Receptor visualization and the atomic bomb. A historical account of the development of the chemical neuroanatomy of receptors for neurotransmitters and drugs during the Cold War.

This is a historical account of how receptors for neurotransmitters and drugs got to be seen at the regional, cellular, and subcellular levels in brain, in the years going from the end of the World War II until the collapse of the Soviet Union, the Cold War (1945-1991). The realization in the US of the problem of mental health care, as a consequence of the results of medical evaluation for military service during the war, let the US Government to act creating among other things the National Institute for Mental Health (NIMH). Coincident with that, new drug treatments for these disorders were introduced. War science also created an important number of tools and instruments, such as the radioisotopes, that played a significant role in the development of our story. The scientific context was marked by the development of Biochemistry, Molecular Biology and the introduction in the early 80's of the DNA recombinant technologies. The concepts of chemical neurotransmission in the brain and of receptors for drugs and transmitters, although proposed before the war, where not generally accepted. Neurotransmitters were identified and the mechanisms of biosynthesis, storage, release and termination of action by mechanisms such as reuptake, elucidated. Furthermore, the synapse was seen with the electron microscope and more important for our account, neurons and their processes visualized in the brain first by fluorescence histochemistry, then using radioisotopes and autoradiography, and later by immunohistochemistry (IHC), originating the Chemical Neuroanatomy. The concept of chemical neurotransmission evolved from the amines, expanded to excitatory and inhibitory amino acids, then to neuropeptides and finally to gases and other "atypical" neurotransmitters. In addition, coexpression of more than one transmitter in a neuron, changed the initial ideas of neurotransmission. The concept of receptors for these and other messengers underwent a significant evolution from an abstract chemical concept to their physical reality as gene products. Important steps were the introduction in the 70's of radioligand binding techniques and the cloning of receptor genes in the 80's. Receptors were first visualized using radioligands and autoradiography, and analyzed with the newly developed computer-assisted image analysis systems. Using Positron Emission Tomography transmitters and receptors were visualized in living human brain. The cloning of receptor genes allowed the use of in situ hybridization histochemistry and immunohistochemistry to visualize with the light and electron microscopes the receptor mRNAs and proteins. The results showed the wide heterogeneity of receptors and the diversity of mode of signal transmission, synaptic and extra-synaptic, again radically modifying the early views of neurotransmission. During the entire period the interplay between basic science and Psychopharmacology and Psychiatry generated different transmitter or receptor-based theories of brain drug action. These concepts and technologies also changed the way new drugs were discovered and developed. At the end of the period, a number of declines in these theories, the use of certain tools and the ability to generate new diagnostics and treatments, the end of an era and the beginning of a new one in the research of how the brain functions.

A preliminary investigation of phoshodiesterase 7 inhibitor VP3.15 as therapeutic agent for the treatment of experimental autoimmune encephalomyelitis mice.

cAMP plays a significant role in signal transduction pathways controlling multiple cellular processes such as inflammation and immune regulation. cAMP levels are regulated by a family of phosphodiesterases (PDEs). We have studied the effects of a novel PDE7 inhibitor (PDE7i) treatment on mice with experimental autoimmune encephalomyelitis (EAE) a model of multiple sclerosis (MS) and compared it with another PDE7i. EAE was induced by immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) peptide. Mice were treated daily either from disease onset or from disease peak with each PDE7i and with fingolimod (used in therapy for MS patients) and disease evolution was followed by clinical symptoms. We examined neuropathology of spinal cord, ex vivo lymphocyte proliferation by [3H]-thymidine incorporation, TNFα by ELISA and cAMP-PDE mRNAs expression by in situ hybridization histochemistry (ISHH) in spinal cord of EAE mice treated with both PDE7 inhibitors. Treatment of EAE mice with the novel PDE7i, VP3.15 showed more efficacy in reducing clinical signs at 10mgkg-1 than the other PDE7i, BRL50481 and similar to fingolimod. VP3.15 acts on peripheral lymphocytes inhibiting their proliferation and TNFα secretion in a dose-dependent manner. PDE7i treatment alters the levels of PDE4B and PDE7 mRNA expression in EAE mice spinal cord. Given the interest in the development of new drugs for MS, including PDE7i as anti-inflammatory drugs, it is important to study the role played by PDE7 in neurodegenerative diseases with inflammatory component to better understand the beneficial and detrimental effects of a future therapy.

