Capsaicin 8% patch for treprostinil subcutaneous infusion site pain in pulmonary hypertension patients.

BACKGROUND: Treprostinil sodium improves haemodynamics and symptoms in pulmonary arterial hypertension (PAH) patients, but its subcutaneous (s.c.) administration can produce severe local site pain, and lead to discontinuation of vital treatment. Treprostinil is a prostacyclin analogue which stimulates prostacyclin receptors in skin nociceptor terminals, resulting in pain and cutaneous hypersensitivity, for which current pain remedies have limited effect. Capsaicin 8% patch relieves neuropathic pain for 3 months after a single 60 min cutaneous application; we investigated whether its pre-application can reduce s.c. trepostinil-induced pain. METHODS: A single-centre, double-blind, randomized, placebo-controlled, crossover study was conducted to assess the safety and efficacy of a single capsaicin 8% patch pre-application for s.c. treprostinil pain in 11 PAH patients, relative to control patch with low-dose capsaicin 0.075% cream. RESULTS: The primary efficacy endpoint, mean difference between the two treatment arms in an 11-point numerical pain rating scale from baseline to 2 weeks after patch applications, was significantly lower on the capsaicin 8% patch treatment arm [P=0.01, mean difference=-1.47 units, 95% credible interval (CI): -2. 59 to -0.38] in the patients who completed the study per protocol, although intention-to-treat analysis did not show significant difference (P=0.28). Heat pain thresholds were decreased (P=0.027, mean difference=5.43°C, 95% CI: 0.71-10.21) and laser Doppler flux increased (P=0.016, mean difference=370 units, 95% CI: 612 to 127.9) at the application site immediately after capsaicin 8% patch, confirming activity. CONCLUSIONS: Further investigation of the efficacy of capsaicin 8% patch in this indication is warranted.

Pittsburgh compound B (11C-PIB) and fluorodeoxyglucose (18 F-FDG) PET in patients with Alzheimer disease, mild cognitive impairment, and healthy controls.

Amyloid load in the brain using Pittsburgh compound B ((11)C-PIB) positron emission tomography (PET) and cerebral glucose metabolism using fluorodeoxyglucose ((18)F-FDG) PET were evaluated in patients with mild Alzheimer disease (AD, n = 18), mild cognitive impairment (MCI, n = 24), and controls (CTR, n = 18). ( 11)C-PIB binding potential (BP(ND)) was higher in prefrontal cortex, cingulate, parietal cortex, and precuneus in AD compared to CTR or MCI and in prefrontal cortex for MCI compared to CTR. For (18)F-FDG, regional cerebral metabolic rate for glucose (rCMRGlu) was decreased in precuneus and parietal cortex in AD compared to CTR and MCI, with no MCI-CTR differences. For the AD-CTR comparison, precuneus BP(ND) area under the receiver operating characteristic (ROC) curve (AUC) was 0.938 and parietal cortex rCMRGlu AUC was 0.915; for the combination, AUC was 0.989. ( 11)C-PIB PET BP(ND) clearly distinguished diagnostic groups and combined with (18)F-FDG PET rCMRGlu, this effect was stronger. These PET techniques provide complementary information in strongly distinguishing diagnostic groups in cross-sectional comparisons that need testing in longitudinal studies.

PIB is a non-specific imaging marker of amyloid-beta (Abeta) peptide-related cerebral amyloidosis.

The in vivo imaging probe [11C]-PIB (Pittsburgh Compound B, N-methyl[11C]2-(4'-methylaminophenyl-6-hydroxybenzathiazole) is under evaluation as a key imaging tool in Alzheimer's disease (AD) and to date has been assumed to bind with high affinity and specificity to the amyloid structures associated with classical plaques (CPs), one of the pathological hallmarks of the disease. However, no studies have systematically investigated PIB binding to human neuropathological brain specimens at the tracer concentrations achieved during in vivo imaging scans. Using a combination of autoradiography and histochemical techniques, we demonstrate that PIB, in addition to binding CPs clearly delineates diffuse plaques and cerebrovascular amyloid angiopathy (CAA). The interaction of PIB with CAA was not fully displaceable and this may be linked to the apolipoprotein E-epsilon4 allele. PIB was also found to label neurofibrillary tangles, although the overall intensity of this binding was markedly lower than that associated with the amyloid-beta (Abeta) pathology. The data provide a molecular explanation for PIB's limited specificity in diagnosing and monitoring disease progression in AD and instead indicate that the ligand is primarily a non-specific marker of Abeta-peptide related cerebral amyloidosis.

