Modulation of memory processes and cellular excitability in the dentate gyrus of freely moving rats by a 5-HT4 receptors partial agonist, and an antagonist.

Firstly, olfactory association learning was used to determine the modulating effect of 5-HT4 receptor involvement in learning and long-term memory. Secondly, the effects of systemic injections of a 5-HT4 partial agonist and an antagonist on long-term potentiation (LTP) and depotentiation in the dentate gyrus (DG) were tested in freely moving rats. The modulating role of the 5-HT4 receptors was studied by using a potent, 5-HT4 partial agonist RS 67333 [1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-n-butyl-4-piperidinyl)-1-propanone] and a selective 5-HT4 receptor antagonist RS 67532 [1-(4-amino-5-chloro-2-(3,5-dimethoxybenzyloxyphenyl)-5-(1-piperidinyl)-1-propanone]. Agonist or antagonist systemic chronic injections prior to five training sessions yielded a facilitatory effect on procedural memory during the first session only with the antagonist. Systemic injection of the antagonist only before the first training session improved procedural memory during the first session and associative memory during the second session. Similar injection with the 5-HT4 partial agonist had an opposite effect. The systemic injection of the 5-HT4 partial agonist prior to the induction of LTP in the dentate gyrus by high-frequency stimulation was followed by a population spike increase, while the systemic injection of the antagonist accelerated the depotentiation 48 h later. The behavioural and physiological results pointed out the involvement of 5-HT4 receptors in processing related to the long-term hippocampal-dependent memory system, and suggest that specific 5-HT4 agonists could be used to treat amnesic patients with a dysfunction in this particular system.

GPCR-interacting proteins (GIPs): nature and functions.

The simplistic idea that seven transmembrane receptors are single monomeric proteins that interact with heterotrimeric G-proteins after agonist binding is definitively out of date. Indeed, GPCRs (G-protein-coupled receptors) are part of multiprotein networks organized around scaffolding proteins. These GIPs (GPCR-interacting proteins) are either transmembrane or cytosolic proteins. Proteomic approaches can be used to get global pictures of these 'receptosomes'. This approach allowed us to identify direct but also indirect binding partners of serotonin receptors. GIPs are involved in a wide range of functions including control of the targeting, trafficking and signalling of GPCRs. One of them, Shank, which is a secondary and tertiary partner of metabotropic and ionotropic glutamate receptors, respectively, can induce the formation of a whole functional glutamate 'receptosome' and the structure to which it is associated, the dendritic spine.

5-HT4 receptors.

Serotonin 4 receptors (5-HT(4)Rs) were discovered 15 years ago. They are coded by a very complex gene (700Kb, 38 exons) which generates eight carboxy-terminal variants (a, b, c, d, e, f, g, n). Their sequences differ after position L(358). Another variant is characterized by a 14 residue insertion within the extracellular loop 2. Highly selective potent 5-HT(4) receptor antagonists and partial agonists which cross the blood-brain barrier have been synthesized, but a specific full agonist for brain studies is still missing. Based on physiological and behavioral experiments, 5-HT(4)Rs may be targets to treat cognitive deficits, abdominal pain and feeding disorders. One 5-HT(4)R-directed drug (SL65.0155) is already in phase II to treat patients suffering from memory deficits or dementia.

Constitutively active mutants of 5-HT4 receptors are they in unique active states?

Somatic mutations leading to constitutively active G-protein coupled receptors (GPCRs) are responsible for certain human diseases. A consistent structural description of the molecular change underlying the conversion of GPCRs from an inactive R state to an active R* state is lacking. Here, we show that a series of constitutively active 5-HT4 receptors (mutated or truncated in the C-terminal and the third intracellular loop) were characterized by an increase in their denaturation rate at 55 degrees C. The thermal denaturation kinetics were monophasic, suggesting that we were measuring mainly the denaturation rate of R*. Analysis of these kinetics revealed that constitutively active C-terminal domain mutants, were due to a change in the J constant governing the R/R* equilibrium. However, the constitutive activity of the receptor mutated within the third intracellular loop was the result of both a change in the allosteric J constant and a change in the R* conformation.

