Inhibition of [3H]catecholamine release and Ca2+ currents by prostaglandin E2 in rabbit carotid body chemoreceptor cells.

Basal release of [3H]catecholamine ([3H]CA) from rabbit carotid bodies (CBs), previously incubated in the presence of [3H]tyrosine, was not significantly modified by prostaglandin E2 (PGE2). On the contrary, PGE2 (3-300 nM) produced a dose-dependent inhibition of the low PO2-evoked release of [3H]CA. The inhibition was greatest (55%) at a low intensity of hypoxic stimulation (incubating solution PO2 approximately 66 mmHg) and decreased with increasing intensities of hypoxia. Chronic denervation of the CB did not modify the response to PGE2. The release of [3H]CA induced by incubating the CBs in a hypercapnic-acidic solution (PCO2 approximately 132 mmHg; pH = 6.60) and by dinitrophenol (100 microM) was not significantly modified by 300 nM PGE2. PGE2 (300 nM) inhibited the release of [3H]CA elicited by incubating the CBs in a high K+ (35 mM)-containing solution. The release response elicited by high K+ (25 mM) was strongly augmented by a dihydropyridine agonist of Ca2+ channels, Bay K 8644, at a concentration of 1 microM. The Bay K 8644 effect was partly inhibited by PGE2 (300 nM). Using whole-cell recordings in freshly dispersed or short-term cultured chemoreceptor cells from adult rabbits it was found that Ca2+ currents (ICa) were reversibly inhibited by bath application of PGE2. A good parallelism exits between the dose-response curves for PGE2 inhibition of ICa in isolated chemoreceptor cells and high extracellular [K+]- or hypoxia-evoked release of [3H]CA from the whole CB.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro activation of cyclo-oxygenase in the rabbit carotid body: effect of its blockade on [3H]catecholamine release.

The release of prostaglandin E2 (PGE2) from rabbit carotid bodies (CBs) incubated in basal conditions (PO2 approximately 132 mmHg; PCO2 approximately 33 mmHg; pH = 7.42) amounts to 94.4 +/- 10.1 pg (mg protein)-1 (10 min)-1 (mean +/- S.E.M.). Incubation of the CB in a hypoxic solution (PO2 approximately 46 mmHg) produced a significant 40% increase (P < 0.05) in the release of PGE2. Indomethacin (2 microM) prevented the hypoxia-induced release of PGE2. Sensory plus sympathetic denervation of the CB 4 days prior to the experiments did not modify either basal or low PO2-induced PGE2 release, indicating that intraglomic nerve endings are not significant sources for the PGE2 released. Incubation of the CB in an acidic-hypercapnic solution (PO2 approximately 132 mmHg; PCO2 approximately 132 mmHg; pH = 6.60) or in a high K(+)-containing solution (35 mM) was also effective in promoting an increase in the outflow of PGE2 from the organs. The release of [3H]catecholamines ([3H]CA) from the CB elicited by incubating the organs in low PO2 solutions (PO2 ranged between 66 and 13 mmHg) was potentiated by two inhibitors of cyclo-oxygenase, acetylsalicylic acid (ASA, 100 microM) and indomethacin (2 microM). The effect persisted after chronic denervation of the organ. The secretory response elicited by acidic stimuli was also augmented by cyclo-oxygenase inhibitors. Thus, [3H]CA release elicited by incubating the CBs in the acidic-hypercapnic solution increased by 300% in the presence of indomethacin (2 microM), and ASA (100 microM) more than doubled the release induced by dinitrophenol (100 microM), a protonophore that mimics an acidic stimulus. Indomethacin, but not ASA, moderately increased the high K(+)-evoked [3H]CA release. The effect of indomethacin on the release of [3H]CA elicited by acidic and hypoxic stimuli was reversed by PGE2 in a dose-dependent manner (0.3-300 nM). These results show that low PO2 and high PCO2-low pH, the natural stimuli to the CB, as well as high extracellular [K+], activate the cyclo-oxygenase pathway in the CB, promoting an increase in the outflow of PGE2. The data also show that the blockade of this pathway activates the stimulus-induced [3H]CA release from the CB, indicating that naturally released prostanoids exert an inhibitory control on chemoreceptor cells. The data lend support to the notion that the hyper-reactivity of the ventilatory response to hypoxia in subjects under anti-inflammatory drug treatment results from CB cycloxygenase inhibition.     