The artificial zinc finger protein 'Blues' binds the enhancer of the fibroblast growth factor 4 and represses transcription.

The design of novel genes encoding artificial transcription factors represents a powerful tool in biotechnology and medicine. We have engineered a new zinc finger-based transcription factor, named Blues, able to bind and possibly to modify the expression of fibroblast growth factor 4 (FGF-4, K-fgf), originally identified as an oncogene. Blues encodes a three zinc finger peptide and was constructed to target the 9 bp DNA sequence: 5'-GTT-TGG-ATG-3', internal to the murine FGF-4 enhancer, in proximity of Sox-2 and Oct-3 DNA binding sites. Our final aim is to generate a model system based on artificial zinc finger genes to study the biological role of FGF-4 during development and tumorigenesis.

Effect of the adenosine A1 receptor agonist GR79236 on trigeminal nociception with blink reflex recordings in healthy human subjects.

GR79236 is a highly potent and selective adenosine A1 receptor agonist that has analgesic and anti-inflammatory actions in humans and animals. In animal models it inhibits trigeminal nerve firing and calcitonin gene-related peptide release which play a pivotal role in migraine pathophysiology. Thus GR79236 may have therapeutic potential in migraine. Although there are no validated human models of migraine, the trigeminal nociceptive pathways may be studied with a novel electrode to elicit nociception-specific blink reflex responses. Twelve healthy female volunteers were randomized in a double-blind, placebo-controlled, cross-over trial to investigate the effect of GR79236 on trigeminal nociceptive pathways, as measured by the blink reflex. A secondary objective was to compare the use of two types of electrode, the standard (SE) and nociception-specific electrodes (NE), to investigate human trigeminal pharmacology. Blink reflexes were elicited with SE and NE before and 30 min after GR79236 (10 microg/kg i.v.) or placebo. The median area under the curve of repeated sweeps of the R2 component of the blink reflex was analysed using analysis of covariance with baseline as covariate. Using NE, GR79236 produced a non-significant reduction of the ipsilateral R2 compared with placebo (P = 0.097) and a significant reduction contralaterally (P = 0.008). No significant changes were observed using SE. There were no significant adverse events. The results suggest that NE is more sensitive than SE to detect pharmacological effects in the trigeminal nociceptive system. Furthermore, the adenosine A1 receptor agonist GR79236 inhibits trigeminal nociception in humans. These results support a possible therapeutic role for GR79236 in primary headache disorders.

Single-nucleotide polymorphism alleles in the insulin receptor gene are associated with typical migraine.

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.

The artificial zinc finger coding gene 'Jazz' binds the utrophin promoter and activates transcription.

Up-regulation of utrophin gene expression is recognized as a plausible therapeutic approach in the treatment of Duchenne muscular dystrophy (DMD). We have designed and engineered new zinc finger-based transcription factors capable of binding and activating transcription from the promoter of the dystrophin-related gene, utrophin. Using the recognition 'code' that proposes specific rules between zinc finger primary structure and potential DNA binding sites, we engineered a new gene named 'Jazz' that encodes for a three-zinc finger peptide. Jazz belongs to the Cys2-His2 zinc finger type and was engineered to target the nine base pair DNA sequence: 5'-GCT-GCT-GCG-3', present in the promoter region of both the human and mouse utrophin gene. The entire zinc finger alpha-helix region, containing the amino acid positions that are crucial for DNA binding, was specifically chosen on the basis of the contacts more frequently represented in the available list of the 'code'. Here we demonstrate that Jazz protein binds specifically to the double-stranded DNA target, with a dissociation constant of about 32 nM. Band shift and super-shift experiments confirmed the high affinity and specificity of Jazz protein for its DNA target. Moreover, we show that chimeric proteins, named Gal4-Jazz and Sp1-Jazz, are able to drive the transcription of a test gene from the human utrophin promoter.