Interaction of serotonin 5-hydroxytryptamine type 2C receptors with PDZ10 of the multi-PDZ domain protein MUPP1.

By using the yeast two-hybrid system, we previously isolated a cDNA clone encoding a novel member of the multivalent PDZ protein family called MUPP1 containing 13 PDZ domains. Here we report that the C terminus of the 5-hydroxytryptamine type 2C (5-HT(2C)) receptor selectively interacts with the 10th PDZ domain of MUPP1. Mutations in the extreme C-terminal SSV sequence of the 5-HT(2C) receptor confirmed that the SXV motif is critical for the interaction. Co-immunoprecipitations of MUPP1 and 5-HT(2C) receptors from transfected COS-7 cells and from rat choroid plexus verified this interaction in vivo. Immunocytochemistry revealed an SXV motif-dependent co-clustering of both proteins in transfected COS-7 cells as well as a colocalization in rat choroid plexus. A 5-HT(2C) receptor-dependent unmasking of a C-terminal vesicular stomatitis virus epitope of MUPP1 suggests that the interaction triggers a conformational change within the MUPP1 protein. Moreover, 5-HT(2A) and 5-HT(2B), sharing the C-terminal EX(V/I)SXV sequence with 5-HT(2C) receptors, also bind MUPP1 PDZ domains in vitro. The highest MUPP1 mRNA levels were found in all cerebral cortical layers, the hippocampus, the granular layer of the dentate gyrus, as well as the choroid plexus, where 5-HT(2C) receptors are highly enriched. We propose that MUPP1 may serve as a multivalent scaffold protein that selectively assembles and targets signaling complexes.

Differential modulation of the 5-HT(4) receptor agonists and antagonist on rat learning and memory.

Recent data suggest that activation of 5-HT(4) receptors may modulate cognitive processes such as learning and memory. In the present study, the effects of two potent and selective 5-HT(4) agonists, RS 17017 [1-(4-amino-5-chloro-2-methoxyphenyl)-5- (piperidin-1-yl)-1-pentanone hydrochloride] and RS 67333 [1(4-amino-5-chloro-2-methoxyphenyl)-3- (1-n-butyl-4-piperidinyl)-1-propanone], were studied in an olfactory associative discrimination task. The implication of 5-HT(4) receptors in the associative discriminative task was suggested by the following observation. Injection of a selective 5-HT(4) receptor antagonist RS 67532 [1-(4-amino-5-chloro-2-(3, 5-dimethoxybenzyloxyphenyl)-5-(1-piperidinyl)-1-pentanone; 1 mg/kg: i.p.] before the third training session induced a consistent deficit in associative memory during the following training sessions. This deficit was absent when the antagonist was injected together with either a specific hydrophilic 5-HT(4) (RS 17017, 1 mg/kg) or a specific hydrophobic (RS 67333, 1 mg/kg) 5-HT(4) receptor agonist. RS 67333 was more potent than RS 17017. This difference in potency certainly reflects a difference in their capacity to enter into the brain. This is also likely to be the reason why, injected alone, the hydrophobic 5-HT(4) agonist (RS 67333) but not the hydrophilic 5-HT(4) agonist (RS 17017) improved learning and memory performance.

Pharmacological properties of 5-Hydroxytryptamine(4) receptor antagonists on constitutively active wild-type and mutated receptors.