Comparison of the monoamine and catabolite content in the cat and rabbit carotid bodies

The monoamine and catabolite contents of a large number of rabbit (n = 95) and cat (n = 32) carotid bodies (CBs) have been measured by high performance liquid chromatography with electrochemical detection (HPLC-ED). The dopamine (DA) content as well as that of its precursors tyrosine (TYR), dihydroxyphenylalanine (DOPA) and catabolites dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA) were approximately equal in both species. The noradrenaline (NA) content was 10 times larger in the cat than in the rabbit CBs. Twenty-nine out of the 32 cat CBs contained more NA than DA while the reverse was true in 92 out of 95 rabbit CBs. In 11 cats the right CB was sympathectomized and its DA and NA contents were compared to those of intact contralateral organs.     

The effect of a nitric oxide donor, sodium nitroprusside, on the release of acetylcholine from the in vitro cat carotid body

The purpose of the present study was to determine the impact of a nitric oxide (NO) donor, sodium nitroprusside (SNP), on the release of acetylcholine (ACh), an essential excitatory neurotransmitter, from the in vitro cat carotid body (CB). Bilateral CBs were harvested from five deeply anesthetized cats according to the regulations contained in the policies of the Johns Hopkins Animal Care and Use Committee. After recovering from the surgical procedures for extraction and cleaning, the CBs were taken through a 15-step protocol in which they were exposed to a hyperoxic gas mixture (40% O2/5% CO2; 20 min), then a hypoxic gas mixture (6% O2/5% CO2; 20 min), and a final 10 min hyperoxic mixture. This sequence was applied twice, followed by the same sequence in the presence, first, of 5 microM SNP, and secondly in the presence of 10 microM SNP. After washing and a recovery period the CBs were again exposed to the gases as in the first two non-SNP trials. The SNP exposures significantly reduced the overall release of ACh by about 20% (P=0.039). Further, SNP significantly reduced the hypoxia-induced increase in ACh release (without SNP: 82.4+/-19.1 fmol/20 microL versus with SNP: 49.7+/-15.0 fmol/20 microL; mean+/-S.E.M.; P=0.032). Trials #1 and #2 which preceded the application of SNP and Trial #3 which followed SNP were statistically indistinguishable. The CBs had recovered their original status. The data support the hypothesis that the frequently reported NO-induced reduction in CB neural output during hypoxia is at least in part due to the reduction in ACh release. The results are consistent with a previous report in which l-arginine, an NO precursor, had the same reducing effect. Possible mechanisms are discussed.     

Pancreatic acini in short-term culture: regulation by EGF, carbachol, insulin, and corticosterone

To define the hormonal regulation of the exocrine pancreas, acini were maintained for 24 h in suspension culture under various conditions and then examined morphologically and tested for their ability to synthesize and secrete protein. Acini cultured in the presence of 20% fetal calf serum (control conditions) retained their differentiated morphology as judged by light and electron microscopy. Under control conditions, stimulation with either cholecystokinin octapeptide (CCK8) or carbachol increased the release of amylase in a dose-dependent manner with a maximal threefold increase occurring at 200 pM CCK8 or 6 microM carbachol. Without inclusion of other factors in the medium during the culture period, the rate of protein synthesis measured in cultured acini was one-third the rate measured in freshly prepared acini. Inclusion of epidermal growth factor (EGF) (2 nM), carbachol (1 microM), or insulin (1 microM) during the culture period led to increases, whereas corticosterone (1 microM) led to decreases in functional parameters. Greatest effects were seen with a combination of EGF, carbachol, and insulin. This combination of factors led to a 160% increase over control in CCK8-stimulated amylase release and a 180% increase in [3H]leucine incorporation. These results indicate that isolated pancreatic acini can be maintained for up to 24 h and that EGF, carbachol, insulin, and corticosterone can regulate the long-term function of this tissue.     

Chronic hypoxia enhances endothelin-1-induced intracellular calcium elevation in rat carotid body chemoreceptors and up-regulates ETA receptor expression