Investigation of the role of intracellular Ca(2+) stores in generation of the muscarinic agonist-induced slow afterdepolarization (sADP) in guinea-pig olfactory cortical neurones in vitro.

1. Intracellular recordings were made from guinea-pig olfactory cortical brain slice neurones to assess the possible role of intracellular Ca(2+) stores in the generation of the slow post-stimulus afterdepolarization (sADP) and its underlying tail current (I(ADP)), induced by muscarinic receptor activation. 2. Caffeine or theophylline (0.5 - 3 mM) reduced the amplitude of the I(ADP) (measured under 'hybrid' voltage clamp) induced in the presence of the muscarinic agonist oxotremorine-M (OXO-M, 10 microM) by up to 96%, without affecting membrane properties or muscarinic depolarization of these neurones. 3. The L-type Ca(2+) channel blocker nifedipine (1, 10 microM) also inhibited I(ADP) (by up to 46%), while ryanodine (10 microM) (a blocker of Ca(2+) release from internal stores) produced a small ( approximately 10%) reduction in I(ADP) amplitude; however, neither 10 microM dantrolene (another internal Ca(2+) release blocker) nor the intracellular Ca(2+) store re-uptake inhibitors thapsigargin (3 microM) or cyclopiazonic acid (CPA, 15 microM) affected I(ADP) amplitude. 4. IBMX (100 microM), a phosphodiesterase inhibitor, also had no effect on I(ADP). Furthermore, inhibition of I(ADP) by caffeine was not reversed by co-application of 100 microM adenosine. 5. Caffeine (3 mM) or nifedipine (10 microM) reduced the duration of presumed Ca(2+) spikes revealed by intracellular Cs(+) loading. When applied in combination, nifedipine and caffeine effects were occlusive, rather than additive, suggesting a common site of action on L-type calcium channels. 6. We conclude that Ca(2+)-induced Ca(2+) release (CICR) from internal stores does not contribute significantly to muscarinic I(ADP) generation in olfactory cortical neurones. However caffeine and theophylline, which enhance CICR in other systems, blocked I(ADP) induction. We suggest that this action might involve a combination of L-type voltage-gated Ca(2+) channel blockade, and a direct inhibitory action on the putative I(ADP) K(+) conductance.

Blockade of GABA(B) receptors facilitates muscarinic agonist-induced epileptiform activity in immature rat piriform cortex in vitro.

The effects of the selective GABA(B) receptor antagonist [3-[[(3,4-dichlorophenyl)methyl]aminolpropyl] (diethoxymethyl) phosphinic acid (CGP 52432) on muscarinic (mAChR) and metabotropic glutamate (mGluR) responsiveness were studied in slices of piriform cortex from both immature (P16-P22) and adult (> or =P40) rats, using a conventional intracellular recording technique. In both adult and immature slices, CGP 52432 (1 microM) had no effect on neuronal membrane properties, whereas it selectively abolished the late inhibitory postsynaptic potential (IPSP) evoked by local electrical stimulation of association fibre terminals. Age-related changes in mAChR (but not mGluR) responsiveness were also detected. In adult neurones, bath-application of the mAChR agonist oxotremorine-M (OXO-M; 10 microM), or the selective mGluR agonist 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 10 microM) evoked similar membrane depolarization and inhibition of evoked excitatory postsynaptic potentials (EPSPs). However, while 1S,3R-ACPD and OXO-M produced indistinguishable slow excitatory effects in immature slices, during superfusion with OXO-M, neurones exhibited spontaneous paroxysmal depolarizing shifts (PDSs) that were suppressed in the presence of atropine (1 microM) or the selective GABA(B) receptor agonist beta-parachlorophenyl-gamma-aminobutyric acid [(-)baclofen; 10 microM]. Also, application of OXO-M resulted in a pronounced prolongation (rather than a decrease) of electrically evoked postsynaptic potentials (PSPs) which now exhibited recurrent superimposed spike discharges. In adult slices, in the continuous presence of CGP 52432 (1 microM; 20 min pre-incubation), a subsequent exposure to 10 microM OXO-M or 1S,3R-ACPD failed to induce any spontaneous epileptiform activity, and evoked PSPs were consistently suppressed. In contrast, in immature slices, after incubation in CGP 52432 (1 microM; 20 min), a subsequent application of a low dose of OXO-M (2.5 microM), which was inactive per se, was able to produce spontaneous PDSs and a prolongation of evoked PSPs. We conclude that a reduction in GABA(B)-mediated synaptic inhibition in immature slices (in co-operation with other factors) may contribute to the facilitation of excitatory neurotransmission and therefore play a role in the generation of mAChR-induced epileptiform activity.

