Medium-chain fatty acids modulate myocardial function via a cardiac odorant receptor.

Several studies have demonstrated the expression of odorant receptors (OR) in various human tissues and their involvement in different physiological and pathophysiological processes. However, the functional role of ORs in the human heart is still unclear. Here, we firstly report the functional characterization of an OR in the human heart. Initial next-generation sequencing analysis revealed the OR expression pattern in the adult and fetal human heart and identified the fatty acid-sensing OR51E1 as the most highly expressed OR in both cardiac development stages. An extensive characterization of the OR51E1 ligand profile by luciferase reporter gene activation assay identified 2-ethylhexanoic acid as a receptor antagonist and various structurally related fatty acids as novel OR51E1 ligands, some of which were detected at receptor-activating concentrations in plasma and epicardial adipose tissue. Functional investigation of the endogenous receptor was carried out by Ca2+ imaging of human stem cell-derived cardiomyocytes. Application of OR51E1 ligands induced negative chronotropic effects that depended on activation of the OR. OR51E1 activation also provoked a negative inotropic action in cardiac trabeculae and slice preparations of human explanted ventricles. These findings indicate that OR51E1 may play a role as metabolic regulator of cardiac function.

Functional characterization of the ectopically expressed olfactory receptor 2AT4 in human myelogenous leukemia.

The olfactory receptor (OR) family was found to be expressed mainly in the nasal epithelium. In the last two decades members of the OR family were detected to be functional expressed in different parts of the human body such as in liver, prostate or intestine cancer cells. Here, we detected the expression of several ORs in the human chronic myelogenous leukemia (CML) cell line K562 and in white blood cells of clinically diagnosed acute myeloid leukemia (AML) patients by RT-PCR and next-generation sequencing. With calcium-imaging, we characterized in greater detail the cell biological role of one OR (OR2AT4) in leukemia. In both cell systems, the OR2AT4 agonist Sandalore-evoked strong Ca(2+) influx via the adenylate cyclase-cAMP-mediated pathway. The OR2AT4 antagonist Phenirat prevented the Sandalore-induced intracellular Ca(2+) increase. Western blot and flow cytometric experiments revealed that stimulation of OR2AT4 reduced the proliferation by decreasing p38-MAPK phosphorylation and induced apoptosis via phosphorylation of p44/42-MAPK. Furthermore, Sandalore increased the number of hemoglobin-containing cells in culture. We described for the first time an OR-mediated pathway in CML and AML that can regulate proliferation, apoptosis and differentiation after activation. This mechanism offers novel therapeutic options for the treatment of AML.

Deorphanization and characterization of the ectopically expressed olfactory receptor OR51B5 in myelogenous leukemia cells.

The ectopic expression of olfactory receptors (ORs) in the human body has been of major interest in the past decade. Several studies have reported the expression of ORs not only in healthy tissues such as heart, sperm or skin cells, but also in cancerous tissues of the liver, prostate or intestine. In the present study, we detected the expression of OR51B5 in the chronic myelogenous leukemia (CML) cell line K562 and in white blood cell samples of clinically diagnosed acute myelogenous leukemia (AML) patients by reverse transcription-PCR and immunocytochemical staining. The known OR51B5 ligand isononyl alcohol increased the levels of intracellular Ca(2+) in both AML patient blood cells and K562 cells. With calcium imaging experiments, we characterized in greater detail the OR51B5-mediated signaling pathway. Here, we observed an involvement of adenylate cyclase and the downstream L-type and T-type calcium channels. In addition, the activation of OR51B5 leads to an inhibition of cell proliferation in K562 cells. In western blot experiments, we found that incubation with isononyl alcohol led to a reduction in p38-MAPK (mitogen-activated protein kinase) phosphorylation that might be responsible for the decreased cell proliferation. In the present study, we characterized the OR51B5-mediated signaling pathway downstream of the activation with isononyl alcohol, which leads to reduced proliferation and therefore provide a novel pharmacological target for CML and AML, the latter of which remains difficult to treat.

[Specific anosmia as a principle of olfactory perception]. / Spezifische Anosmie als Prinzip olfaktorischer Wahrnehmung.

Specific anosmia, the inability to perceive a specific odor, while olfactory perception is otherwise intact, is known as a rather seldom phenomenon. By testing the prevalence of specific anosmia to 20 different odors in a sample of 1600 people, we were able to estimate the general prevalence of anosmia. This revealed that specific anosmia is not rare at all. In contrast, the general likelihood for specific anosmia approaches 1. In addition, specific anosmia can be very well reversed by "smell training" during the course of 3 months. To summarize, specific anosmia seems to be a rule, not an exception, of olfactory sensation. The lack of perception of certain odors may constitute a flexible peripheral filter mechanism, which can be adapted by exposure to odors.