Phosphodiesterase-7 inhibition affects accumbal and hypothalamic thyrotropin-releasing hormone expression, feeding and anxiety behavior of rats.

Thyrotropin-releasing hormone (TRH) has anorexigenic and anxiolytic functions when injected intraventricularly. Nucleus accumbens (NAcc) is a possible brain region involved, since it expresses proTRH. TRH from hypothalamic paraventricular nucleus (PVN) has a food intake-regulating role. TRHergic pathways of NAcc and PVN are implicated in anxiety and feeding. Both behaviors depend on cAMP and phosphorylated-cAMP response element binding protein (pCREB) intracellular levels. Intracellular levels of cAMP are controlled by the degrading activity of phosphodiesterases (PDEs). Since TRH transcription is activated by pCREB, a specific inhibitor of PDE7B may regulate TRH-induced effects on anxiety and feeding. We evaluated the effectiveness of an intra-accumbal and intraperitoneal (i.p.) administration of a PDE7 inhibitor (BRL-50481) on rats' anxiety-like behavior and food intake; also on TRH mRNA and protein expression in NAcc and PVN to define its mediating role on the PDE7 inhibitor-induced behavioral changes. Accumbal injection of 4µg/0.3µL of PDE7 inhibitor decreased rats' anxiety. The i.p. injection of 0.2mg/kg of the inhibitor was able to increase the PVN TRH mRNA expression and to decrease feeding but did not change animals' anxiety levels; in contrast, 2mg/kg b.w inhibitor enhanced accumbal TRH mRNA, induced anxiolysis with no change in food intake. PDE7 inhibitor induced anxiolytic and anorexigenic like behavior depending on the dose used. Results supported hypothalamic TRH mediated feeding-reduction effects, and accumbal TRH mediation of inhibitor-induced anxiolysis. Thus, an i.p dose of this inhibitor might be reducing anxiety with no change in feeding, which could be useful for obese patients.

From unilateral to bilateral parkinsonism: Effects of lateralization on dyskinesias and associated molecular mechanisms.

The mechanisms underlying lateralization and progression of motor symptoms from unilateral to bilateral in Parkinson's disease (PD) remain to be elucidated. In addition, the molecular mechanisms involved in levodopa-induced dyskinesias (LIDs) depending on lateralization and disease progression from unilaterally to bilateral have not been described yet. We investigated motor symptoms, LIDs and associated striatal molecular markers expression after unilateral left or right, and after a sequential bilateral 6-hydroxydopamine (6-OHDA)-induced nigrostriatal lesions in rats. Sequentially bilateral lesioned animals showed a bilateral increase in striatal preproenkephalin (PPE) mRNA without changes in pre-prodynorphin (PDyn) mRNA expression. The increase in dyskinesias when parkinsonism becomes bilateral was mostly due to an increase in orolingual dyskinesias associated to a increase in PDyn mRNA expression. Right lesion induces, or facilitates when first-done, a greater level of LIDs and an increase in striatal PPE and PDyn mRNAs in the second lesioned side. We describe a new striatal molecular pattern that appears when parkinsonism becomes bilateral and the relevance of the lateralization for the development of LIDs.

Early L-dopa, but not pramipexole, restores basal ganglia activity in partially 6-OHDA-lesioned rats.