3,5-Dihydro-4H-2,3-benzodiazepine-4-thiones: a new class of AMPA receptor antagonists.

Synthesis and evaluation of anticonvulsant activity of a series of 2,3-benzodiazepin-4-ones (2) chemically related to 1-(4'-aminophenyl)-4-methyl-7,8-(methylenedioxy)-5H-2,3-benzodiazepine (1, GYKI 52466) have been reported in our recent publications. Compounds 2 manifested marked anticonvulsant properties acting as 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptor antagonists. In an attempt to better define the structure-activity relationships (SAR) and to obtain more potent and selective anticonvulsant agents, 1-aryl-3,5-dihydro-4H-2, 3-benzodiazepine-4-thiones 3 were synthesized from the corresponding isosteres 2. The evaluation is reported of their anticonvulsant effects, both in the audiogenic seizures test with DBA/2 mice and against the maximal electroshock- and pentylenetetrazole-induced seizures in Swiss mice. New derivatives 3 showed higher potency, less toxicity and longer-lasting anticonvulsant action than those of the parent compounds 2 in all tests employed. Analogous to derivatives 2, new compounds 3 do not affect the benzodiazepine receptor (BZR) while they do antagonize AMPA-induced seizures; their anticonvulsant activity is reversed by pretreatment with aniracetam but not with flumazenil, thus suggesting a clear involvement of AMPA receptors. Electrophysiological data indicate a noncompetitive blocking mechanism at the AMPA receptor sites for 3i, the most active of the series and over 5-fold more potent than 1.

Muscarinic agonist-induced burst firing in immature rat olfactory cortex neurons In vitro.

Age-related changes in pre-/postsynaptic muscarinic (mAChR) and metabotropic-glutamate (mGluR) responsiveness were studied in slices of olfactory cortex from both immature [postnatal day 16-22 (P16-P22)] and adult (>/=P40) rats, using a conventional intracellular recording technique. In adult neurons, bath application of the mAChR agonist oxotremorine-M (OXO-M; 10 microM), or the selective mGluR agonist 1-aminocyclopentane-1S-3R-dicarboxylic acid (1S,3R-ACPD; 10 microM) evoked sustained membrane depolarizations, increases in input resistance, intense repetitive firing, and the appearance of a slow poststimulus afterdepolarizing potential (sADP). Excitatory postsynaptic potentials (EPSPs) evoked by local electrical stimulation of association fiber terminals were also depressed. In contrast, in neurons from immature slices, the 10 microM OXO-M-induced membrane depolarization was followed by the appearance of spontaneous rhythmic epileptiform activity, which was voltage independent and reversible on drug wash out. Epileptiform bursts were abolished or reduced by coapplication of tetrodotoxin (1 microM), atropine (1 microM), pirenzepine (100-200 nM), the N-methyl-D-aspartate (NMDA) receptor antagonist -amino-5-phosphonovaleric acid (-APV; 100 microM), the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5-20 microM), the anesthetic-sedative barbiturate pentobarbitone (100 microM), or by raising the extracellular Mg2+ concentration, whereas a clear facilitatory effect was exhibited by the selective gamma-aminobutyric acid-A (GABAA) receptor blocker (-)-bicuculline methiodide (10 microM). The epileptogenic effects induced by OXO-M were indistinguishable from those produced by 4-aminopyridine (4-AP; 100-200 microM), although these latter actions were unaffected by atropine. In slices from immature animals, electrical stimulation of layer III association fibers in the presence of 10 microM OXO-M was accompanied by a dramatic prolongation of evoked depolarizing postsynaptic potentials (PSPs), with the appearance of recurrent superimposed spike discharges. This effect was readily reversed on wash out of OXO-M. No comparable age-dependent differences were observed in the nature or time course of 1S,3R-ACPD-evoked pre- (or post)synaptic responses, even in immature cells where muscarinic epileptiform activity had previously been demonstrated. We suggest that the overall susceptibility toward muscarinic-induced epileptiform discharge in immature olfactory cortical neurons may depend on the functional integrity of presynaptic inhibitory mAChRs; additional contributing mechanisms were also considered.