The smelling of Hedione results in sex-differentiated human brain activity.

A large family of vomeronasal receptors recognizes pheromone cues in many animals including most amphibia, reptiles, rhodents, and other mammals. Humans possess five vomeronasal-type 1 receptor genes (VN1R1-VN1R5), which code for proteins that are functional in recombinant expression systems. We used two different recombinant expression systems and identified Hedione as a ligand for the putative human pheromone receptor VN1R1 expressed in the human olfactory mucosa. Following the ligand identification, we employed functional magnetic resonance imaging (fMRI) in healthy volunteers to characterize the in vivo action of the VN1R1 ligand Hedione. In comparison to a common floral odor (phenylethyl alcohol), Hedione exhibited significantly enhanced activation in limbic areas (amygdala, hippocampus) and elicited a sex-differentiated response in a hypothalamic region that is associated with hormonal release. Utilizing a novel combination of methods, our results indicate that the putative human pheromone receptor VN1R1 is involved in extra-olfactory neuronal activations induced by the odorous substance Hedione. The activation of VN1R1 might play a role in gender-specific modulation of hormonal secretion in humans.

Sweet taste and chorda tympani transection alter capsaicin-induced lingual pain perception in adult human subjects.

Sweetness signals the nutritional value of food and may moreover be accompanied by a sensory suppression that leads to higher pain tolerance. This effect is well documented in infant rats and humans. However, it is still debated whether sensory suppression is also present in adult humans. Thus, we investigated the effects of sweet taste on the perception of the painful trigeminal stimulus capsaicin in two groups of healthy adult human subjects. A solution of 100 µM capsaicin was applied to the tip of the subject's tongues in order to stimulate trigeminal Aδ- and C-fiber nociceptors. When swallowed, 1M sucrose reduced the capsaicin-induced burning sensation by 29% (p ≤ 0.05) whereas a solution of similar taste intensity containing 1 µM quinine did not. Similarly, sucrose application to the frontal hemitongue suppressed the perception of the burning sensation induced by contralaterally applied capsaicin by 25% (p ≤ 0.01). We furthermore investigated the effects of documented unilateral transection of the chorda tympani nerve on capsaicin perception. In accordance with the ipsi-to-contralateral effect of sucrose on capsaicin perception in healthy subjects, hemiageusic subjects were more sensitive for capsaicin on the tongue contralateral to the taste nerve lesion (+38%; p ≤ 0.01). Taken together, these results argue I) for the existence of food intake-induced sensory suppression, if not analgesia, in adult humans and II) a centrally mediated suppression of trigeminal sensation by taste inputs that III) becomes disinhibited upon peripheral taste nerve lesion.

[Neuropeptides and their receptors as a molecular explanation for sensitive skin]. / Neuropeptide und ihre Rezeptoren als molekulare Grundlage der empfindlichen Haut.

The concept of sensitive skin represents a symptom of physiological reactions rather than a disease entity. According to epidemiological studies, up to 50% of adults report on sensitivity of the face with various distinctive symptoms such as prickling, burning, tingling, pain or itching. These sensations can be mediated by receptors expressed on neurons and keratinocytes. The heat receptor TRPV1 is for example involved in nociception and mediates not only warmth but also burning. Furthermore, neurotrophins and exogenic factors such as stress may have a biological role as discussed in this review.

Electrical responses to chemosensory stimulation recorded from the vomeronasal duct and the respiratory epithelium in humans.

The physiological significance of the human vomeronasal duct (VND) is still unclear. The aim of the present study was to investigate the question whether mucosal responses obtained from the VND are different from those obtained from the respiratory epithelium. There were 15 healthy subjects (8 male, 7 female; age range 19-45 years; 14 normosmic subjects, 1 anosmic subject). All subjects participated in two sessions whereby the first session was used to acquaint them with the experimental conditions. For chemical stimulation, an olfactometer was used which delivered chemical stimulants without altering mechanical or thermal conditions at the stimulated nasal mucosa. For stimulation we used substances previously reported to produce vomeronasal activation ("estra"=estra-1,3,5(10),16-tetraen-3ol and "andro"=androsta-4,16-dien-3-on); in addition, gaseous CO(2) was used as a non-odorous, relatively specific stimulant of the trigeminal nerve. Placement of electrodes either in the VND or on the respiratory epithelium was performed under endoscopical guidance. Subjects rated the overall intensity of the stimuli, the strength of trigeminally mediated sensations, and the hedonic tone of the stimulants. Responses could not be recorded from all subjects. For the remaining 7 subjects, intensity was strongest for CO(2) stimuli (p<0.001), whereas no significant difference was observed between "andro" and "estra" (p=0.33). All three stimulants produced responses at the respiratory epithelium with largest responses obtained after stimulation with CO(2). Similar findings were made for recordings inside the VND. Due to the small sample size sexual dimorphisms could not be addressed. In summary, these results seem to indicate that the presently used stimulants produce similar responses at the respiratory epithelium and in the VND which argues against a specific responsiveness of the VND epithelium to chemosensory stimuli although it has to be kept in mind that the effective sample size was small.

Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons.

Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase C delta 1 (PLC delta 1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P(2)/IP(3) and PI(3,4,5)P(3), the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2s of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction.

Monoterpenoid agonists of TRPV3.

BACKGROUND AND PURPOSE: Transient receptor potential (TRP) V3 is a thermosensitive ion channel expressed predominantly in the skin and neural tissues. It is activated by warmth and the monoterpene camphor and has been hypothesized to be involved in skin sensitization. A selection of monoterpenoid compounds was tested for TRPV3 activation to establish a structure-function relationship. The related channel TRPM8 is activated by cool temperatures and a number of chemicals, among them the monoterpene (-)-menthol. The overlap of the receptor pharmacology between the two channels was investigated. EXPERIMENTAL APPROACH: Transfected HEK293 cells were superfused with the test substances. Evoked currents were measured in whole cell patch clamp measurements. Dose-response curves for the most potent agonists were obtained in Xenopus laevis oocytes. KEY RESULTS: Six monoterpenes significantly more potent than camphor were identified: 6-tert-butyl-m-cresol, carvacrol, dihydrocarveol, thymol, carveol and (+)-borneol. Their EC(50) is up to 16 times lower than that of camphor. All of these compounds carry a ring-located hydroxyl group and neither activates TRPM8 to a major extent. CONCLUSIONS AND IMPLICATIONS: Terpenoids have long been recognized as medically and pharmacologically active compounds, although their molecular targets have only partially been identified. TRPV3 activation may be responsible for several of the described effects of terpenoids. We show here that TRPV3 is activated by a number of monoterpenes and that a secondary hydroxyl-group is a structural requirement.

Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay.

1. TRPM8 (CMR1) is a Ca(2+)-permeable channel, which can be activated by low temperatures, menthol, eucalyptol and icilin. It belongs to the transient receptor potential (TRP) family, and therefore is related to vanilloid receptor type-1 (VR1, TRPV1). We tested whether substances which are structurally related to menthol, or which produce a cooling sensation, could activate TRPM8, and compared the responses of TRPM8 and VR1 to these ligands. 2. The effects of 70 odorants and menthol-related substances on recombinant mouse TRPM8 (mTRPM8), expressed in HEK293 cells, were examined using a FLIPR assay. In all, 10 substances (linalool, geraniol, hydroxycitronellal, WS-3, WS-23, FrescolatMGA, FrescolatML, PMD38, CoolactP and Cooling Agent 10) were found to be agonists. 3. The EC(50) values of the agonists defined their relative potencies: icilin (0.2+/-0.1 microM)>FrescolatML (3.3+/-1.5 microM) > WS-3 (3.7+/-1.7 microM) >(-)menthol (4.1+/-1.3 microM) >frescolatMAG (4.8+/-1.1 microM) > cooling agent 10 (6+/-2.2 microM) >(+)menthol (14.4+/-1.3 microM) > PMD38 (31+/-1.1 microM) > WS-23 (44+/-7.3 microM) > Coolact P (66+/-20 microM) > geraniol (5.9+/-1.6 mM) > linalool (6.7+/-2.0 mM) > eucalyptol (7.7+/-2.0 mM) > hydroxycitronellal (19.6+/-2.2 mM). 4. Known VR1 antagonists (BCTC, thio-BCTC and capsazepine) were also able to block the response of TRPM8 to menthol (IC(50): 0.8+/-1.0, 3.5+/-1.1 and 18+/-1.1 microM, respectively). 5. The Ca(2+) response of hVR1-transfected HEK293 cells to the endogenous VR1 agonist N-arachidonoyl-dopamine was potentiated by low pH. In contrast, menthol- and icilin-activated TRPM8 currents were suppressed by low pH. 6. In conclusion, in the present study, we identified 10 new agonists and three antagonists of TRPM8. We found that, in contrast to VR1, TRPM8 is inhibited rather than potentiated by protons.

Modulation of the olfactory CNG channel by Ptdlns(3,4,5)P3.