Endothelin-1 (ET-1) excites carotid body (CB) chemoreceptors and induces mitosis of the chemoreceptors in chronic hypoxia. The aim of the present study was to examine the hypothesis that up-regulation of both ETA receptor and endogenous ET-1 expression in CB chemoreceptors enhances the response of intracellular Ca2+ to ET-1 following adaptation to chronic hypoxia (10% inspired O2 for 3-4 weeks). Cytosolic free [Ca2+] ([Ca2+]i) in type-I (glomus) cells freshly dissociated from rat CBs was measured by spectrofluorometry. Application of exogenous ET-1 (1-100 nM) concentration-dependently elevated [Ca2+]i in the glomus cells. This response to ET-1 (100 nM) was 49% greater in the chronically hypoxic (CH) group. The ET-1 response was abolished completely by the ETA receptor antagonist BQ610 (1 microM), but not by the ETB antagonist BQ788 (1 microM). The transient [Ca2+]i elevation induced by caffeine (30 mM) in the normoxic group was similar to that in the CH group, suggesting no differences in the intracellular Ca2+ stores. In situ hybridization with a digoxigenin-labelled antisense ETA receptor mRNA oligonucleotide probe revealed very intense and ubiquitous specific expression of ETA receptors in the lobules of glomus cells in the CH group, whereas staining in normoxic controls was light. Immunohistochemical studies revealed intense cytoplasmic staining for ET-1-immunoreactivity in most of the cell clusters in glomera in the CBs of CH rats but was faint in normoxic CBs. These findings indicate increased expression of both the ETA receptor and ET-1 in CB chemoreceptors during chronic hypoxia. Taken together, our results suggest that the [Ca2+]i response to ET-1 in rat CB chemoreceptors is augmented by up-regulation of ETA receptors and ET-1 expression. The enhancement of the paracrine/autocrine effect of ET-1 on the chemoreceptors is consistent with an excitatory and mitogenic role of the ET-1 and ETA receptor in the CB during chronic hypoxia.     

Cholinergic modulation of synaptic transmission and plasticity in entorhinal cortex and hippocampus of the rat

Effects of cholinergic agents on synaptic transmission and plasticity were examined in entorhinal cortex and hippocampus. Bath application of carbachol (0.25-0.75 microM) induced transient depression of field potential responses in all cases tested (24/24 in layer III of medial entorhinal cortex slices and 24/24 in CA1 of hippocampal slices; 11.0+/-1.9% and 7.8+/-2.5%, respectively) and long-lasting potentiation in some cases (4/24 in entorhinal cortex and 12/24 in hippocampus; 33.7+/-3.7% and 32.1+/-9.9%, respectively, in successful cases). Carbachol (0.5 microM) induced transient depression, but not long-lasting potentiation, of N-methyl-D-aspartate receptor-mediated responses in entorhinal cortex. At 5 microM, carbachol induced transient depression only (55. 9+/-4.7% in entorhinal cortex and 41.4+/-2.9% in hippocampus), which was blocked by atropine. Paired-pulse facilitation was not altered during carbachol-induced potentiation but enhanced during carbachol-induced depression. These results suggest that the underlying mechanisms of carbachol-induced depression and potentiation are decreased transmitter release and selective enhancement of non-N-methyl-D-aspartate receptor-mediated responses, respectively. Long-term potentiation could be induced in the presence of 10 microM atropine by theta burst stimulation. The magnitude was significantly lower (15.2+/-5.2%, n=9) compared with control (37.2+/-6.1%, n=8) in entorhinal cortex, however.These results demonstrate similar, but not identical, cholinergic modulation of synaptic transmission and plasticity in entorhinal cortex and hippocampus.     

Ventilatory responses and carotid body function in adult rats perinatally exposed to hyperoxia

Hypoxia increases the release of neurotransmitters from chemoreceptor cells of the carotid body (CB) and the activity in the carotid sinus nerve (CSN) sensory fibers, elevating ventilatory drive. According to previous reports, perinatal hyperoxia causes CSN hypotrophy and varied diminishment of CB function and the hypoxic ventilatory response. The present study aimed to characterize the presumptive hyperoxic damage. Hyperoxic rats were born and reared for 28 days in 55%–60% O₂; subsequent growth (to 3.5–4.5 months) was in a normal atmosphere. Hyperoxic and control rats (born and reared in a normal atmosphere) responded with a similar increase in ventilatory frequency to hypoxia and hypercapnia. In comparison with the controls, hyperoxic CBs showed (1) half the size, but comparable percentage area positive to tyrosine hydroxylase (chemoreceptor cells) in histological sections; (2) a twofold increase in dopamine (DA) concentration, but a 50% reduction in DA synthesis rate; (3) a 75% reduction in hypoxia-evoked DA release, but normal high [K⁺]₀-evoked release; (4) a 75% reduction in the number of hypoxia-sensitive CSN fibers (although responding units displayed a nearly normal hypoxic response); and (5) a smaller percentage of chemoreceptor cells that increased [Ca²⁺]₁ in hypoxia, although responses were within the normal range. We conclude that perinatal hyperoxia causes atrophy of the CB–CSN complex, resulting in a smaller number of chemoreceptor cells and fibers. Additionally, hyperoxia damages O₂-sensing, but not exocytotic, machinery in most surviving chemoreceptor cells. Although hyperoxic CBs contain substantially smaller numbers of chemoreceptor cells/sensory fibers responsive to hypoxia they appear sufficient to evoke normal increases in ventilatory frequency.     