Binding properties of the artificial zinc fingers coding gene Sint1.

On the basis of the recognition "code" that suggests specific rules between zinc finger's primary structure and the finger's potential DNA binding sites, we have constructed a new three-zinc finger coding gene to target the nine base pair DNA sequence: 5'-TGG-ATG-GAC-3'. This artificial gene named "Sint1" belongs to the Cys2-His2 zinc finger type. The amino acid positions, crucial for DNA binding, have been specifically chosen on the basis of the amino acid/base contacts more frequently represented in the available list of the proposed recognition "code". Here we demonstrate that Sint1 protein binds specifically the double strand "code" DNA target, with a dissociation constant (Kd) comparable to the Kd of the well known Zif268 protein. Sint1 "code" deduced and the "experimental" selected DNA binding sites share five nucleotide positions. Interestingly, Sint1 shows both high affinity and specificity toward the single strand "code" DNA binding site, with a Kd comparable to the corresponding double strand DNA target. Moreover, we prove that Sint1 is able to bind RNA similarly to several natural zinc finger proteins.

Inhibition of nitric oxide synthase prevents magnesium-free-induced epileptiform activity in guinea-pig piriform cortex neurones in vitro.

The effects of N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, were examined on Mg2+-free-induced epileptiform activity, in guinea-pig piriform cortex slices in vitro. L-NAME (0.1-1 mM) had no effect on neuronal membrane properties or electrically-evoked postsynaptic potentials (PSPs). In contrast, during superfusion of the slices with Mg2+-free solution neurones exhibited spontaneous and stimulus-evoked epileptiform potentials that were suppressed in the presence of L-NAME (100 microM) or the selective NMDA receptor antagonist DL-APV (100 microM). The inhibitory effects induced by L-NAME were reversibly reduced by L-arginine (1 mM), but not D-arginine (1 mM), the latter drug not being a substrate for NO formation. It was concluded that L-NAME can suppress epileptiform activity induced by Mg2+-free exposure primarily through a decrease in presynaptic transmitter release, although additional actions on the NMDA-receptor complex were also considered.

1-Aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-ones: novel AMPA receptor antagonists.

Our previous publication (Eur. J. Pharmacol. 1995, 294, 411-422) reported preliminary chemical and biological studies of some 2,3-benzodiazepines, analogues of 1-(4-aminophenyl)-4-methyl-7,8-(methylenedioxy)-5H-2,3-benzodiazepine (1, GYKI 52466), which have been shown to possess significant anticonvulsant activity. This paper describes the synthesis of new 1-aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-ones and the evaluation of their anticonvulsant effects. The observed findings extend the structure-activity relationships previously suggested for this class of anticonvulsants. The seizures were evoked both by means of auditory stimulation in DBA/2 mice and by pentylenetetrazole or maximal electroshock in Swiss mice. 1-(4'-Aminophenyl)- (38) and 1-(3'-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin- 4-one (39), the most active compounds of the series, proved to be more potent than 1 in all tests employed. In particular, the ED50 values against tonus evoked by auditory stimulation were 12.6 micromol/kg for derivative 38, 18.3 micromol/kg for 39, and 25.3 micromol/kg for 1. Higher doses were necessary to block tonic extension induced both by maximal electroshock and by pentylenetetrazole. In addition these compounds exhibited anticonvulsant properties that were longer lasting than those of compound 1 and were less toxic. The novel 2,3-benzodiazepines were also investigated for a possible correlation between their anticonvulsant activities against convulsions induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and their affinities for benzodiazepine receptors (BZR). The 2,3-benzodiazepines did not affect the binding of [3H]flumazenil to BZR, and conversely, their anticonvulsant effects were not reversed by flumazenil. On the other hand the 2,3-benzodiazepines antagonized seizures induced by AMPA and aniracetam in agreement with an involvement of the AMPA receptor. In addition, both the derivative 38 and the compound 1 markedly reduced the AMPA receptor-mediated membrane currents in guinea-pig olfactory cortical neurons in vitro in a noncompetitive manner. The derivatives 25 and 38-40 failed to displace specific ligands from N-methyl-D-aspartate (NMDA), AMPA/kainate, or metabotropic glutamate receptors.

