Association of neuropeptide Y Y1 receptors with glutamate-positive and NPY-positive neurons in rat hippocampal cultures.

The hippocampus is particularly enriched with neuropeptide tyrosine (NPY) and NPY receptors including the Y1, Y2 and Y5 subtypes. We have previously reported on the enrichment of cultured rat hippocampal neurons in specific [125I][Leu31, Pro34]PYY/BIBP3226-sensitive (Y1) binding sites and Y1 receptor mRNAs [St-Pierre et al. (1998) Br. J. Pharmacol., 123, p183]. We have now identified which cell types express the Y1 receptor. The majority of Y1 receptors, visualized using either the radiolabeled probe [125I][Leu31,Pro34]PYY or two antibodies directed against distinct domains of the Y1 receptor, was expressed in neurons as revealed by neuron-specific enolase (NSE) immunostaining. One antibody was directed against the second extracelllular loop of the Y1 receptor (amino acids 185-203) whereas the second was directed against the intracellular C-terminal loop (amino acids 355-382). The labelling was evident over both perikarya and processes. Neurons labelled by the various Y1 receptor probes were mostly glutamate-positive as revealed by double immunostaining. Most interestingly, a number of NPY-positive cultured hippocampal neurons were also enriched with the Y1 receptor, suggesting that this subtype may act as an autoreceptor to regulate NPY release in the hippocampus. These results thus provide an anatomical basis for the modulation of glutamate and NPY release by the Y1 receptor in the hippocampus.

Sub-population of cultured hippocampal astrocytes expresses neuropeptide Y Y(1) receptors.

The expression and pharmacological characterization of neuropeptide Y (NPY) receptors of the Y(1) subtype on cultured hippocampal neurons was reported using radioreceptor assays and immunohistochemical approaches (St-Pierre et al., 1998). The present study aimed to establish the presence of NPY Y(1) receptors on cultured hippocampal astrocytes using similar strategies. Immunocytochemical experiments were carried out using three antisera directed against distinct domains (amino acids sequence 185-203, 198-213 and 355-382) of the Y(1) receptor. Double-labeling experiments and confocal microscopy with these Y(1) receptor antisera demonstrated their recognition of the same sub-population (20%) of GFAP-positive astrocytes in culture. The immunostaining seen with all three Y(1) receptor antisera took the form of large irregular clusters distributed throughout cell bodies and processes. Further experiments using radioactive ligands confirmed the presence of NPY receptors on cultured hippocampal astrocytes. Emulsion receptor autoradiography using a newly developed ligand, [(125)I]GR231118 in the presence of PYY, hPP or BIBP3226 (1 microM), pharmacologically established the Y(1) nature of these receptors. Specific [(125)I]GR231118 binding was competed by PYY and the selective Y(1) antagonist BIBP3226 but not by hPP (a Y(4)/Y(5) agonist). Similar autoradiographic labeling patterns were obtained using [(125)I][Leu(31).Pro(34)]PYY (a Y(1)/Y(4)/Y(5) agonist) whereas [(125)I]PYY(3-36) (a Y(2)/Y(5) agonist) failed to generate any specific signal. Hence, rat cultured hippocampal astrocytes express a typical Y(1) receptor without evidence for the presence of Y(2), Y(4) or Y(5) subtypes. These data suggest a preferential regulation by NPY, acting via the Y(1) receptors, of astrocytic function.

Multiple receptors for neuropeptide Y in the hippocampus: putative roles in seizures and cognition.

Neuropeptide Y (NPY) is widely distributed throughout the central nervous system (CNS) and is one of the most conserved peptides in evolution, suggesting an important role in the regulation of basic physiological functions, including learning and memory. In addition, experimental studies have suggested that NPY, together with its receptors, may have a direct implication in several pathological disorders, including epilepsy/seizure. NPY-like immunoreactivity and NPY receptors have been shown to be present throughout the brain, but is concentrated in the hippocampus. The hippocampal formation has been repeatedly implicated in the modulation of cognition, as well as the pathogenesis of seizure. This review will concentrate on the hippocampal distribution of NPY, its receptors and the putative role played by this peptide in seizure, together with the regulation of cognitive function associated with learning and memory.

Preferential expression of the neuropeptide Y Y1 over the Y2 receptor subtype in cultured hippocampal neurons and cloning of the rat Y2 receptor.

