BP 897, a selective dopamine D3 receptor ligand with therapeutic potential for the treatment of cocaine-addiction.

BP 897 is a potent (K(i) = 0.92 nM) dopamine D(3) receptor compound developed for the treatment of cocaine abuse and craving. BP 897 has a high selectivity for the dopamine D(3) versus D(2) receptors (70-fold) and a moderate affinity for 5-HT(1A) receptors, (K(i) = 84 nM), adrenergic-alpha(1) (K(i) = 60 nM) and -alpha(2) adrenoceptors (K(i) = 83 nM). BP 897 displays significant intrinsic activity at the human dopamine D(3) receptor by decreasing forskolin-stimulated cAMP levels and by stimulating mitogenesis of dopamine D(3)-expressing NG108-15 cells. Although these findings suggest that BP 897 is a partial agonist, recent studies in Chinese Hamster Ovary (CHO) cells with expressed dopamine D(3) receptors demonstrated that BP 897 is devoid of any intrinsic activity but potently inhibits dopamine agonist effects (pIC(50) = 9.43 and 9.51) in agonist-induced acidification rate or increase of GTPgammaS binding, respectively. In addition, BP 897 inhibits in vivo (EC(50) = 1.1 mg/kg, i.v.) agonist-induced decrease of firing rate of dopaminergic neurons in the substantia nigra. It has been clearly shown that BP 897, 1 mg/kg, i.p., reduces cocaine-seeking behavior in rats, without producing reinforcement on its own. In rhesus monkeys, BP 897 is not self-administered (up to 30 microg/kg, i.v.) but reduces cocaine self-administration. The potential usefulness of BP 897 in the treatment of drug-seeking behavior is further supported by its effects in drug conditioning models. Although BP 897 reduces L-DOPA-induced dyskinesia in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, it provokes a return of parkinsonian symptoms. At high doses BP 897 has been reported to produce catalepsy in rats. Pharmacokinetic and toxicological data have not yet been published. These interesting preclinical findings with BP 897 provide additional validation for dopamine D(3) receptor as a therapeutic target for the treatment of cocaine abuse and its associated central nervous system (CNS) disorders. BP 897 recently entered phase II clinical studies.

Oligodendrocytes express different isoforms of beta-amyloid precursor protein in chemically defined cell culture conditions: in situ hybridization and immunocytochemical detection.

The expression of beta-amyloid precursor protein (betaAPP) by astrocytes is well documented; however, data concerning oligodendrocytes remain controversial. The main goal of the present study was to determine whether or not oligodendrocytes in culture constitutively express the different betaAPP isoforms. Oligodendrocytes were cultured in a chemically defined medium that avoids putative effects of unknown serum factors on oligodendrocyte development. We have employed immunocytochemistry and in situ hybridization with antibodies and synthetic oligonucleotides recognizing, respectively, specific protein epitopes and mRNA transcripts of rat betaAPP isoforms. Oligodendrocytes, in both mixed primary cultures in the presence of serum or in secondary cultures in defined medium, were clearly labeled by antibodies directed to different betaAPP sequences. Antibodies against the serine protease inhibitor domain of betaAPP, also strongly labelled oligodendrocytes. Immunohistochemistry and in situ hybridization were combined to determine precisely the expression of different isoforms of betaAPP. In situ hybridization revealed the presence in oligodendrocytes of mRNA transcripts coding not only for betaAPP695 but also for betaAPP770 and betaAPP751. This indicates that betaAPP immunoreactivity found in oligodendrocytes corresponds to constitutive expression of betaAPP. Oligodendrocyte cultured in chemically defined medium are able to express not only betaAPP695 but also betaAPP770, betaAPP751 isoforms containing the Kunitz protease inhibitor domain. Although the role of betaAPP in the pathological processes of Alzheimer's disease (AD) remains unknown, possible disturbances of betaAPP processing and/or synthesis in oligodendrocytes may account for some myelin disorders observed in AD and other senile dementias.

Ventral mesencephalic grafts in the neostriatum of the weaver mutant mouse: structural molecule and receptor studies.