The most appropriate time for the initiation of dopaminergic symptomatic therapy in Parkinson's disease remains debatable. It has been suggested that early correction of basal ganglia pathophysiological abnormalities may have long-term beneficial effects. To test this hypothesis, we investigated the early and delayed actions of L-dopa and pramipexole, using a delayed-start protocol of treatment. The effects of early and delayed administration of these drugs on motor response, development of dyskinesias, neurogenesis and molecular markers in basal ganglia were studied in rats with a unilateral and partial 6-hydroxydopamine-induced nigrostriatal lesion. Ten days after lesioning, rats received treatment with: a) L-dopa methyl ester (25mg/kg with 6.25mg/kg of benserazide, i.p., twice a day); b) pramipexole (0.5mg/kg, sc, twice a day) or c) saline for 4weeks. Four weeks after treatment initiation, rats from the saline group were distributed in three groups that then received the following treatments: d) L-dopa, e) pramipexole or f) saline, for 4weeks more. Three animals in each treatment arm received 5-bromo-2-deoxyuridine injections (200mg/kg) 3days before starting treatment. When compared with delayed-start L-dopa, early-start L-dopa treatment induced a lower rotational response (p<0.01), an improvement in limb akinesia (p<0.05), a lower level of dyskinesia (p<0.01) and a normalization of lesion-induced molecular changes in basal ganglia. When compared with delayed-start pramipexole, early-start pramipexole induced a higher rotational response (p<0.01), but did not improve limb akinesia, induce dyskinesia nor normalize lesion-induced molecular changes. Neither significant modifications of striatal dopamine D1-D3 receptor heteromerization nor subventricular zone neurogenesis was found after any L-dopa or pramipexole treatments. Our data support a possible restoration of basal ganglia physiological mechanisms by early-start L-dopa therapy.

Subthalamic 6-OHDA-induced lesion attenuates levodopa-induced dyskinesias in the rat model of Parkinson's disease.

The subthalamic nucleus (STN) receives direct dopaminergic innervation from the substantia nigra pars compacta that degenerates in Parkinson's disease. The present study aimed to investigate the role of dopaminergic denervation of STN in the origin of levodopa-induced dyskinesias. Rats were distributed in four groups which were concomitantly lesioned with 6-OHDA or vehicle (sham) in the STN and in the medial forebrain bundle (MFB) as follows: a) MFB-sham plus STN-sham, b) MFB-sham plus STN-lesion, c) MFB-lesion plus STN-sham, and d) MFB-lesion plus STN-lesion. Four weeks after lesions, animals were treated with levodopa (6mg/kg with 15mg/kg benserazide i.p.) twice daily for 22 consecutive days. Abnormal involuntary movements were measured. In situ hybridization was performed measuring the expression of striatal preproenkephalin, preprodynorphin, STN cytochrome oxidase (CO) and nigral GAD67 mRNAs. STN 6-OHDA denervation did not induce dyskinesias in levodopa-treated MFB-sham animals but attenuated axial (p<0.05), limb (p<0.05) and orolingual (p<0.01) dyskinesias in rats with a concomitant lesion of the nigrostriatal pathway. The attenuation of dyskinesias was associated with a decrease in the ipsilateral STN CO mRNA levels (p<0.05). No significant differences between MFB-lesion plus STN-sham and MFB-lesion plus STN-lesion groups in the extent of STN dopaminergic denervation were observed. Moreover, intrasubthalamic microinfusion of dopamine in the MFB-lesion plus STN-lesion group triggered orolingual (p<0.01), but not axial or limb, dyskinesias. These results suggest that dopaminergic STN innervation influences the expression of levodopa-induced dyskinesias but also the existence of non dopaminergic-mediated mechanisms. STN noradrenergic depletion induced by 6-OHDA in the STN needs to be taken in account as a possible mechanism explaining the attenuation of dyskinesias in the combined lesion group.

Anxiolytic effects of ethanol are partially related to a reduced expression of adenylyl cyclase 5 but not to µ-opioid receptor activation in rat nucleus accumbens.