We studied the pharmacological properties of twenty-four 5-hydroxytryptamine (5-HT)(4) receptor ligands known to act as antagonists on 5-HT(4) receptors positively coupled to adenylyl cyclase endogenously expressed in mouse colliculi neurons. In COS-7 cells expressing human or mouse 5-HT(4(a)) receptors (100-8000 fmol/mg of protein), we found neutral antagonists, partial agonists, and inverse agonists. The majority of neutral antagonists belong to the benzodioxanyl ketone class, whereas partial agonists belong to different chemical classes. We found only two inverse agonists, GR 125487 and SB 207266, which are both indoles. Analysis of pharmacological characteristics of the constitutively active wild-type and constitutively active mutated receptors revealed that 1) the ratio between the efficiencies of the full agonist 5-HT and the partial agonist RS 23597 was invariable when the receptor density increased, but was dependent on receptor structure; 2) similarly, the efficacy of the inverse agonist SB 207266 was not dependent on receptor density but was dependent on receptor structure; 3) when the receptor concentration increased, the EC(50) values of the full agonist 5-HT were not modified and the increase in basal constitutive activity, as well as its stimulation by 5-HT, followed a parallel evolution; and 4) the stimulation of basal constitutive activity by 5-HT was not modified by the overexpression of Galphas. All these results indicate that in COS-7 cells, the coupling of the 5-HT(4) receptor to adenylyl cyclase was linear with no indication of spare receptors even at high receptor density (8 pmol/mg). These results are also in accordance with a precoupling between the activated receptor (f(R*)) and adenylyl cyclase. Such observations allowed us to use the two-state model to calculate the constant J, i.e., the equilibrium allosteric constant denoting the ratio of the receptor in the inactive versus active state (J = [R]/[R*]). We found that J was a receptor structural characteristic, independent of receptor density.

Novel brain-specific 5-HT4 receptor splice variants show marked constitutive activity: role of the C-terminal intracellular domain.

We have cloned new 5-Hydroxytryptamine 4 (5-HT4) receptor splice variants from mouse (m5-HT4(e)R and m5-HT4(f)R), rat (r5-HT4(e)R), and human brain tissue (h5-HT4(e)R) which differ, as do the previously described 5-HT4 receptor variants, in the length and composition of their intracellular C termini after the common splicing site (L358). These new variants have a unique C-terminal sequence made of two PV repeats and are only expressed in brain tissue. All of the 5-HT4 receptor splice variants have a high constitutive activity when expressed at low and physiological densities (<500 fmol/mg protein). At similar density, they showed a much higher constitutive activity than the native and the mutated beta2-adrenergic receptors. The constitutive activity of the new splice variants with short C-terminal sequences (m5-HT4(e)R and m5-HT4(f)R) was higher than that of the long C-terminal sequence variants (m5-HT4(a)R and m5-HT4(b)R). This may indicate that the short variants have a higher capacity for isomerization from the inactive to the active conformation. Moreover, we further identified a sequence within the C-terminal tail upstream of L358, rich in serine and threonine residues, that played a crucial role in maintaining 5-HT4R under its inactive conformation.

Neurotransmitter regulation of MAP kinase signaling in striatal neurons in primary culture.

Glutamate and dopamine are important neurotransmitters in the basal ganglia. Dopamine can act via D1 receptors to activate adenylyl cyclase in striatal neurons, while glutamate stimulation of NMDA receptors leads to an increase in intracellular calcium. Increases in intracellular calcium or cAMP can induce immediate early gene expression in striatal neurons. In the present study, NMDA receptor stimulation or adenylyl cyclase activation resulted in the activation of MAP kinase in striatal neurons in primary culture. The effect of cAMP appeared to involve cAMP-dependent protein kinase, in addition to a tyrosine kinase and MEK. NMDA-induced MAP kinase activation was also dependent on a tyrosine kinase and MEK. The EGF receptor, which has been implicated in calcium- and G protein-induced MAP kinase activation, did not mediate the effects of NMDA or forskolin on MAP kinase. Furthermore, the src kinase inhibitor, herbimycin A, and the phosphoinositol-3-kinase inhibitor, wortmannin, did not prevent MAP kinase activation by these stimuli. However, the ability of both NMDA and forskolin to activate MAP kinase in striatal neurons was blocked by SB 203580, an inhibitor of p38 reactivating kinase. These results indicate that both NMDA receptor activation and elevations in cAMP can result in MEK-induced MAP kinase activation in striatal neurons. However, the signal transduction pathways mediating these responses appear to be distinct from those known to mediate MAP kinase activation by other stimuli.