Mesencephalic cell suspensions were prepared from E12 wild-type (+/+) mouse embryos and stereotaxically implanted into the dorsal neostriatum of weaver mutant mice (wv/wv), which have a genetic mesostriatal dopamine (DA) deficiency. Survival of DA neurons in the grafts was documented by tyrosine hydroxylase (TH) immunocytochemistry. Axon growth was monitored by immunocytochemistry using a battery of antibody markers, and the cellular localization of structural protein and receptor RNA transcripts was studied by in situ hybridization histochemistry using [32P]oligonucleotide probes. The cell suspension grafts exhibited strong immunoreactivity for neural cell adhesion molecule (N-CAM), growth-associated phosphoprotein GAP-43, microtubule-associated protein 2 (MAP2), beta-amyloid protein precursor (beta APP), and phosphorylated neurofilament epitopes (clone SMI-31); intermediate-to-high levels of immunoreactivity were seen for synaptophysin. High levels of hybridization were found in the grafts for the RNA transcripts of GAP-43, MAP2, and isoforms beta APP695, beta APP714 and beta APP751 of the beta APP. No hybridization signal was detected in the grafts for DA D2 or neurotensin receptor mRNAs, both of which are normally expressed by nigral DA neurons. DA receptor autoradiography using the D2/D3 agonist [3H]CV 205-502 as a ligand showed no binding in the transplants, indicating an apparent abnormality of grafted cells; neurotensin binding sites, labeled with [125I]neurotensin, were visualized in the suspensions, indicating the possibility that receptors could be present but that RNA message levels might be too low to allow detection. These findings offer a molecular correlate of axonal, dendritic and structural protein expression by transplanted mesencephalic neurons; further, they suggest that specific functional properties of grafted nigral cells are maintained after transplantation, while other aspects of their cellular biology may be compromised.

Excitatory amino acid AMPA receptor mRNA localization in several regions of normal and neurological disease affected human brain. An in situ hybridization histochemistry study.

In situ hybridization histochemistry was used to localize the mRNAs coding for four alpha-aminoisoxazole propionic acid-sensitive glutamate receptor subunits in human brain (age range 51-95 years, postmortem delay 4.5-10 h). High levels of the B receptor subunit mRNA were present in all the studied regions, followed by the A-subunit and the C-subunit. Only very low levels of the D-subunit mRNA were detected. In hippocampus, the mRNA coding for the B-subunits of the glutamate receptor was observed in granule cells of dentate gyrus and in the pyramidal cells of Ammon's horn. In cortex, the highest levels of glutamate receptor subunit mRNAs were found in layer I and layers III-IV of entorhinal and temporal cortex, although significant levels were also observed in the other cell layers. A differential distribution was seen in cerebellum where the A-subunit mRNA is expressed mainly by Purkinje cells, while the B-subunit mRNA is present in the internal granule cell layer. These results correlate well with previous data from autoradiographic studies on the localization of excitatory amino acid binding sites in human brain and pinpoint the cells where these receptors are synthesized. In situ hybridization in the hippocampus of patients affected by Alzheimer's disease (age range 77-82 years, postmortem delay 19-25.5 h) revealed a decrease on the content of the mRNAs coding for these excitatory amino acid receptors, while an increase was detected in surgically dissected epileptic human hippocampi. These results corroborate and extend the previous data from in vitro autoradiography and suggest alteration of the excitatory amino acid disfunction during these neurodegenerative processes.

Autoradiographic localization of [3H]-L-glutamate binding sites in a model of cerebellar granule cell ectopia generated by methylazoxymethanol treatment.

The distribution of [3H]glutamate binding sites was studied in a model of altered cerebellar development obtained by injecting methylazoxymethanol (MAM) in 5-day-old mice. In these mice, at the 25th postnatal day, cerebella were smaller than normal, stratification was normal except for the presence in some lobes of a thin ectopic granule cell layer in the middle of the molecular layer, the proportion of the distribution of [3H]glutamate binding sites between molecular and internal granule cell layers was maintained but site density of both quisqualate- and NMDA-sensitive types was increased in the two layers. In the molecular layer, this increase was uniform in spite of the presence of the ectopic cell layer. In the internal granular layer, the increase of quisqualate-sensitive and NMDA-sensitive [3H]glutamate binding sites is topographically segregated and the first corresponds to areas of lesser cellular density. These results show that MAM treatment induces persistent alterations of the cerebellar glutamatergic system, which consist of receptor over-expression, possibly due to deficit of innervation, reactive gliosis and immaturity of surviving granule cells.

Beta APP gene expression is increased in the rat brain after motor neuron axotomy.

The response of the beta APP gene to neuronal injury was studied in the facial and hypoglossal nerve nuclei of the rat after corresponding nerve axotomy. Increased levels of beta APP 695, 714, 751 and 770 mRNAs were observed after either facial or hypoglossal nerve axotomy in the parent ipsilateral motor neurons. The increase was gradual, with maximal values 7 days after axotomy. beta APP mRNA expression returned to normal values 60 days after the lesion. Increased beta APP immunostaining was also detected in ipsilateral chromatolytic motor neurons. No change in beta APP immunoreactivity was observed in oligodendrocytes, another cell type expressing beta APP under normal conditions. A rapid increase in the expression of the GFAP gene was observed in reactive astrocytes surrounding chromatolytic neurons in the ipsilateral facial or hypoglossal nuclei. Thus, in contrast with other models of neuronal injury, where only the Kunitz protease inhibitor-containing beta APP mRNA isoforms are increased, all beta APP mRNAs are increased in the axotomy model. Furthermore, although beta APP expression has been shown to be increased in reactive astrocytes following neuronal injury, in the present study the increase was essentially found in the motor neurons reacting to axotomy.