Anxiolytic effects of alcohol participate in the reinforcing properties of the drug, in which nucleus accumbens (NAcc) is implicated. The opioidergic system in NAcc is considered a main pathway involved in the emotional responses of animals: rats microinjected with morphine in NAcc and the systemic administration of µ-opioid receptors (MOR) agonists yield low anxiety scores in the elevated plus maze (EPM), a behavioral test of anxiety. However, the specific participation of NAcc MOR in the anxiolytic effect of ethanol has not been studied. AC5, a cAMP-synthezising adenylyl-cyclase, is highly expressed in NAcc; it is negatively coupled to MOR and has been implicated in anxiety levels of animals. We evaluated the anxiolytic effects of an intra-gastric administration of ethanol (2.5 g/kg) in animals subjected to EPM at 1, 4, and 8 h after drug or water exposure. Locomotion was assayed with the open-field test; we also measured accumbal AC5 and MOR mRNA levels by RT-PCR. After 1 h, ethanol-exposed animals showed anxiolytic-like behavior, as well as decreased and increased AC5 and MOR expression in NAcc, respectively. Intra-accumbal injection of ß-funaltrexamine (FNA), a MOR antagonist, did not block ethanol-induced anxiolysis, rather it induced a tendency to increase anxiety levels in the water-exposed group. FNA partially decreased accumbal AC5 expression in ethanol-treated rats. We concluded that AC5 in NAcc is participating in the emotional effects of ethanol; that MOR was not mediating the drug-induced AC5 reduction in NAcc nor the ethanol-induced anxiolysis. MOR only might be involved in basal levels of anxiety of animals.

D2 and D4 dopamine receptor mRNA distribution in pyramidal neurons and GABAergic subpopulations in monkey prefrontal cortex: implications for schizophrenia treatment.

D2 and D4 dopamine receptors play an important role in cognitive functions in the prefrontal cortex and they are involved in the pathophysiology of neuropsychiatric disorders such as schizophrenia. The eventual effect of dopamine upon pyramidal neurons in the prefrontal cortex depends on which receptors are expressed in the different neuronal populations. Parvalbumin and calbindin mark two subpopulations of cortical GABAergic interneurons that differently innervate pyramidal cells. Recent hypotheses about schizophrenia hold that the root of the illness is a dysfunction of parvalbumin chandelier cells that produces disinhibition of pyramidal cells. In the present work we report double in situ hybridization histochemistry experiments to determine the prevalence of D2 receptor mRNA and D4 receptor mRNA in glutamatergic neurons, GABAergic interneurons and both parvalbumin and calbindin GABAergic subpopulations in monkey prefrontal cortex layer V. We found that around 54% of glutamatergic neurons express D2 mRNA and 75% express D4 mRNA, while GAD-positive interneurons express around 34% and 47% respectively. Parvalbumin cells mainly expressed D4 mRNA (65%) and less D2 mRNA (15-20%). Finally, calbindin cells expressed both receptors in similar proportions (37%). We hypothesized that D4 receptor could be a complementary target in designing new antipsychotics, mainly because of its predominance in parvalbumin interneurons.

The human area postrema and other nuclei related to the emetic reflex express cAMP phosphodiesterases 4B and 4D.

Phosphodiesterase 4 (PDE4) inhibitors, i.e. rolipram, are being extensively investigated as therapeutic agents in several diseases. Emesis is one of the most common side effects of PDE4 inhibitors. Given the fact that the area postrema is considered the chemoreceptor trigger zone for vomiting, the present study investigates the regional distribution and cellular localization of the four gene transcripts of the PDE4 subfamily (PDE4A, PDE4B, PDE4C and PDE4D) in human brainstem. In situ hybridization histochemistry was used to locate the mRNA distribution of the four PDE4 subfamilies in the area postrema and related nuclei of human postmortem brainstem. We have found that in the brainstem PDE4B and PDE4D mRNA expression is abundant and distributed not only in neuronal cells, but also in glial cells, and on blood vessels. The hybridization signals for PDE4B and PDE4D mRNAs in the area postrema were stronger than those in any other nuclei in the brainstem. They were also found in vomiting-related nuclei such as the nucleus of the solitary tract and the dorsal vagal motor nucleus. These findings suggest that cAMP signaling modification in the area postrema could mediate the emetic effects of PDE4 inhibitors in human brainstem.