5-HT4 receptors: gene, transduction and effects on olfactory memory.

In this paper we discuss 1) the primary structures, pharmacology, and brain distribution of cloned 5-HT4 receptors; 2) the chromosomal localization of the h5-HT4 receptor; 3) whether benzamides are full or partial agonists because of a species or a coupling difference; 4) the intrinsic activity of 5-HT4 receptors and inverse agonism of GR125487 in COS-7 cells but not in colliculi neurons; 5) the modulation of 5-HT4 receptor binding and activity; and 6) the long-term blockade of K+ channels by 5-HT4 agonists and its effect on olfactory memory. We conclude that 1) the cloning of 5-HT4 receptors in different species using RT-PCR from different tissues reveals the presence of several splice variants for 5-HT4 receptors differing in the C-terminal part, downstream from the amino acid L358; 2) the pharmacological properties of 5-HT4 receptors are dependent on the cellular context in which they are expressed; and 3) 5-HT4 agonists can be added to the list of compounds having pro-cognitive properties.

5-HT4 receptors: cloning and expression of new splice variants.

On the basis of differences in the potencies and intrinsic activity of 5-HT4 receptor agonists in different biological models it has been suggested that there is heterogeneity among 5-HT4 receptors. Here, we report the molecular cloning of several 5-HT4 receptor splice variants in mouse, rat, and human brain. Our data suggest that the differences in efficacy of 5-HT4 ligands on 5-HT4 receptor-mediated responses in several tissues is due to differences in coupling efficiency rather than to the presence of different 5-HT4 receptor isoforms.

Cloning and expression of human 5-HT4S receptors. Effect of receptor density on their coupling to adenylyl cyclase.

We have isolated a cDNA encoding the 5-HT4S receptor by RT-PCR on poly (A)+ RNA from both human heart and brain. The sequence homology with the rat and mouse 5-HT4 receptors was high: 93.8% of identity in the amino acid sequence. None of the 24 amino acid substitutions observed between rat and human receptors are at positions likely to modify their pharmacology. Comparing the pharmacological properties of six agonists and five antagonists on rat and human 5-HT4S receptors revealed no significant differences. We have analyzed the behavior of renzapride, a full and a partial agonist on mouse colliculi neurons and human heart biological responses respectively. The coupling efficiency of renzapride was two-fold lower than that of 5-HT for the stimulation of 5-HT4S receptors transfected in two different cell lines (LLC-PK1 and COS-7), but increasing the receptor density suppressed the partial agonist effect of renzapride.

5-HT4 receptors improve social olfactory memory in the rat.

Serotonin (5-HT) is involved in a large variety of physiological functions and it appears now that it could play a role in cognitive processes through the activation of 5-HT4 receptors. The present study was conducted to determine the effect of BIMU1, a mixed 5-HT4 agonist/5-HT3 antagonist on social olfactory recognition in rats, a behaviour test which has previously been shown to access short-term memory and to be sensitive to cholinergic drugs. This test is based on the investigation of an unfamiliar juvenile by an adult rat during two distinct 5-min presentations. At a 30-min delay after each presentation adults recognized the juvenile, whereas after a 2-hr delay all the adults had forgotten it. When administered intraperitoneally immediately after the first presentation, BIMU1 (10 mg/kg) enhanced short-term memory (i.e. recognition of the juvenile after a 2-hr delay). Ondansetron (10 and 100 micrograms/kg injected intraperitoneally), a 5-HT3 antagonist, had no significant effect on this form of memory. The effect of BIMU1 was antagonized by intraperitoneal injection of GR 125487, a very selective and potent 5-HT4 antagonist. The antagonistic effect was obtained at 1 and 10 mg/kg of GR 125487, but not at 0.1 mg/kg. It is certainly a specific effect on brain 5-HT4 receptors, since we determined a brain concentration of GR 125487 equal to 3.8 x 10(-7) M after the intraperitoneal injection of 10 mg/kg of this drug. This GR 125487 concentration is certainly sufficient to occupy all the 5-HT4 brain receptors (Kd = 10(-10) M) but not to occupy 5-HT3 receptors (Kd > 10(-6) M). The 5-HT4 specificity of the blockade by GR 125487 is further demonstrated by the fact that a 10-fold lower dose of GR 125487 (1 mg/kg) is also effective to inhibit the BIMU1 effect.

