue ([Tyr-O-Me21]hNPY) produced analogues demonstrating an apparent preference for the brain receptor site. This suggests that modifications of tyrosine residues at positions 20 and/or 21 may eventually lead to the development of NPY analogues distinguishing between the most abundant class of sites present in the brain and vas deferens, respectively.

Structural study of the N-terminal segment of neuropeptide tyrosine.

A series of analogues of neuropeptide tyrosine (NPY) was synthesized by solid-phase peptide synthesis using BOP as a coupling reagent for the complete synthesis. A structure-activity study of the N-terminal portion of the molecule was performed with the analogues obtained by the successive replacement of the first 10 amino acids by the residue L-alanine. NPY and its analogues [Ala1-10]hNPY were tested for their potency on rat vas deferens and for their affinity to central nervous system receptors on a rat brain membrane preparation. The results suggest that the hypothetical polyproline type II helix structure of the N-terminal segment is involved in both potency and affinity. Indeed, the substitution by L-Ala of proline residues in position 2, 5, or 8 showed important losses of activity and affinity. The more important losses were observed with the replacement of Pro-5 or Pro-8. A critical loss of potency of hNPY was also observed after the substitution of the Tyr-1 residue by L-Ala, thus confirming the important role played by this residue for the full expression of the biological activity of NPY.

Neuropeptide Y receptor binding sites in human brain. Possible alteration in Alzheimer's disease.

Neuropeptide Y (NPY) and peptide YY (PYY) receptor sites were studied in human brain using saturation binding experiments and receptor autoradiography. Additionally, the affinities and densities of [3H]NPY binding sites were compared in the temporal cortex, hippocampus and putamen of patients dying from Alzheimer's disease (AD) and aged matched controls. High densities of [3H]NPY binding sites were found in the putamen (192 +/- 32 fmol/mg protein), followed by the hippocampus (165 +/- 42 fmol/mg protein) and temporal cortex (118 +/- 19 fmol/mg protein). Receptor autoradiography revealed that these sites were especially concentrated in certain layers of the hippocampus, laminae I and IV-V of the temporal cortex and the amygdalo-hippocampal area. No significant changes in [3H]NPY binding affinities were seen between the AD and aged-matched groups (Kd ranges: 2.5-6.8 nM). However, significant decreases in [3H]NPY receptor densities (Bmax) were found in temporal cortex (-43%) and hippocampus (-49%) in AD brains. No significant change in [3H]NPY Bmax values was found in the putamen. It is therefore possible that decreases in [3H]NPY receptor densities may be associated to the degenerative process taking place in certain brain regions in AD, although further work will be necessary to confirm this hypothesis. Part of this work was presented at the 17th Annual Meeting of the Society for Neuroscience.

Neuropeptide Y: localization in the central nervous system and neuroendocrine functions.

Neuropeptide Y (NPY) is a 36-amino acid peptide first isolated and characterized from porcine brain extracts. A number of immunocytochemical investigations have been conducted to determine the localization of NPY-containing neurons in various animal species including both vertebrates and invertebrates. These studies have established the widespread distribution of NPY in the brain and in sympathetic neurons. In the rat brain, a high density of immunoreactive cell bodies and fibers is observed in the cortex, caudate putamen and hippocampus. In the diencephalon, NPY-containing perikarya are mainly located in the arcuate nucleus of the hypothalamus; numerous fibers innervate the paraventricular and suprachiasmatic nuclei of the hypothalamus, as well as the paraventricular nucleus of the thalamus and the periaqueductal gray. At the electron microscope level, using the pre- and post-embedding immunoperoxidase techniques, NPY-like immunoreactivity has been observed in neuronal cell body dendrites and axonal processes. In nerve terminals of the hypothalamus, the product of the immunoreaction is associated with large dense core vesicles. In lower vertebrates, including amphibians and fish, neurons originating from the diencephalic (or telencephalic) region innervate the intermediate lobe of the pituitary where a dense network of immunoreactive fibers has been detected. At the ultrastructural level, positive endings have been observed in direct contact with pituitary melanotrophs of frog and dogfish. These anatomical data suggest that NPY can act both as a neurotransmitter (or neuromodulator) and as a hypophysiotropic neurohormone. In the rat a few NPY-containing fibers are found in the internal zone of the median eminence and high concentrations of NPY-like immunoreactivity are detected in the hypothalamo-hypophyseal portal blood, suggesting that NPY may affect anterior pituitary hormone secretion. Intrajugular injection of NPY causes a marked inhibition of LH release but does not significantly affect other pituitary hormones. Passive immunoneutralization of endogenous NPY by specific NPY antibodies induces stimulation of LH release in female rats, suggesting that NPY could affect LH secretion at the pituitary level. However, NPY has no effect on LH release from cultured pituitary cells or hemipituitaries. In addition, autoradiographic studies show that sites for 125I-labeled Bolton-Hunter NPY or 125I-labeled PYY (2 specific ligands of NPY receptors) are not present in the adenohypophysis, while moderate concentrations of these binding sites are found in the neural lobe of the pituitary. It thus appears that the inhibitory effect of NPY on LH secretion must be mediated at the hypothalamic level.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative autoradiographic distribution of [125I]Bolton-Hunter neuropeptide Y receptor binding sites in rat brain. Comparison with [125I]peptide YY receptor sites.