1 Neuropeptide Y (NPY) and NPY receptors are most abundant in the hippocampal formation where they modulate cognitive functions. Expression of NPY receptors in rat cultured primary hippocampal cells was investigated in the present study by use of combined molecular, pharmacological and immunohistochemical approaches, including the cloning of the rat Y2 receptor described here for the first time. 2 More than 70% of the hippocampal neurones were endowed with [125I]-[Leu31,Pro34]PYY Y1-like receptor silver grain accumulations and Y1 receptor immunostaining. These radio- and immuno-labelling signals were distributed over cell bodies and processes of bipolar, stellate and pyramidal-like neuronal cells, as confirmed by neurone-specific enolase and MAP-2 staining. 3 Competition binding profiles revealed that specific [125I]-[Leu31,Pro34]PYY binding was competitively displaced according to a ligand selectivity pattern prototypical of the Y1 receptor sub-type with [Leu31,Pro34]substituted NPY/PYY analogues >> C-terminal fragments = pancreatic polypeptides, with the non-peptide antagonist BIBP3226 being most potent. This profile excludes the possible labelling by [125I]-[Leu31,Pro34]PYY of the newly cloned Y4, Y5 and Y6 receptors. 4 The expression of the genuine Y1 receptor was confirmed by RT-PCR in hippocampal cultures. In contrast, negligible levels of Y2-like/[125I]-PYY3-36 binding were detected in these cultures in spite of the presence of its mRNA, as characterized by RT-PCR. The expression of both the Y1 and the Y2 receptor mRNAs was also noted in normal embryonic hippocampal tissues showing that signals expressed in cultured neurones were also present in utero. 5 Taken together, these results suggest that the Y1 receptor subtype may be of critical importance in the normal functioning of the rat hippocampus, especially during brain development and maturation.

Differential binding profile and internalization process of neurotensin via neuronal and glial receptors.

Two G-protein-coupled receptors for the tridecapeptide neurotensin (NT) have been identified and cloned in mammalian brain: a high-affinity (Kd = 0.3 nM) receptor, sensitive to the antagonist SR 48692 but insensitive to levocabastine, and a lower-affinity (Kd = 2-4 nM) receptor, sensitive to levocabastine but with poor affinity for SR 48692. Although there is good evidence that the high-affinity site is predominantly expressed in neurons, little is known of the cellular localization of the low-affinity receptor. In the present study, we identify by confocal microscopy selective levocabastine-sensitive, SR 48692-resistant binding of a fluorescent derivative of NT (fluo-NT) to a subpopulation of glial fibrillary acidic protein-immunoreactive glial cells grown in culture from the midbrain and cerebral cortex of embryonic and neonatal rats, respectively. We also demonstrate, by combining fluo-NT detection with tyrosine hydroxylase immunofluorescence, that these glial binding sites are differentially regulated from the SR 48692-sensitive NT receptor expressed in the same cultures by mesencephalic dopamine neurons. Whereas the latter undergoes rapid ligand-induced internalization followed by centripetal mobilization of ligand-receptor complexes from processes to perikarya and from perikaryal periphery to cell center, the former induces the formation of cell-surface clusters that fail to internalize. It is concluded that NT may exert its effects on both neurons and astrocytes in the CNS. Whereas NT neural signaling is exerted through high-affinity receptors and may be partly effected through internalization of receptor-ligand complexes, glial signaling is exerted through low-affinity NT receptors and appears to be transduced exclusively at the level of the plasma membrane.

Comparative autoradiographic distribution of neuropeptide Y Y1 receptors visualized with the Y1 receptor agonist [125I][Leu31,Pro34]PYY and the non-peptide antagonist [3H]BIBP3226.

BIBP3226, a non-peptide neuropeptide Y Y1 receptor antagonist, was used in its tritiated form to investigate the autoradiographic distribution of its binding sites in the rat brain. [125I][Leu31,Pro34]pPYY a recently developed Y1 agonist radioligand was used for comparison. High levels of specific binding for both radioligands were seen in various areas of the rat brain, including superficial cortical laminae, anterior olfactory nucleus, claustrum, geniculate nuclei and medial mamilliary nucleus. However, a few regions, such as the external plexiform layer of the olfactory bulb, lateral septum nucleus tractus, solitarius and area postrema were only enriched with specific [125I][Leu31,Pro34]pPYY binding sites. These data suggest the possible heterogeneity of [125I][Leu31,Pro34]pPYY binding sites in the rat brain, a sub-population of which apparently fails to be recognized by the newly developed Y1 receptor antagonist, BIBP3226.

Systemic administration of the NMDA receptor antagonist MK-801 potentiates circling induced by intrastriatal microinjection of dopamine.

Systemic administration of the non-competitive antagonist of NMDA receptors MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) potentiates the circling response induced by direct stimulation of the striatal dopaminergic receptors through intracerebral application of dopamine. Microinjection of dopamine (1, 5, 25 or 50 micrograms/1.0 microliters) induced a dose-dependent contralateral circling response, when injected directly into the lesioned side of unilaterally 6-hydroxydopamine-lesioned rats. Interestingly, intrastriatal application of dopamine (1, 5, 25 or 50 micrograms/1.0 microliters) followed by a systemic administration of MK-801 (100 micrograms/kg i.p.) produced a potentiated contralateral circling response in unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. This motor effect is reversed compared to the marked ipsilateral circling response produced by MK-801 when given alone. Moreover, the potentiated responses persist 4-fold longer compared to the circling induced by dopamine alone. The results suggest that the potentiation by NMDA receptor antagonists of motor activity induced by dopaminergic agonists in animal models of Parkinson's disease cannot be ascribed simply to increased release of dopamine. Other mechanisms including increased sensitivity of dopamine D1 receptors or blockade or glutamatergic transmission in output structures must be considered.

GABAA receptor-mediated inhibition of N-methyl-D-aspartate-evoked [3H]dopamine release from mesencephalic cell cultures.