BIMU1 increases associative memory in rats by activating 5-HT4 receptors.

Olfactory association learning was used to investigate the involvement of 5-HT4 receptors in learning and long-term memory. The behavioral role of the 5-HT4 receptors was studied by using BIMU1 (3-ethyl-2,3-dihydro-N-[endo-8-methyl-8-azabicyclo(3.2.1)oct-3-yl]-2-oxo -1 H-benzimidazole-1-carboxamide, hydrochloride (Boehringer Ingelheim, Italy); a mixed 5-HT4 agonist/5-HT3 antagonist, and GR125487 (1-[2-[methyl sulphonyl)-amino]ethyl]-4-piperidinyl-methyl 5-fluro-2-methoxy-1H-indole-3- carboxylate; Glaxo Group Research, Hertfordshire, U.K.), a specific 5-HT4 antagonist. The intraperitoneal injections of BIMU1 at 1, 5, and 10 mg/kg were followed by an substantial improvement (> 15% in percentage of correct responses at the dose of 10 mg/kg) in associative memory. Difficulty rapidly reversing behavioral responses to previously learned association, 1 month later indicated that the BIMU1 effect at 10 mg/kg was not transient, but correlated to long-term memory. The effects of BIMU1 are most likely to be mediated by 5-HT4 receptors since they were blocked by GR125487 at 10 mg/kg. These data suggest that activation of 5-HT4 receptors may modulate cognitive processes like learning and memory.

Lesion study of the distribution of serotonin 5-HT4 receptors in rat basal ganglia and hippocampus.

The regional distribution of 5-hydroxytryptamine (5-HT4) receptors labelled with [3H]GR113808 was examined in rat basal ganglia and hippocampus after specific lesions. Lesion of serotonin neurons induced by injections of 5,7-dihydroxytryptamine into the dorsal and medial raphe nuclei resulted in increased 5-HT4 receptor binding in most regions examined, compared with controls. More precisely, there was a 78% increase in the rostral but no change in the caudal part of caudate-putamen, and 83% and 54% increases in the shell and core of the nucleus accumbens respectively. In the substantia nigra, the increase in 5-HT4 binding was larger (72%) than that in the globus pallidus (32%). In the hippocampus, 63%, 30% and 28% increases were measured in CA2, CA1 and CA3 respectively. Following lesion of dopamine neurons by intranigral injection of 6-hydroxydopamine, increased 5-HT4 receptor binding was observed in the caudal (59%), but not the rostral part of caudate-putamen, as well as in the globus pallidus (93%). Since no decreases in 5-HT4 receptor density were detected after the dopamine lesion, it was concluded that these receptors are not expressed in dopamine neurons. Kainic acid lesions of the caudate-putamen were associated with dramatic local decreases in 5-HT4 receptor binding on the injected side (-89%), which suggested that striatal neurons express 5-HT4 receptors. Corresponding decreases of 72 and 20% in receptor density were detected in globus pallidus and substantia nigra, consistent with a presumed localization of 5-HT4 receptors on striatal GABA neurons projecting to these regions. In the substantia nigra, the decrease in [3H]GR113808 binding was localized to the pars lateralis, indicating that striatal neurons belonging to the cortico-striato-nigro-tectal pathway, and containing GABA and dynorphin, express 5-HT4 receptors.

