Pharmacological characterization of the receptor mediating the adrenergic inhibition of responses to substance P in the cingulate cortex

The excitatory responses of neurones in the anterior cingulate cortex of the rat to iontophoretically applied substance P (SP) are reduced by noradrenaline (NA) applied iontophoretically or released from noradrenergic pathways. In order to determine the receptor involved in this inhibitory effect we have studied the effects of a number of receptor-specific adrenergic agonists and antagonists on responses of cingulate neurones to SP in rats anaesthetized with chloral hydrate. Low iontophoretic currents (0-15 nA) of NA, adrenaline and the beta-agonist, clenbuterol, all strongly reduced responses to SP. Isoprenaline was also effective but less consistently so, although problems were experienced with its iontophoretic release from micropipettes. The alpha 1-agonists, phenylephrine and methoxamine were also able to reduce responses to SP. However, this reduction required higher iontophoretic currents (15-60 nA) and was associated with depressant effects on baseline firing rate. The alpha 2-agonist clonidine was only weakly active at high currents and this too was associated with depression of baseline firing. Similar weak effects were noted with dopamine. The inhibitory effects of NA on SP responses were convincingly blocked or reversed by the beta-antagonist, practolol, but not by the alpha 1-antagonist, prazosin. The reduction of SP responses by phenylephrine was also blocked by practolol but unaffected by prazosin. Finally, reduction of SP excitations by activation of the coeruleocortical pathway was also blocked by practolol applied iontophoretically to the cortical cells. These results are consistent with the hypothesis that the effect of NA on SP responsiveness in the cingulate cortex is mediated by beta-adrenoreceptors.     

Electrophysiological characterization of a novel potent and orally active NMDA receptor antagonist: CGP 37849 and its ethylester CGP 39551

The selectivity and potency of the novel competitive N-methyl-D-aspartate (NMDA) receptor antagonists, CGP 37849 and CGP 39551, were investigated in vitro and in vivo using electrophysiological approaches. Like the reference blocker DL-AP5, both compounds acted in vitro (hippocampus, substantia nigra, spinal cord) to antagonize the excitatory actions of exogenously administered NMDA as well as the synaptically elicited, physiological NMDA receptor responses in hippocampus and spinal cord. In all isolated preparations CGP 37849 was more potent than CGP 39551, and 5- to 10-fold more potent than DL-AP5. Neither compound showed any marked effect on responses evoked by quisqualate and kainate. NMDA excited dopaminergic cells in the pars compacta region of the substantia nigra in a concentration-dependent manner. This effect also could be selectively antagonized by CGP 37849 and CGP 39551. In the anaesthetized rat, excitatory responses of hippocampal pyramidal cells evoked by iontophoretic application of NMDA were antagonized by CGP 37849 and CGP 39551 following their oral administration without reducing quisqualate or kainate responses. In contrast to the in vitro situation, CGP 39551 was more potent than CGP 37849 in vivo. Effective doses were 30 mg/kg p.o. for CGP 39551 and 100 mg/kg p.o. for CGP 37849. In conclusion, it is demonstrated that CGP 37849 and CGP 39551 selectively antagonize NMDA evoked neuronal responses in vivo and in vitro and that the drugs are centrally active following their oral administration.     

CGP 37849 and CGP 39551: novel and potent competitive N-methyl-D-aspartate receptor antagonists with oral activity

1. The pharmacological properties of CGP 37849 (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid; 4-methyl-APPA) and its carboxyethylester, CGP 39551, novel unsaturated analogues of the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoate (AP5), were evaluated in rodent brain in vitro and in vivo. 2. Radioligand binding experiments demonstrated that CGP 37849 potently (Ki 220 nM) and competitively inhibited NMDA-sensitive L-[3H]-glutamate binding to postsynaptic density (PSD) fractions from rat brain. It inhibited the binding of the selective NMDA receptor antagonist, [3H]-((+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), with a Ki of 35 nM, and was 4, 5 and 7 fold more potent than the antagonists [+/-)-cis-4-phosphonomethylpiperidine-2-carboxylic acid) (CGS 19755), CPP and D-AP5, respectively. Inhibitory activity was associated exclusively with the trans configuration of the APPA molecule and with the D-stereoisomer. CGP 39551 showed weaker activity at NMDA receptor recognition sites and both compounds were weak or inactive at 18 other receptor binding sites. 3. CGP 37849 and CGP 39551 were inactive as inhibitors of L-[3H]-glutamate uptake into rat brain synaptosomes and had no effect on the release of endogenous glutamate from rat hippocampal slices evoked by electrical field stimulation. 4. In the hippocampal slice in vitro, CGP 37849 selectively and reversibly antagonized NMDA-evoked increases in CA1 pyramidal cell firing rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vagal afferents innervating the gastrointestinal tract and CCKA-receptor immunoreactivity.