Effect of carbachol on luminal release of somatostatin from isolated perfused rat duodenum.

The dynamic release of somatostatin-like immunoreactivity (SLI) from duodenum into the lumen was studied in the isolated, vascularly perfused rat duodenum. The luminal release of duodenal SLI was stimulated by a cholinergic agonist, carbachol, and the carbachol-induced release of SLI was completely blocked by atropine, but not by hexamethonium. These data suggest that the luminal release of SLI from rat duodenum is under the control of a cholinergic muscarinic stimulation. The ratio of somatostatin-14 to somatostatin-28 in picograms was about 1 during basal release but increased to approximately 2 during carbachol stimulation.     

Adenosine as a modulator of sympathetic nerve-stimulation-induced release of noradrenaline from the isolated rabbit heart

The ability of adenosine to inhibit sympathetic nerve-stimulation-induced overflow of noradrenaline was studied in isolated rabbit hearts with intact sympathetic innervation. Noradrenaline in the heart effluents was measured by HPLC with electrochemical detection. The drugs used, adenosine, theophylline, and 8-parasulphophenyltheophylline, were administered via the perfusion fluid. Adenosine (1-100 microM) dose-dependently inhibited stimulation-evoked outflow of noradrenaline from the heart, by up to 47%: the inhibition was completely antagonized by theophylline (200 microM), and by 8-parasulphophenyltheophylline (100 microM). Neither theophylline nor 8-parasulphophenyltheophylline did per se affect basal or stimulation-evoked outflow of noradrenaline. Simultaneous infusion of adenosine (100 microM) and theophylline (200 microM) significantly increased the outflow of noradrenaline during nerve stimulation, by almost 40%. No such effect was observed by adenosine (100 microM) and 8-parasulphophenyltheophylline (100 microM), indicating that theophylline may facilitate transmitter release by an action dissociated from purinoceptor antagonism. It is concluded that (a) adenosine inhibits depolarization-induced liberation of sympathetic transmitter in the rabbit heart, (b) this inhibition is mediated by activation or purinoceptors, probably located on the presynaptic nerve terminals, and (c) brief periods of sympathetic stimulation in the normoxic heart does not release sufficient amounts of adenosine to cause significant inhibition of transmitter release.     

Retrograde signaling changes the venue of postsynaptic inhibition in rat substantia nigra

Both endocannabinoids through cannabinoid receptor type I (CB1) receptors and dopamine through dopamine receptor type D1 receptors modulate postsynaptic inhibition in substantia nigra by changing GABA release from striatonigral terminals. By recording from visually identified pars compacta and pars reticulata neurons we searched for a possible co-release and interaction of endocannabinoids and dopamine. Depolarization of a neuron in pars reticulata or in pars compacta transiently suppressed evoked synaptic currents which were blocked by GABA(A) receptor antagonists (inhibitory postsynaptic currents [IPSCs]). This depolarization-induced suppression of inhibition (DSI) was abrogated by the cannabinoid CB1 receptor antagonist AM251 (1 microM). A correlation existed between the degree of DSI and the degree of reduction of evoked IPSCs by the CB1 receptor agonist WIN55,212-2 (1 microM). The cholinergic receptor agonist carbachol (0.5-5 microM) enhanced DSI, but suppression of spontaneous IPSCs was barely detectable pointing to the existence of GABA release sites without CB1 receptors. In dopamine, but not in GABAergic neurons DSI was enhanced by the dopamine D1 receptor antagonist SCH23390 (3-10 microM). Both the antagonist for CB1 receptors and the antagonist for dopamine D1 receptors enhanced or reduced, respectively, the amplitudes of evoked IPSCs. This tonic influence persisted if the receptor for the other ligand was blocked. We conclude that endocannabinoids and dopamine can be co-released. Retrograde signaling through endocannabinoids and dopamine changes inhibition independently from each other. Activation of dopamine D1 receptors emphasizes extrinsic inhibition and activation of CB1 receptors promotes intrinsic inhibition.     