The autoradiographic distribution of [125I]Bolton-Hunter neuropeptide Y receptor binding sites was quantified in rat brain. The highest level of [125I]Bolton-Hunter neuropeptide Y binding sites was seen in the hippocampus (ventral stratum radiatum, CA3 subfield: 6029 +/- 250 fmol/g tissue). The distribution of these sites was clearly laminated, being particularly concentrated in the oriens layer (dorsal CA3 subfield: 2562 +/- 147 fmol/g tissue) and stratum radiatum (dorsal CA3 subfield: 2577 +/- 95 fmol/g tissue). Lower levels of sites were seen in the pyramidal cell layer (1708 +/- 105 fmol/g tissue) and the molecular layer (1155 +/- 116 fmol/g tissue). The cortical distribution of [125I]Bolton-Hunter neuropeptide Y receptor sites was also laminated, being particularly enriched in superficial laminae (occipital cortex, layers I-II, 4038 +/- 148 fmol/g tissue; layers III-IV, 1392 +/- 97 fmol/g tissue and layers V-VI, 1522 +/- 138 fmol/g tissue). Other areas containing high amounts of sites included the anterior olfactory nuclei (ventral part, 4935 +/- 119 fmol/g tissue; lateral part, 4530 +/- 149 fmol/g tissue; dorsal part, 3378 +/- 140 fmol/g tissue and medial part, 2601 +/- 150 fmol/g tissue); anteromedial (5168 +/- 211 fmol/g tissue), medial (4611 +/- 107 fmol/g tissue) and lateral posterior thalamic nuclei (4465 +/- 189 fmol/g tissue); medial mammillary nucleus (5555 +/- 241 fmol/g tissue); medial geniculate nucleus (4747 +/- 56 fmol/g tissue); claustrum (4123 +/- 235 fmol/g tissue); posteromedial cortical amygdaloid nucleus (3524 +/- 138 fmol/g tissue), tenia tecta (2540 +/- 195 fmol/g tissue); lateral septum (1785 +/- 90 fmol/g tissue); suprachiasmatic hypothalamic nucleus (1604 +/- 115 fmol/g tissue), and substantia nigra, pars compacta (1846 +/- 142 fmol/g tissue) and pars lateralis (1750 +/- 165 fmol/g tissue). Areas moderately enriched with [125I]Bolton-Hunter neuropeptide Y binding sites included the zonal layer of the superior colliculus (1347 +/- 71 fmol/g tissue); anterior pretectal nucleus (1172 +/- 113 fmol/g tissue); ventral tegmental area (1090 +/- 97 fmol/g tissue); periventricular fibre system (1026 +/- 48 fmol/g tissue); core of nucleus accumbens (948 +/- 29 fmol/g tissue) and area postrema (799 +/- 87 fmol/g tissue). These results are discussed with regard to some of the suggested biological effects of neuropeptide Y in the central nervous system such as effects on learning, locomotion and circadian rhythms. Moreover, we also compared the distribution of [125I]Bolton-Hunter neuropeptide Y receptor sites with that of [125I]peptide YY sites in rat brain. The resolution of the autoradiographic image is better with [125I]peptide YY most likely because of higher affinity and percentage of specific labelling.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative radioautographic study of somatostatin receptors heterogeneity in the rat extrahypothalamic brain.

The possible heterogeneity of extrahypothalamic somatostatin receptors was studied in rat brain by quantitative radioautography. The respective distribution and relative proportion of two somatostatin receptor sub-types (SS1 and SS2) were assessed by using two radioligands, the non-selective probe [125I]Tyr3-D-Trp8-somatostatin14 and the SS1 selective analogue [125I]Tyr3-SMS 201-995. For both ligands, adjacent brain sections were processed in the presence of micromolar concentrations of either a non-discriminative competitor (somatostatin14) or SS1-selective analogue (SMS 201-995). The comparative analysis of the specific binding remaining in the presence of each non-radioactive competitor permitted a semi-quantitative analysis of the proportion of SS1 and SS2 receptor sub-types in each brain region examined. Data obtained correlate well with homogenate binding results reported previously [Reubi J. C. (1984) Neurosci. Lett. 49, 259-263]. Although the distribution patterns obtained with both radioligands were similar, [125I]Tyr3-SMS 201-995 labelled only a fraction of [125I]Tyr0-D-Trp8-somatostatin14-labelled sites in certain brain regions. For example, both superficial and deep cortical laminae, as well as the basolateral amygdaloid nucleus and CA1 hippocampal area exhibited different binding densities with [125I]Tyr0-D-Trp8-somatostatin14 depending on the competitor used in the assay (somatostatin14 or SMS 201-995). On the other hand, [125I]Tyr3-SMS 201-995 binding was eliminated in an identical fashion by either competitor in these very same brain areas. This suggests the existence of SS1 and SS2 somatostatin receptor sub-types in these regions. In all other brain areas examined, somatostatin receptor sites are apparently of the SS1 sub-type. The heterogeneity of somatostatin receptors observed in certain regions may have relevance for the various biological effects induced by somatostatin in the central nervous system.

Radioautographic analysis of somatostatin receptor sub-type in rat hypothalamus.

Hypothalamic somatostatin (SRIF) receptors were examined in a qualitative and quantitative radioautographic study using [125I-Tyr0,D-Trp8]SRIF14 and the stable octapeptide analog [125I-Tyr3]SMS 201-995 as radioligands. The latter has been shown to bind selectively to the high-affinity SS1 receptor subtype. Both radioligands labeled specifically and with high resolution various hypothalamic nuclei. In addition, the labeling patterns obtained with the two probes were identical; in both cases specific binding density was highest in the preoptic area and lowest in the ventromedial hypothalamic nucleus. Inhibition of the specific binding of each radioligand by either SRIF14 or the SS1-selective (SMS 201-995) unlabeled competitor was assessed on serial sections throughout the hypothalamus. The proportions of both non-selective and SS1-selective binding, remaining in the presence of either SRIF14 or SMS 201-995 (micromolar concentrations) were identical. These results indicate the existence of a homogeneous class of SRIF binding sites of the SS1 type in the hypothalamus.