A large body of evidence derived from electrophysiological recording and pharmacological/behavioral experiments suggests the presence of CCKA-receptors on vagal primary afferent fibers innervating the gastrointestinal tract. With the availability of antibodies specific for the CCKA-receptor, we wanted to demonstrate its presence and distribution on identified vagal afferent fibers and different types of terminals in the mucosa, myenteric plexus, and external muscle layers of the stomach and duodenum. In the duodenal mucosa, neither a C-terminal (Ab-1) nor an N-terminal (Ab-2) specific antibody produced any specific staining; in the myenteric plexus, non-vagal enteric neurons and their processes, but not vagal intraganglionic laminar endings (IGLEs), exhibited CCKAR-immunoreactivity. Similarly, in the gastric myenteric plexus, a population of enteric neurons and their processes, but not identified vagal IGLEs, were labeled by both antibodies. In both external muscle layers of the stomach, CCKAR-immunoreactive axons were in close register with labeled vagal afferent intramuscular arrays, but the two labels were not contained in the same varicosities. Ab-1 immunoreactivity was found in the cell membrane of vagal afferent perikarya in the nodose ganglia and in pancreatic acinar cells. The failure to detect CCKAR-immunoreactivity in peripheral vagal afferent terminals cannot be due to methodological problems because it was present in enteric neurons in the same sections, and because it did not stain structures resembling IGLEs in material without the potentially masking vagal afferent label. We conclude that CCKA-receptors on vagal afferent terminals: 1) are below the immunohistochemical detection threshold, 2) exhibit a conformation or affinity state inaccessible to the two antibodies, or 3) are not transported to the peripheral terminals.     

GABAB receptors in various in vitro and in vivo models of epilepsy: a study with the GABAB receptor blocker CGP 35348.

The effect of the GABAB receptor blocker CGP 35348 on epileptic processes in vitro and in vivo was studied. In hippocampal slices of the rat maintained in vitro, CGP 35348 (100 microM) induced a moderate increase in the frequency of extracellularly recorded spontaneous epileptiform burst discharges induced in CA3 by penicillin (1.2 mM), bicuculline (5 microM) and low Mg(2+) (0.1 mM). This effect was observed in 50-75% of the slices. A similar but less consistent increase was also observed in CA1 in bicuculline and low Mg2+. Data obtained by intracellular recordings from CA1 pyramidal cells in the presence of bicuculline (10 microM) demonstrated that CGP 35348 (100 microM) increased the duration of the paroxysmal depolarization underlying an evoked epileptiform burst and reduced the early component of the after hyperpolarization which followed the burst. In mice pretreated with isoniazid, CGP 35348 (300 mg/kg, i.p.) significantly increased the number of convulsing mice. However, convulsions induced by submaximal doses of pentylenetetrazol, picrotoxin or strychnine were not facilitated by CGP 35348. We conclude that GABAB receptors appear to exert a suppressant effect on various kinds of epileptiform discharges of hippocampal neurons in vitro. In vivo, however, the role of GABAB receptors in regulating convulsions is less prominent since only isoniazid-induced convulsions were facilitated by GABAB receptor blockade.     

Stereoselectivity of L-baclofen in hippocampal slices of the rat

Extra- and intracellular recording from hippocampal slices of the rat revealed the following effects when baclofen (BF) (0.1-10 microM) was added to the perfusion fluid: a block of synaptic potentials evoked by stimulation of stratum radiatum; a direct hyperpolarization and a conductance increase (for potassium ions) of CA1 pyramidal cells. All this activity was found in the L- none in the D-enantiomer. D-BF did not antagonize the action of L-BF.     

Glutamate-induced activation of rat locus coeruleus increases CA1 pyramidal cell excitability

In anesthetized rats, injections of a 0.5 mM glutamate solution into the locus coeruleus (LC) reversibly increased the amplitude of the population spike evoked in CA1 by stimulation of the Schaffer-commissural fiber tract. This effect was absent in N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)-treated, noradrenaline (NA)-depleted animals. The excitatory postsynaptic potential recorded in the stratum radiatum was unaffected following the glutamate injections. Systemic administration of the NA-uptake inhibitor desipramine also produced an increase in population spike amplitude. The findings demonstrate that activation of LC neurons increases pyramidal cell excitability in vivo.     

An Organotypic Spinal Cord - Dorsal Root Ganglion - Skeletal Muscle Coculture of Embryonic Rat. II. Functional Evidence for the Formation of Spinal Reflex Arcs In Vitro

Electrical properties of motoneurons, muscle fibres and dorsal root ganglion (DRG) cells were studied in an organotypic coculture of embryonic rat spinal cord, dorsal root ganglia and skeletal muscle. The motoneurons were identified by their morphology and position in culture. Their size and input conductance were significantly larger than those of spinal interneurons. Intracellular current injection evoked action potentials in all motoneurons, but only evoked stable repetitive firing patterns in some. Excitability was correlated to somatic size and the rate of spontaneous excitatory input. It is suggested that the somatic growth and the increase in excitability is regulated by the excitatory afferents. The motoneurons showed spontaneous excitatory and inhibitory postsynaptic potentials and action potentials which disappeared with the application of various agents known to inhibit excitability or excitatory synaptic transmission. Excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs respectively) were distinguished by their shape, reversal potential and pharmacology. IPSPs could be depolarizing or hyperpolarizing in different cells. A higher percentage of cells with hyperpolarizing IPSPs was found in older cultures and in the presence of skeletal muscle, suggesting a reversal of the polarity of IPSPs with development. The spontaneous muscle contractions observed in the cultures could be due either to innervation, spontaneous oscillations of the membrane potential, or electrical coupling between neighbouring fibres. A small percentage of DRG cells showed spontaneous action potentials, all of which were found in cultures with spontaneous muscle contractions. The electrical stimulation of DRG afferents evoked mono- and polysynaptic EPSPs in motoneurons, endplate potentials and muscle contractions. The stimulation of the ventral horns evoked endplate potentials and muscle contractions via mono- or polysynaptic pathways. Together these results indicate that appropriate and functional contacts were established in the culture between myotubes and DRG cells, between DRG cells and motoneurons, and between motoneurons and muscle fibres.