Denervation hypersensitivity of histamine H1-receptors in rat brain cortex.

We have studied the effects of the unilateral electrolytic lesion of the medial forebrain bundle at the level of the lateral hypothalamus on the density and functionality of histamine H1 receptors in rat brain cortex. The treatment resulted, after two and four weeks, in an increase in the maximal phosphoinositide breakdown induced by histamine, which can be accounted for by the appearance of a higher potency component for the response. On the other hand, the density of cortical histamine H1 receptors, determined by the specific binding of [3H]mepyramine to membranes, remained unchanged two weeks after the lesion but after four weeks a small but significant increase was also found. These results suggest that the denervation hypersensitivity developed may initially be the result of a more efficient coupling of the H1 receptors to the effector system prior to the subsequent increase in receptor numbers.

Regional distribution of transient [3H]kainic acid-binding sites in the central nervous system of the developing mouse: an autoradiographic study.

[3H]kainate-binding site distribution in mouse brain was studied by in vitro autoradiography during postnatal development. Sites, highly concentrated at early postnatal ages and undetectable at adult ages, were observed in deep cerebellar nuclei, inferior olive, pontine nuclei, inferior colliculus and stratum lacunosum moleculare of the area CA1 in the hippocampus as well as in previously described rat brain areas. It is suggested that the molecules carrying these sites play a role in the development of the regions where they are transiently expressed.

Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and low-affinity nerve growth factor receptor during the postnatal development of the rat cerebellar system.

The spatio-temporal pattern of expression of neurotrophin-3 (NT3), brain-derived neurotrophic factor (BDNF) and low-affinity nerve growth factor receptor (LNGFR) genes was analyzed in the postnatal developing cerebellar system of the rat by in situ hybridization histochemistry. Different ontogenetic patterns of expression were observed for these three genes. In agreement with previously published results (Neuron, 5 (1990) 501-509; Dev. Brain Res., 55 (1990) 288-292) we found that NT3 and LNGFR mRNA peaked early, during the first 2 postnatal weeks, whereas BDNF mRNA peaked later, around postnatal day 20, in the cerebellar cortex. High levels of NT3 mRNA were found in the internal granule cell layer as early as postnatal day 5. NT3 mRNA was also present in the external-premigratory granule cell layer at postnatal day 10. From postnatal day 5 on, LNGFR mRNA was present in the proliferative area of the external granule cell layer and in the Purkinje cells. NT3 mRNA level decreased and BDNF mRNA increased in granule cells concomitantly with their migration and maturation, suggesting a sequential stimulation of these two genes during this developmental process. LNGFR mRNA levels decreased along the same period. Although practically undetectable in the cerebellar granule cell layer in the first two postnatal weeks, BDNF mRNA was transiently expressed in the deep cerebellar nuclei during this time and it was very abundant in the inferior olivary system from postnatal day 5 on. LNGFR mRNA was transiently expressed in the inferior olivary system, in the first postnatal week. These data are discussed in relation to the coordinated postnatal maturation of the different cells of the cerebellar system.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased levels of the Kunitz protease inhibitor-containing beta APP mRNAs in rat brain following neurotoxic damage.

Deposits of beta-amyloid are one of the main pathological characteristics of Alzheimer's disease. The beta-amyloid peptide (or beta/A4) constituent of these deposits is derived from the beta-amyloid precursor protein (beta APP), which is expressed in several isoforms. It has been suggested that an imbalance in the normal ratio between the Kunitz protease inhibitor (KPI)-containing beta APPs versus the non containing forms could result in altered processing of beta APP and progressive beta/A4 deposition. We have studied the expression of four beta APP isoforms in the rat brain after intracerebroventricular application of kainic acid. Increased levels of the KPI-containing beta APP and GFAP mRNAs were observed in tissues surrounding areas of neuronal damage. A parallel increase of beta APP and GFAP immunoreactivity was observed in reactive astrocytes in these areas. These results suggest that the normal ratio of beta APP isoforms may be profoundly altered as a result of neuronal damage and that non-neuronal cells may respond to neuronal injury by increased expression of the KPI-containing beta APP isoforms.

Ectopic granule cells in mouse cerebellum molecular layer express high affinity GABAA receptor.