Recent data suggest that the 3-phosphoinositides can modulate cyclic nucleotide signaling in rat olfactory receptor neurons (ORNs). Given the ability of diverse lipids to modulate ion channels, we asked whether phosphatidylinositol 3,4,5-trisphosphate (PIP3) can regulate the olfactory cyclic nucleotide-gated (CNG) channel as a possible mechanism for this modulation. We show that applying PIP3 to the intracellular side of inside-out patches from rat ORNs inhibits activation of the olfactory CNG channel by cAMP. The effect of PIP3 is immediate and partially reversible, and reflects an increase in the EC50 of cAMP, not a reduction in the single-channel current amplitude. The effect of PIP3 is significantly stronger than that of PIP2; other phospholipids tested have no appreciable effect on channel activity. PIP3 similarly inhibits the recombinant heteromeric (A2/A4) and homomeric (A2) olfactory CNG channel expressed in HEK293 cells, suggesting that PIP3 acts directly on the channel. These findings indicate that 3-phosphoinositides can be functionally important regulators of CNG channels.

A novel function for chemokines: downregulation of neutrophil migration.

Migration is a key function of stem cells during ontogenesis, of fibroblasts in wound healing and of immune cells in host defence. The signals that initiate migration are as important as signals that terminate migration, once the destination has been reached. We now show that formyl-methionyl-leucyl-phenylalanine (fMLP)-induced migration of neutrophils was inhibited by increasing concentrations of interleukin-8 (IL-8). IL-8 dose dependently increased the frequency and the duration of stop-periods, whereas the percentage of cells of a population that was locomotory active remained constant. The stop-signal delivered by IL-8 was intracellularly transduced by a dichotomic pathway: (i) the activation of the adenylyl cyclase leads to an increase of cytosolic cyclic adenosine monophosphate, which results in an activation of the sarcoplasmatic/endoplasmatic reticulum calcium ATPase pump and a calcium sequestration; (ii) the activation of the phospholipase Cbeta (PLCbeta) generates inositol-1,4,5-phosphate (IP3) and diacylglycerol (DAG), which results in IP3-mediated release of intracellularly stored calcium in the endoplasmatic reticulum and DAG-mediated activation of protein kinase C. Thus, we show for the first time that a chemokine, IL-8, in concert with fMLP, downregulates the neutrophil migration through the regulation of the intracellular calcium concentration via the adenylyl cyclase and the PLCbeta2.

Cloning and functional characterization of two glycine receptor alpha-subunits from the perch retina.

Glycine receptors are ligand-gated ion channel proteins mediating synaptic inhibition in the spinal cord, retina and brain of vertebrates. We have cloned and functionally characterized two glycine receptor alpha-subunits from the perch (Roccus americana) retina. Based on sequence homology with the mammalian counterparts, we termed these subunits alpha 1 and alpha 3. RT-PCR revealed the presence of both subunits in retina and brain, whereas alpha1 was predominant in spinal cord. A short splice variant of alpha1 was detected in the brain but not in the retina. Functional expression of the perch subunits in HEK-293 cells yielded robust glycine-gated currents sensitive to strychnine. The perch receptors displayed a high efficacy for taurine and GABA and thus differ from the mammalian counterparts. Because the retina is a rich source for taurine, this finding could be of physiological importance. The structural features of the ligand binding domain strongly support the notion of increased glycine/GABA discrimination in higher vertebrates.

Electrophysiological and morphological properties of cell types in the chick neostriatum caudolaterale.

The neostriatum caudolaterale, in the chick also referred to as dorsocaudal neostriatal complex, is a polymodal associative area in the forebrain of birds that is involved in sensorimotor integration and memory processes. We have used whole-cell patch-clamp recordings in chick brain slices to characterize the principal cell types of the neostriatum caudolaterale. Electrophysiological properties distinguished four classes of neurons. The morphological characteristics of these classes were examined by intracellular injection of Lucifer Yellow. Type I neurons characteristically fired a brief burst of action potentials. Morphologically, type I neurons had large somata and thick dendrites with many spines. Type II neurons were characterized by a repetitive firing pattern with conspicuous frequency adaptation. Type II neurons also had large somata and thick dendrites with many spines. There was no clear morphological distinction between type I and type II neurons. Type III neurons showed high-frequency firing with little accommodation and a prominent time-dependent inward rectification. They had thin, sparsely spiny dendrites and extensive local axonal arborizations. Electrophysiological and morphological properties indicated them as being interneurons. Type IV neurons had a longer action potential duration, a larger input resistance, and a longer membrane time constant than the other classes. Type IV neurons had small somata and short dendrites with few spines. The long axon collaterals of neurons in all spiny cell classes (types I, II, IV) followed similar patterns, suggesting that neurons from all these types can contribute to the projections of the neostriatum caudolaterale to sensory, limbic and motor areas. The electrophysiological and anatomical characterization of the major classes of neurons in the caudal forebrain of the chick provides a framework for the investigation of sensorimotor integration and learning at the cellular level in birds.