Metabotropic glutamate receptor subtypes mediating slow inward tail current (IADP) induction and inhibition of synaptic transmission in olfactory cortical neurones.

1. The pharmacological features of the pre- and postsynaptic metabotropic glutamate receptors (mGluRs) present in the guinea-pig olfactory cortex, were examined in brain slices in vitro by use of a conventional intracellular current clamp/voltage clamp recording technique. 2. Bath-application of trans-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) (50 microM) produced a sustained membrane depolarization, increase in cell excitability and induction of a post-stimulus inward (after depolarizing) tail current (IADP) (measured under 'hybrid' voltage clamp) similar to those evoked by the muscarinic receptor agonist oxotremorine-M (OXO-M, 2 microM). 3. L-Glutamate (0.25 1 mM. in the presence of 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 microM-DL-amino-5-phosphono valeric acid (DL-APV)) or the broad spectrum mGluR agonists 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 10 microM), 1S,3S-ACPD (50 microM), ibotenate (Ibo; 25 microM. in the presence of 100 microM DL-APV), the selective mGluR I agonists (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG, 10 microM), (S)-3-hydroxyphenylglycine ((S)-3HPG, 50 microM), or quisqualate (10 microM, in the presence of 20 microM CNQX), but not the mGluR II agonist 2S,1'S,2'S-2-(2'-carboxycyclopropyl)-glycine (L-CCG1,1 microM) or mGluR III agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 1 mM), were all effective in producing membrane depolarization and inducing a post-stimulus IADP. Unexpectedly, the proposed mGluR II-selective agonist (2S,1'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)-glycine (DCG-IV, 10 microM, in the presence of 100 microM DL-APV) was also active. 4. The excitatory effects induced by 10 microM 1S,3R-ACPD were reversibly antagonized by the mGluR I/II antagonist (1)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG, 0.5 1 mM), as well as the selective mGluR I antagonists (S)-4-carboxyphenylglycine ((S)-4CPG) and (S)-4-carboxy-3-hydroxyphenyl glycine ((S)-4C3HPG) (both at 1 mM), but not the nonselective mGluR antagonist L(+)-2-amino-3-phosphonopropionic acid (L-AP3, 1 mM) or the selective mGluR III antagonist (S)-alpha-methyl-L-AP4 (MAP4, 1 mM). 5. The excitatory postsynaptic potentials (e.p.s.ps), induced by single focal stimulation of cortical excitatory fibre tracts, were markedly reduced by 1S,3R-ACPD or L-AP4 (both at 10 microM), and by the selective mGluR II agonists (mGluR 1 antagonists) (S)-4CPG or (S)-4C3HPG (both at 1 mM) but not (S)-3,5-DHPG or (S)-3HPG (both at 100 microM). 6. The inhibitory effects of 1S-3R-ACPD, but not L-AP4, were reversibly blocked by (+)-MCPG (1 mM), whereas those produced by L-AP4, but not 1S,3R-ACPD, were blocked by the selective mGluR III antagonist MAP4 (1 mM). 7. It is concluded that a group I mGluR is most likely involved in mediating excitatory postsynaptic effects, whereas two distinct mGluRs (e.g. group II and III) might serve as presynaptic inhibitory autoreceptors in the guinea-pig olfactory cortex.