Effects of early vs. late initiation of levodopa treatment in hemiparkinsonian rats.

We investigated the effect of early vs. late initiation of levodopa treatment on dyskinetic movements, rotational behavior and molecular markers in hemiparkinsonian rats. Male Sprague-Dawley rats received a unilateral 6-hydroxydopamine (6-OHDA) administration in the nigrostriatal pathway. Rats were divided into three groups treated with: (i) levodopa (6 mg/kg) twice daily for 22 days starting at 4 weeks after 6-OHDA (Early group); (ii) levodopa at the same dose, regimen and duration but starting at 12 weeks after 6-OHDA (Late group), and (iii) saline starting at 4 weeks after 6-OHDA and continuing until the Late group finished treatment. Dyskinesias were quantified on days 1 and 22 of levodopa treatment. Striatal expression of preproenkephalin and preprodynorphin mRNAs, subthalamic cytochrome oxidase mRNA, and glutamate decarboxylase 67 mRNA in the pars reticulata of the substantia nigra was measured by in-situ hybridization. After 22 days of levodopa treatment, the percentage of rats showing dyskinesia was lower in the Early group than in the Late group (60% vs. 100%, respectively). No significant differences in total dyskinesia score were observed between both groups with the exception of the orolingual dyskinesias that were significantly less frequent in the Late group (P < 0.01). No significant differences were observed in the molecular markers between the Early and Late groups. Prompt initiation of levodopa treatment might be able to delay some of the basal ganglia molecular and circuitry changes underlying the development of dyskinesia but, once developed, they are behaviorally and molecularly similar to those appearing after late initiation of levodopa.

Entacapone potentiates the long-duration response but does not normalize levodopa-induced molecular changes.

Coadministration of entacapone with levodopa attenuates motor complications in experimental models of Parkinson's disease. The mechanisms underlying entacapone effects are unknown. We investigated the effect of entacapone, on: long-duration response (LDR) to levodopa, levodopa-induced postsynaptic pharmacodynamic mechanisms and molecular changes in hemiparkinsonian rats. 6-Hydroxydopamine-unilaterally lesioned rats were treated with levodopa (25 mg/kg)+vehicle; levodopa+entacapone (30 mg/kg) or saline, twice daily for 22 days. The LDR and the apomorphine-induced rotations were measured. In situ hybridization was performed measuring the expression of striatal preproenkephalin, preprodynorphin and dopamine D-3 receptor mRNAs, subthalamic cytochrome oxidase mRNA and nigral glutamic acid decarboxylase mRNA. Entacapone potentiated the LDR but did not modify either the apomorphine-induced rotational behavior or the molecular changes. Our results suggest that the effects of entacapone on levodopa-induced motor response are not mediated by postsynaptic mechanisms and that administration of entacapone is not able to normalize the molecular alterations induced by levodopa in the basal ganglia.

Quantitative analysis of glutamatergic and GABAergic neurons expressing 5-HT(2A) receptors in human and monkey prefrontal cortex.