The antimitotic/mutagenic agent methylazoxymethanol when injected in mice pups at postnatal days 3 and 4 produces hypogranular adult cerebella with subpial cells clusters and with a supplementary, ectopic granule cells layer in the molecular layer. The internal granular layer of these treated mice displayed a much lower density of [3H]muscimol binding sites than in controls. At the same time these binding sites are expressed in the ectopic granule cells in the middle of the molecular layer, in spite of the unusual localization of the cells and of the alteration of their nerve inputs. The subpial cells do not express these sites. Our autoradiographic data confirm the suggestion that during ontogeny the GABAA receptors in the granule cells appear when these cells leave the subpial region and indicate that the expressed receptor subtype is the same whether granule cells are in the internal granular layer or in the middle of the molecular layer.

Ectopic granule cell layer in mouse cerebellum after methyl-azoxy-methanol (MAM) treatment.

Previous results from our laboratory (Bejar et al. 1985) indicated that a single injection in mouse pups of the antimitotic/mutagenic agent methylazoxymethanol at postnatal day 5 typically produces hypogranular cerebella with no changes in foliation, in contrast to the severe alterations observed after the more usual injection on the day of birth. Here we report that injection of a higher dose (30 mg/kg) of methylazoxymethanol, always at postnatal day 5, leads to the additional presence of a ectopic cell layer in adult cerebellum. Immunostaining with several antibodies recognizing cell specific proteins ruled out the possibility that these ectopic cells were glial and electron microscopy indicated that they were morphologically mature granule cells. In the molecular layer of other cerebellar areas and apparently unrelated with granule cell ectopia, ectopic Golgi epithelial cells were observed. The reason for the presence of these ectopic cells of different type in the molecular layer was discussed in relation with analogous ectopias obtained by other means.

Autoradiographic characterization of [3H]L-glutamate binding sites in developing mouse cerebellar cortex.

Postnatal changes of [3H]L-glutamate binding sites in mouse cerebellum were studied by in vitro autoradiography. These sites were already present at birth, their density globally increased until postnatal day 25, and at all ages it was higher when Cl- and Ca2+ were present in the incubation buffer. At birth, these binding sites were diffused through the whole cerebellar mass, but became distinctly concentrated in the molecular and the internal granular layers by postnatal day 10. From this age on, binding site sensitivity to ions and glutamate analogues takes a different course in each layer. The external granular layer and the white matter never displayed significant amounts of binding. In the molecular layer the Cl-/Ca2+ effect increased during ontogeny until, in adults, the ion-dependent binding was threefold higher than the ion-independent binding. Quisqualate-sensitive sites accounted for 80% of the total binding sites already at postnatal day 15, while displacement by alpha-amino-3-hydroxy-methyl-4-isoxazolepropionic and ibotenic acids attained the maximum (68%) at postnatal day 60. N-Methyl-D-aspartate displaced glutamate binding (50%) only in the presence of Cl- and Ca2+. Starting from postnatal day 15, binding site density in the molecular layer of lobules VIb and VII of the vermis was lower than in other lobules. In the internal granular layer, the Cl-/Ca2+ effect observed in young animals decreased during development. These transient binding sites were sensitive to quisqualic and ibotenic acid. In adults, the majority of glutamate binding sites were ion-independent and mainly sensitive to D,L-amino-5-phospho-valeric acid and N-methyl-D-aspartate. Throughout development and in both layers, sites displaced by kainate were present at low density and sites displaced by D,L-2-amino-4-phosphonobutyric acid were not detected. The localized postnatal changes of the [3H]L-glutamate binding sites were correlated with the events occurring during growth and maturation of cerebellar structures. The increase of the Cl-/Ca(2+)-dependent binding in the molecular layer is simultaneous with the growth of Purkinje cell dendrites and of parallel fibres and with the formation of the synapses between them. This suggests that these binding sites are localized in these synapses. The changing pattern of sensitivity to different agonists during development might correspond to the maturation of these synapses. The low density of [3H]L-glutamate binding in the molecular layer of lobules VIb and VII probably indicates the presence of specific nerve projections to these areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmentally regulated changes of glutamate binding sites in mouse deep cerebellar nuclei.

The expression of L-[3H]glutamate binding sites of different ionic and pharmacological sensitivities was studied in mouse deep cerebellar nuclei during early postnatal development by means of in vitro autoradiography. Ca2+/Cl(-)-dependent, quisqualate/AMPA/ibotenate-sensitive, and APB-insensitive binding sites are present at high density in the deep cerebellar nuclei of young animals, but greatly decrease between the 10th and 25th postnatal day and remain low in the adult. The density of Ca2+/Cl(-)-independent binding sites remains low and constant during the whole of postnatal development. The possible involvement of the Ca2+/Cl(-)-dependent binding sites in brain development is discussed.