Cloning, expression and pharmacology of the mouse 5-HT(4L) receptor.

Since most of our knowledge on pharmacological properties of brain 5-HT4 receptors have been discussed for mouse colliculi neurons, we cloned the corresponding receptor using the RT-PCR approach. As expected, the homology with the already cloned rat 5-HT(4L) receptor was high, revealing only 16 differences at the amino-acid level. One of the differences, proline75 in mouse, alanine75 in the already published rat sequences was not confirmed. Therefore this proline is part of the consensus sequence present in all 5-HT receptor transmembrane domain II (LVMP). Comparing the affinities of 11 agonists and five antagonists for the cloned mouse receptor (5-HT(4L))expressed in LLCPK1 and the corresponding receptor in mouse colliculi shows an excellent correlation. The transfected mouse 5-HT(4L) receptor stimulated cAMP production. When expressed at high density, it exhibited intrinsic activity. In contrast to the previously described distribution, we found that mRNA encoding for both the short (5-HT(4S))and the long form (5-HT(4L)) of 5-HT4 receptors are expressed in all mouse and rat brain areas.

Alternative splicing in the N-terminal extracellular domain of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor modulates receptor selectivity and relative potencies of PACAP-27 and PACAP-38 in phospholipase C activation.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides of the vasoactive intestinal peptide/secretin/glucagon family. We previously described alternative splicing of the region encoding the third intracellular loop of the PACAP receptor generating six isoforms with differential signal transduction properties (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170-175). In addition, we demonstrated that the potencies of the two forms of PACAP are similar for adenylate cyclase stimulation, whereas PACAP-38 is more potent than PACAP-27 in phospholipase C activation. In the present work, we document the existence of a new splice variant of the PACAP receptor that was characterized by a 21-amino-acid deletion in the N-terminal extracellular domain. We demonstrate that this domain modulates receptor selectivity with respect to PACAP-27 and -38 binding and controls the relative potencies of the two agonists in phospholipase C stimulation.

Pharmacological comparison between [3H]GR 113808 binding sites and functional 5-HT4 receptors in neurons.

5-HT4 receptors positively coupled to adenylyl cyclase and possessing unique pharmacological properties were first described in mouse colliculi neurons using functional studies. The recent introduction of a radiolabeled 5-HT4 receptor antagonist, [3H]GR 113808 [1-[2-(methylsulphonylamino)ethyl]4-piperidinyl]methyl-1-methyl-in dole-3 carboxylate] having high specificity and affinity allowed the pharmacological comparison between the specific binding sites identified with this compound and the functional 5-HT4 receptors in the same preparation, the colliculi neurons. We show here that [3H]GR 113808 binding is saturable in this preparation and reveals a homogeneous population of sites with a pKd value of 9.5 +/- 0.2 and a Bmax of 75 +/- 23 fmol/mg protein. Seventeen agonists and six antagonists with molecules structurally related either to indoles, benzamides or benzimidazolones and previously known as 5-HT4 receptor ligands, were tested for their ability to compete with [3H]GR 113808 binding sites and to stimulate or inhibit 5-HT-stimulated adenylyl cyclase activity. Highly significant correlations were obtained between the affinities of either agonists or antagonists for [3H]GR 113808 binding sites and their potencies for functional 5-HT4 receptors (r = 0.87 and 0.99, respectively). In addition, we also found good correlations between the Kd of several 5-HT4 receptor ligands determined in cell membranes of mouse colliculi neurons and their Kd determined in previous studies in guinea-pig striatum (0.95) and in human caudate (0.97). [3H]GR 113808 binding studies demonstrated that the 50% decrease in 5-HT-stimulated cAMP accumulation which followed a 5 min exposure period with 5-HT (10 microM) was not accompanied by any significant decrease in the number of binding sites. Longer exposure periods with 5-HT resulted in a decrease in [3H]GR 113808 binding sites which started to be significant after 30 min.