5-hydroxytryptamine (5-HT) or serotonin 2A receptors play an important role in modulation of prefrontal cortex (PFC) activity and have been implicated in the physiopathology of psychiatric disorders. There is no quantitative information on the percentage of glutamatergic and GABAergic cells that express 5-HT(2A) receptors in human and monkey PFC. We have used double in situ hybridization to quantify the mRNA co-localization of 5-HT(2A) receptor with the glutamatergic transporter vesicular glutamate transporter 1, and with the GABAergic marker glutamic acid decarboxylase 65/67 and in parvalbumin and calbindin GABAergic cell populations. Our results show that nearly every glutamatergic cell (86-100%) in layers II-V expressed 5-HT(2A) receptor mRNA in both species. This percentage was lower in layer VI (13-31%). In contrast, not all the GABAergic interneurons (13-46%) expressed 5-HT(2A) receptor mRNA. This receptor was expressed in 45-69% of parvalbumin and in 61-87% of calbindin positive cells. These results indicate that, while the majority of glutamatergic neurons can be sensitive to 5-HT action via 5-HT(2A) receptors, this modulation occurs only in a limited population of GABAergic interneurons and provides new neuroanatomical information about the role played by serotonin through 5-HT(2A) receptors in the PFC and on the sites of action for drugs such as antipsychotics and antidepressants used in treatment of psychiatric disorders.

Concomitant short- and long-duration response to levodopa in the 6-OHDA-lesioned rat: a behavioural and molecular study.

The long-duration response (LDR) is a sustained improvement in parkinsonism due to chronic levodopa therapy and lasts after discontinuation of treatment. We have investigated the molecular changes that underlie the LDR in rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion. Animals were treated for 22 days with levodopa or saline. Forelimb akinesia was evaluated prior and following a test dose of levodopa. Rotational behaviour was weekly evaluated. Levodopa induced an improvement in the parkinsonian limb akinesia that lasted for 48 h after withdrawal. A shortening in the duration of rotational behaviour was observed. After 3 days of washout, levodopa treatment maintained elevated striatal preproenkephalin mRNA expression, also inducing an increase in preprodynorphin (PDyn) and dopamine D-3 receptor mRNAs, but without any modification of the adenosine A(2A) mRNA expression induced by 6-OHDA. Levodopa reversed the lesion-induced increase in the expression of cytochrome oxidase mRNA in the subthalamic nucleus and glutamate decarboxylase mRNA in the pars reticulata of the substantia nigra. After 7 days of levodopa washout, the molecular markers show a decline in the basal ganglia evolving towards the parkinsonian state, being statistically significant for the striatal PDyn mRNA. This study characterizes the concomitant presence of the short-duration response and LDR to levodopa in the 6-OHDA model of parkinsonism and shows that the molecular changes induced by levodopa in the basal ganglia are not permanent and that this reversal after levodopa washout may be responsible for the gradual motor deterioration that characterize the LDR.

Species differences in the localization of soluble guanylyl cyclase subunits in monkey and rat brain.

Nitric oxide (NO) exerts most of its physiological effects through activation of a predominantly soluble guanylyl cyclase (sGC). In mammalian cells sGC exists as a heterodimer of alpha and beta subunits. Currently, four subunits (alpha1, alpha2, beta1, and beta2) have been characterized. We used in situ hybridization with subunit-specific 33P-labeled oligonucleotide probes to compare the anatomical distribution of sGC subunit mRNAs in rat and monkey brains. Message for all subunits except beta2 was detected in both species. The sGC subunit showing the highest expression and widest distribution was beta1. High expression for all subunits was found in basal ganglia, olfactory system, hippocampus, cortex, and cerebellum. Minor species differences in the relative distribution of alpha subunits were observed. In general, the alpha1 message was more prominent in monkey brain and the alpha2 message in rat brain. This was more evident in limbic areas and cerebellar cortex. Some differences were also observed in subunit laminar distribution in cerebral cortex. The limited species differences in sGC subunit distribution suggest that in primates, as occurs in rodents, the NO-cGMP signaling pathway will be involved in important brain functions such as memory formation, sensory processing, and behavior.

Reversion of levodopa-induced motor fluctuations by the A2A antagonist CSC is associated with an increase in striatal preprodynorphin mRNA expression in 6-OHDA-lesioned rats.