Effects of felbamate on muscarinic and metabotropic-glutamate agonist-mediated responses and magnesium-free or 4-aminopyridine-induced epileptiform activity in guinea pig olfactory cortex neurons in vitro.

The effects of the anticonvulsant agent felbamate (FBM) were examined on muscarinic and metabotropic-glutamate receptor agonist-induced responses and chemically induced epilepti-form activity, in guinea pig olfactory cortex slices in vitro. FBM (100-500 microM) had little effect on neuronal membrane properties and on postsynaptic potentials evoked by electrical stimulation of lateral olfactory tract terminals, whereas it reduced the duration of presumed Ca++ spikes induced by intracellular Cs+ loading. In contrast, the muscarinic receptor agonist oxotremorine-M (10 microM) or the metabotropic glutamate receptor agonist 1-aminocyclopentane-1S-3R-dicarboxylic acid (10 microM) induced a sustained membrane depolarization with repetitive firing, an increase in input resistance and the appearance of a slow poststimulus afterdepolarizing potential. These effects were reversibly reduced in the presence of FBM (100-500 microM). After preincubation of slices with Mg+(+)-free solution or 200 microM 4-aminopyridine, neurons exhibited spontaneous and stimulus-evoked epileptiform potentials that were suppressed by FBM (1 mM). We conclude that FBM can interfere with muscarinic and metabotropic-glutamate response generation and slow after-depolarization induction in olfactory cortical neurons, most likely by blocking Ca++ influx through voltage-sensitive Ca++ channels. A possible interaction of FBM with other voltage-insensitive Ca++ conductances is also considered. We also suggest that FBM can suppress epileptiform activity induced by Mg+(+)-free or 4-aminopyridine exposure primarily through inhibition of N-methyl-D-aspartate-gated ion channels, although additional actions on non-N-methyl-D-aspartate receptor sites and/or presynaptic transmitter release mechanisms cannot be excluded.

Chronic (-)baclofen or CGP 36742 alters GABAB receptor sensitivity in rat brain and spinal cord.

Administration of the GABAB receptor agonist, (-)-baclofen 10 mg kg-1, i.p. daily for 21 days to rats prevented (-)-baclofen-induced hyperpolarizing responses and synaptically-evoked late inhibitory potentials (IPSPs) in olfactory cortical neurones recorded intracellularly from 450 microns brain slices. In contrast, pre-treatment with CGP 36742 induced a significant increase in (-)-baclofen-mediated post-synaptic responses and late IPSP amplitude. In the spinal cord, the potency of (-)-baclofen in inhibiting electrically-evoked substance P-like immunoreactivity or amino acid release was significantly reduced or increased in slices from rats pre-treated with the GABAB agonist or antagonist, respectively. These data suggest that functional responses to GABAB receptor activation in the mammalian central nervous system can be up- or down-regulated.

Selective blockade of the hyperpolarization-activated cationic current (Ih) in guinea pig substantia nigra pars compacta neurones by a novel bradycardic agent, Zeneca ZM 227189.

The effects of a novel bradycardic agent Zeneca ZM 227189 (4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) triazinium iodide) were tested on the inward rectifying properties of guinea-pig substantia nigra pas compacta (SNC) and guinea-pig olfactory cortical cells recorded in vitro. In SNC neurones, ZM 227189 (10-100 microM) produced a dose-dependent block of the slow anomalous rectifier; under voltage clamp, a clear reduction was seen in the amplitude of the slow inward current (Ih) relaxation evoked by negative voltage commands from a holding potential of -60 mV. ZM 227189 (50-100 microM) induced an irreversible block of the Ih current after 10-15 min exposure. A similar block of Ih was observed following application of 5 mM Cs+. ZM 227189 had little effect on other membrane properties. By contrast, in olfactory cortical neurones, ZM 227189 (100 microM) induced an increase in the input resistance (approximately 20%) and cell excitability, accompanied by a small (< 2 mV) hyperpolarization; these effects were also not reversible. Activation of the fast (K(+)-mediated) inward rectifier at negative membrane potentials remained unaffected. Lower concentrations (1-10 microM) of ZM 227189 had no obvious effect on cortical cell properties. Our data indicate that ZM 227189 is a potent and apparently selective blocker of Ih in substantia nigra neurones, but has no effect on the fast-type inward rectifier in olfactory cortical cells.