Phosphorylation of voltage-gated ion channels in rat olfactory receptor neurons.

In olfactory receptor neurons (ORNs), ligand-odorant receptor interactions cause G protein-mediated activation of adenylate cyclase and a subsequent increase in concentration of the intracellular messenger cAMP. Odorant-evoked elevation in cAMP is thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca2+ influx, leading to membrane depolarization and the generation of action potentials. Our data show that in freshly dissociated rat ORNs, odorant-induced elevation in cAMP also activates cAMP-dependent protein kinase (PKA), which is then able to phosphorylate various protein targets in the olfactory signal transduction pathway, specifically voltage-gated sodium and calcium channels. The presence of PKI (PKA inhibitor peptide) blocked the modulatory action of cAMP on voltage-gated ion channels. By modulating the input/output properties of the sensory neurons, this mechanism could take part in the complex adaptation process in odorant perception. In addition, we found modulation of voltage-gated sodium and calcium channel currents by 5-hydroxytryptamine and the dopamine D1 receptor agonist SKF 38393. These findings suggest that in situ ORNs might also be a target for efferent modulation.

Functional expression and characterization of odorant receptors using the Semliki Forest virus system.

The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants starts with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have expressed and characterized different olfactory receptors with several expression systems. Here we provide the first documentation of functional expression of odorant receptors using the Semliki Forest virus system. The human odorant OR 17-40 receptor and the rat 17 receptor were functionally expressed in vertebrate kidney cells (HEK293) using recombinant Semliki Forest viruses. Receptors were expressed as a fusion protein with the N-terminal membrane import sequence of the guinea pig serotonin receptor. Experiments employing the Ca2+-sensitive dye fura-2 revealed a fast, transient increase in the [Ca2+]i after application of the specific agonists helional and octanal to HEK293 cells infected with viruses containing RNA for the human odorant OR 17-40 receptor and the rat 17 receptor, respectively.

Functional expression and characterization of a Drosophila odorant receptor in a heterologous cell system.

Odorant receptors (ORs) constitute the molecular basis for the detection of volatile odorous molecules and the perception of smell. Our understanding of chemical senses has been greatly expanded by the discovery of the OR gene families in vertebrates and in the nematode Caenorhabditis elegans. Recently, candidate Drosophila OR genes have been identified. The putative ORs do not possess any primary sequence identity with known vertebrate or C. elegans receptors, but belong to the family of G protein-coupled receptors according to their predicted seven transmembrane topology. To prove olfactory function of these proteins, we expressed a member of the putative Drosophila OR gene family, Or43a, in Xenopus laevis oocytes. Using two-electrode voltage-clamp recording we identified four odors (cyclohexanone, cyclohexanol, benzaldehyde, and benzyl alcohol) that activated the receptor at low micromolar concentration and structurally related substances that did not. This report shows the function and specificity of a member of the recently identified family of Drosophila ORs expressed in a heterologous system.

A single amino-acid in the TM1 domain is an important determinant of the desensitization kinetics of recombinant human and guinea pig alpha-homomeric 5-hydroxytryptamine type 3 receptors.

Desensitization of ligand-gated ion channels shapes synaptic responses and provides critical neuroprotection at central synapses, yet the molecular mechanisms underlying the desensitization process are poorly understood. Using the whole-cell voltage-clamp technique, we investigated desensitization kinetics of recombinant human and guinea pig alpha-homomeric 5-hydroxytryptamine type 3 (5-HT(3A)) receptors heterologously expressed in human embryonic kidney 293 cells. Human 5-HT(3A) receptors desensitize 3.5 times faster than does the homologous receptor from guinea pigs. By constructing various chimeras and through site-directed mutagenesis, we have identified a single serine in the M1 region of the human 5-HT(3A) receptor sequence (S248) that, when substituted with threonine found in the equivalent guinea pig sequence (T254), conferred guinea pig-like kinetics on the time course of desensitization of the human receptor. Correspondingly, the reverse mutation (guinea pig T254S) resulted in a fast, human-like time constant of desensitization. Thus, the primary structure of the M1 region is an important determinant of desensitization kinetics of recombinant 5-HT(3A) receptors.

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons.

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.