The molecular mechanisms involved in the reversion of levodopa-induced motor fluctuations by the adenosine A2A antagonist 8-(3-chlorostryryl) caffeine (CSC) were investigated in rats with a 6-hydroxydopamine (6-OHDA)-induced lesion and compared with the ones achieved by the kappa-opioid agonist, U50,488. Animals were treated with levodopa (50 mg/kg/day) for 22 days and for one additional week with levodopa + CSC (5 mg/kg/day), levodopa + U50,488 (1 mg/kg/day), or levodopa + vehicle. The reversion of the decrease in the duration of levodopa-induced rotations by CSC, but not by U50,488, was maintained until the end of the treatment and was associated with a further increase in levodopa-induced preprodynorphin mRNA in the lesioned striatum, being higher in the ventromedial striatum. The increase in striatal preprodynorphin expression, particularly in the ventromedial striatum, may be related to the reversion of levodopa-induced motor fluctuations in the CSC-treated animals, suggesting a role of the direct striatal output pathway activity in the ventromedial striatum in the pathophysiology of motor fluctuations.

Neuronal expression of cAMP-specific phosphodiesterase 7B mRNA in the rat brain.

cAMP plays an important role as second messenger molecule controlling multiple cellular processes in the brain. cAMP levels depend critically on the phosphodiesterases (PDE) activity, enzymes responsible for the clearance of intracellular cAMP. We have examined the regional distribution and cellular localization of mRNA coding for the cAMP-specific phosphodiesterase 7B (PDE7B) in rat brain by in situ hybridization histochemistry. PDE7B mRNA is specifically distributed in rat brain, preferentially in neuronal cell populations. The highest levels of hybridization are observed in olfactory tubercle, islands of Calleja, dentate gyrus, caudate-putamen and some thalamic nuclei. Positive hybridization signals are also detected in other areas, such as cerebral cortex, Purkinje cells of the cerebellum and area postrema. By double in situ hybridization histochemistry, we found that 74% and 79% of the cells expressing PDE7B mRNA in striatum and olfactory tubercle, respectively, were GABAergic cells (expressing glutamic acid decarboxylase mRNA), in contrast with the lack of expression in the few cholinergic cells (expressing choline acetyltransferase mRNA) present in those two areas (around 0.4% in olfactory tubercle). In the thalamic nuclei, a majority of cells containing PDE7B mRNA also expresses a glutamatergic marker (76.7% express vesicular glutamate transporter vGluT1 and 76% express vGluT2 mRNAs). Almost all PDE7B expressing cells in dentate gyrus (93%) were glutamatergic. These results offer a neuroanatomical and neurochemical base that will support the search for specific functions for cAMP dependent PDEs and for the development of specific PDE7 inhibitors.

The kappa opioid agonist U50,488 potentiates 6-hydroxydopamine-induced neurotoxicity on dopaminergic neurons.

Several observations support the hypothesis that kappa opioid (kappa-opioid) receptor agonism may contribute to neurotoxicity, but other reports have suggested that certain kappa-agonists can attenuate neurological dysfunction. Degeneration of dopaminergic neurons in the substantia nigra is the pathological hallmark of Parkinson's disease. Therefore, it is of particular interest to study whether kappa-opioid receptor agonism has an influence on the progressive degeneration of dopaminergic neurons. We have investigated the effect exerted by the selective kappa-agonist U50,488 on the neurotoxicity induced by intrastriatal 6-hydroxydopamine (6-OHDA) administration on dopaminergic neurons. Male Sprague-Dawley rats received an acute (0.5 mg/kg) or subacute (0.5 mg/kg, twice at day, for 7 days) administration of U50,488, receiving the last dose 30 min before intrastriatal 6-OHDA administration. Acute or subacute U50,488 pretreatment potentiated the 6-OHDA-induced decrease in the number of nigral tyrosine hydroxylase immunoreactive neurons (P < 0.05). Acute U50,488 pretreated animals showed a tendency, although not statistically significant to increase striatal mRNA encoding for enkephalin (PPE mRNA). Subacute U50,488 significantly potentiated the increase in PPE mRNA induced by 6-OHDA (P < 0.05). The present results show a neurotoxic effect of the kappa agonist U50,488 on dopaminergic neurons in rats with a striatal lesion induced by 6-OHDA. This neurotoxic effect is associated to an increase in striatal PPE mRNA levels, suggesting that an increase in the indirect pathway activity and consequently an increase in the activity of the subthalamo-nigral pathway might be involved in this phenomenon.