A comparison of the muscarinic response and morphological properties of identified cells in the guinea-pig olfactory cortex in vitro.

The electrophysiological and morphological characteristics of neurons in the guinea-pig olfactory cortex brain slice were investigated using a combined intracellular recording and neurobiotin-dye filling technique, in an attempt to show whether a clear relation existed between cell morphology and excitatory muscarinic response profile. Out of 46 sampled neurons, 25 (termed type 1), responded to bath-application of the muscarinic agonist oxotremorine-M (10 microM, 2-3 min) with a strong and persistent excitation coupled with the appearance of a slow depolarizing afterpotential (10-20 mV amplitude) following a large depolarizing stimulus. These neurons were identified as deep pyramidal cells located in cortical layer III, with characteristic pyramidal/ovoid shaped cell bodies, prominent apical dendrites with branches extending to the surface, and extensive basal dendritic trees. The cells showed a regular spiking pattern in response to injected depolarizing current, with no evidence of bursting behaviour. Nine cells (termed type 2), were strongly excited by oxotremorine-M, but only generated a weak depolarizing afterpotential (< 5 mV) following stimulation. These neurons (located in layer III or at layer II-III border) had a variable, non-pyramidal morphology with either a fusiform/tripolar, stellate/multipolar or bipolar/bi-tufted appearance, respectively. Apart from a more prominent post-spike afterhyperpolarization observed in some type 2 cells, their resting membrane properties and firing patterns were indistinguishable from those of type 1 responding cells. Twelve cells (termed type 3) showed little or no excitatory response to oxotremorine-M, and never generated a post-stimulus slow afterdepolarization. These cells (within compact layer II) had the morphological features of superficial pyramidal cells, typified by their short apical trunks and well-developed apical dendritic trees. They could be distinguished electrophysiologically by their ability to show spike fractionation during injection of large depolarizing current pulses. The morphology and laminar position of neurobiotin-filled cells was also compared with those of cells stained by the Golgi-Cox method. Some factors that may have contributed to the observed differences in muscarinic response profile are discussed. It is proposed that the selective muscarinic induction of the slow depolarizing afterpotential phenomenon in deep pyramidal cells may be important in olfactory cortical learning and memory processes.

Persistent muscarinic excitation in guinea-pig olfactory cortex neurons: involvement of a slow post-stimulus afterdepolarizing current.

The persistent excitatory effects of the muscarinic agonist oxotremorine-M were investigated in guinea-pig olfactory cortex neurons in vitro (28-30 degrees C) using a single-microelectrode current-clamp/voltage-clamp technique. In 40% of recorded cells (type 1), bath-application of oxotremorine-M (2-10 microM; 1-2 min) induced a strong membrane depolarization, an increase in input resistance and a sustained neuronal discharge lasting over 30 min following agonist washout. A large depolarizing stimulus applied during the action of oxotremorine-M, evoked a slow post-stimulus afterdepolarization (approximately 10-15 mV) lasting approximately 30 s. Injection of steady negative current at the peak of this response produced a slow repolarization of the membrane potential (half-time approximately 0.6 min) towards a plateau level ("hyperpolarization recovery"); these effects of oxotremorine-M were slowly reversed on washout or by application of atropine (1 microM). In a second population of neurons (type 2; 39% of total), oxotremorine-M produced a large depolarization, a resistance increase and repetitive firing that did not persist after agonist washout; these neurons failed to generate a prominent slow afterdepolarization on stimulation, and showed no hyperpolarization recovery effect. Their resting membrane properties were not significantly different from those of type 1 cells. The remaining proportion of cells (type 3) elicited little or no muscarinic response to oxotremorine-M and no slow afterdepolarization; these cells showed characteristics spike fractionation (pre-potentials) during an evoked train of action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)