Direct activations of both GABAA and GABAB receptors are known to hyperpolarize dopaminergic neurons. However systemic or intra-ventral tegmental administration of a GABAA receptor agonist produces paradoxical depolarization of mesencephalic dopaminergic neurons and increases dopamine release. Thus indirect excitation appears to preclude observation of inhibitory GABAA effects on dopamine release in intact tissue. The present study used cultures of isolated cells from rat ventral mesencephalon to characterize effects of GABAA and GABAB receptor activation on evoked dopamine release. The GABAA receptor agonist, muscimol, produced a potent and complete inhibition of N-methyl-D-aspartate (NMDA)-evoked [3H]dopamine release. This effect was blocked by the GABAA receptor antagonist, picrotoxin, and enhanced by flunitrazepam. Omission of Mg2+ greatly reduced the inhibitory effect of muscimol on NMDA-evoked [3H]dopamine release. Muscimol had little or no effect on [3H]dopamine release evoked by the non-NMDA receptor agonists, quisqualate and kainate. The GABAB receptor agonist, baclofen, slightly inhibited NMDA-evoked [3H]dopamine release and had no effect on release evoked by quisqualate or kainate. Endogenous GABA released by the mesencephalic cells also appeared to inhibit NMDA-evoked [3H]dopamine release mainly via a GABAA receptor-mediated mechanism. This is suggested by the observations that NMDA-evoked [3H]dopamine release was potentiated by picrotoxin but not by the GABAB receptor antagonist, phaclofen, and that blockade of extracellular GABA removal, with amino-oxyacetic acid and beta-alanine, inhibited NMDA-evoked [3H]dopamine release in a picrotoxin-sensitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Intranigral but not intrastriatal microinjection of the NMDA antagonist MK-801 induces contralateral circling in the 6-OHDA rat model.

When systemically administered to unilaterally 6-OHDA lesioned rats, the non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine) produces a marked rotation response toward the lesioned side. In addition, the same type of injection in non-denervated rats increases locomotor activity without direction predominance. These findings suggest that MK-801 interacts with the dopaminergic pathway to induce motor activity. However, intracerebral injection of MK-801 (5 micrograms/0.5 microliter) in the substantia nigra pars reticulata (SNr), either on the lesioned side or the intact side, induces a significant circling response, contraversive to the injection side. A similar injection of MK-801 (5 micrograms/1.0 microliter) in the striatum causes no rotation response. These results show that the non-competitive NMDA antagonist can induce motor activity in the presence or in the absence of the dopaminergic pathway and that in the latter case its action is exerted predominantly at the level of the substantia nigra, perhaps through suppression of the excitatory subthalamic drive.

Excitatory and inhibitory amino acids in the cerebral cortex of nucleus basalis magnocellularis lesioned rats.

It is well known that pathways arising from the nucleus basalis magnocellularis in the basal forebrain which terminate in the cerebral cortex are involved in cognitive function. The cholinergic system is generally thought to play a large part in these processes from lesion, pharmacological and transplantation studies. With increasing evidence suggesting the involvement of amino acid transmitters in learning and memory processes, it is of interest to also evaluate possible changes in the levels of amino acid transmitters in the cortex of nucleus basalis magnocellularis-lesioned rats. In the present study, 9 cortical amino acids were measured in rats with bilateral lesions of the nucleus basalis magnocellularis. We measured significant reductions in aspartate, alanine and gamma-aminobutyric acid; these were 80%, 75%, and 81%, respectively, of control brain values. These results suggest that changes in the amino acid content of the cerebral cortex following lesion of the nucleus basalis magnocellularis-lesioned rat should perhaps also be considered when evaluating behavioral effects in this model.

Transsynaptic effect of an intrastriatal fetal nigral graft on the sensitivity of the substantia nigra pars reticulata to muscimol in the rat.

In the present study, we have investigated the effect of a unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway and of a fetal nigral graft in the striatum on circling induced by unilateral microinjection of muscimol in a target structure of the striatum, the substantia nigra pars reticulata. A group of ovariectomized female rats received a unilateral nigral lesion with 6-hydroxydopamine. The lesioned animals were then tested for apomorphine circling (0.25 mg/kg sc). The animals which displayed circling with this drug were divided into two groups with equivalent circling rate. One group received a graft of 1.5 x 10(6) cells taken from the ventral mesencephalon of 14- to 15-day-old rat embryos and the other was kept as control. Six months after the graft, all animals received one microinjection on each side of the GABAergic agonist muscimol (25 ng/0.5 microliters) in site A (rostral part) or in site B (caudal part) of the substantia nigra pars reticulata using indwelling guide cannulae. Circling was monitored during 90 min after the injection. Our results show that: (1) Unilateral degeneration of the nigrostriatal pathway was associated with a decrease of contralateral rotation when muscimol was injected in site A (rostral part) and an increase when administrated in site B (caudal part) of the substantia nigra pars reticulata on the lesioned side compared to the intact side. (2) A fetal nigral graft implanted into the striatum can normalize the changes in GABAergic sensitivity in site A but not in site B of the substantia nigra pars reticulata.