Alterations on phosphodiesterase type 7 and 8 isozyme mRNA expression in Alzheimer's disease brains examined by in situ hybridization.

Phosphodiesterases (PDEs) play a central role in signal transduction by regulating intracellular levels of cyclic AMP (cAMP) and cGMP. It has been suggested that cAMP pathways could be upregulated in Alzheimer's disease. By in situ hybridization histochemistry we have determined the expression pattern of two newly described cAMP-specific phosphodiesterases families, PDE7 and PDE8, in several brain areas in control subjects. The hybridization levels of PDE8A mRNA were very low in all brain areas examined. High PDE7B and PDE8B mRNA signal intensities were found in the hippocampal formation. PDE7A was found to be present in both neuronal and non-neuronal cell populations. When the expression of these isozymes in control brains was compared with that in Alzheimer's disease brains staged according to Braak and Braak (Acta Neuropathol. (Berl.) 82 (1991), 239), we found that PDE8B was the only isozyme showing a significant increase, in cortical areas and parts of the hippocampal formation, at Braak stages III-VI. Our results show that the expression of specific cAMP PDE isoforms is selectively regulated in Alzheimer's disease and associated with the stages of the disease. The availability of animal models of Alzheimer's disease and of new pharmacological tools such as selective PDE inhibitors will allow study of the therapeutic potential of the control of cAMP levels in AD.

Somatostatin, cholecystokinin and neuropeptide Y mRNAs in normal and weaver mouse brain.

The distribution of mRNAs encoding for somatostatin, cholecystokinin and neuropeptide Y was determined by in situ hybridization histochemistry in the weaver (wv/wv) mouse, a model of dopamine deficiency as well as in normal (+/+) controls. Weaver mutants did not show any appreciable departure from the normal pattern of expression for mRNA encoding for neuropeptide Y. In contrast, an 82% increase in mRNA encoding for somatostatin was observed in the reticular thalamic nucleus, whereas increases in the order of 20-87% were observed in different hypothalamic nuclei of the weaver brain. In addition, a 47-103% increase of the hybridization signal encoding for cholecystokinin was observed in the cerebral cortex, hippocampus and thalamus of the weaver brain. It can be assumed that the elevated and region-specific somatostatin and cholecystokinin levels observed in the weaver brain may be due to a secondary or compensatory response under conditions of altered neurotransmitter levels.

Cloning and characterization of a novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT4 receptor variants.

We have cloned a novel C-terminal splice variant of serotonin 5-HT4 receptors from human hippocampus. The deduced protein extends only one aminoacid past the splicing point. We propose to call the novel variant h5-HT4(n) since it contains none of the C-terminal exons alternatively spliced in other variants. The pharmacological profile of h5-HT4(n) stably expressed in HeLa cells is in agreement with other reported variants. Stably transfected cells showed increased basal levels of intracellular cAMP in absence of agonist, indicating constitutive activity of the expressed receptors. 5-HT induced robust increases of intracellular cAMP. The 5-HT4 receptor antagonist GR 113808 blocked the effects of 5-HT and brought intracellular cAMP below basal constitutive levels, indicating inverse agonism of this compound in this system. The RT-PCR distribution of all known human C-terminal splice variants in human brain regions and periphery showed complex patterns of variant expression, with the novel variant h5-HT4(n) being widely and abundantly expressed.