Nicotinic and muscarinic reduction of unitary excitatory postsynaptic potentials in sensory cortex; dual intracellular recording in vitro

We studied the cholinergic modulation of glutamatergic transmission between neighboring layer 5 regular-spiking pyramidal neurons in somatosensory cortical slices from young rats (P10-P26). Brief bath application of 5-10 microM carbachol, a nonspecific cholinergic agonist, decreased the amplitude of evoked unitary excitatory postsynaptic potentials (EPSPs). This effect was blocked by 1 microM atropine, a muscarinic receptor antagonist. Nicotine (10 microM), in contrast to carbachol, reduced EPSPs in nominally magnesium-free solution but not in the presence of 1 mM Mg+2, indicating the involvement of NMDA receptors. Likewise, when the postsynaptic cell was depolarized under voltage clamp to allow NMDA receptor activation in the presence of 1 mM Mg+2, synaptic currents were reduced by nicotine. Nicotinic EPSP reduction was prevented by the NMDA receptor antagonist D-AP5 (50 microM) and by the nicotinic receptor antagonist mecamylamine (10 microM). Both carbachol and nicotine reduced short-term depression of EPSPs evoked by 10 Hz stimulation, indicating that EPSP reduction happens via reduction of presynaptic glutamate release. In the case of nicotine, several possible mechanisms for NMDAR-dependent EPSP reduction are discussed. As a result of NMDA receptor dependence, nicotinic EPSP reduction may serve to reduce the local spread of cortical excitation during heightened sensory activity.     

Nicotinic receptor-mediated biphasic effect on neuronal excitability in chick lateral spiriform neurons

Local neuronal circuits integrate synaptic information with different excitatory or inhibitory time windows. Here we report that activation of nicotinic acetylcholine receptors (nAChRs) leads to biphasic effects on excitability in chick lateral spiriform (SPL) neurons during whole cell recordings in brain slices. Carbachol (100 microM in the presence of 1 microM atropine) produced an initial short-term increase in the firing rates of SPL neurons (125+/-14% of control) that was mediated by postsynaptic nAChRs. However, after 3 min exposure to nicotinic agonists, the firing rate measured during an 800 ms depolarizing pulse declined to 19+/-7% (100 microM carbachol) or 26+/-8% (10 microM nicotine) of the control rate and remained decreased for 10-20 min after washout of the agonists. Similarly, after 60 s of electrically-stimulated release of endogenous acetylcholine (ACh) from cholinergic afferent fibers, there was a marked reduction (45+/-5% of control) in firing rates in SPL neurons. All of these effects were blocked by the nAChR antagonist dihydro-beta-erythroidine (30 microM). The inhibitory effect was not observed in Ca(2+)-free buffer. The nAChR-mediated inhibition depended on active G-proteins in SPL neurons and was prevented by the GABA(B) receptor antagonist phaclofen (200 microM), while the GABA(B) receptor agonist baclofen (10 microM) decreased firing rate in SPL neurons to 13+/-1% of control. The inhibitory response thus appears to be due to a nAChR-mediated enhancement of presynaptic GABA release, which then activates postsynaptic GABA(B) receptors. In conclusion, activation of nAChRs in the SPL initiates a limited time window for an excitatory period, after which a prolonged inhibitory effect turns off this window. The prolonged inhibitory effect may serve to protect SPL neurons from excessive excitation.     

Increased responsiveness of the cerebral cortical phosphatidylinositol system to noradrenaline and carbachol in senescent rats

The responsiveness of cerebral cortical alpha 1-adrenoceptors and cholinergic muscarinic M1 receptors was assessed in young (3 months) and aged (24 months) male Sprague-Dawley rats. The measure of responsiveness was the accumulation of inositol phosphate (IP) formed in [3H]myo-inositol-preloaded cerebral cortical slices in the presence of lithium, following stimulation with various concentrations of noradrenaline (1-300 microM) and carbachol (5-1000 microM). In old rats the maximum response to noradrenaline was higher by 80%, and that to carbachol by 33%, indicating an increased responsiveness of the investigated receptors in senescence.     

The impact of adenosine on the release of acetylcholine, dopamine, and norepinephrine from the cat carotid body

Exogenously administered adenosine provokes an increase in respiration in both animal models and in man. Administered near the carotid body adenosine increases neural output from the carotid body in rats and cats. Hypoxia has the same effect. Hypoxia also provokes a release of acetylcholine (ACh), dopamine (DA), and norepinephrine (NE) from the carotid body. The present study aimed to determine the effect of exogenous adenosine on the release of ACh, DA, and NE from the carotid bodies of cats. After a recovery period (from surgery) carotid bodies were first incubated for 10 (DA, NE) or 15 (ACh) min in Eppendorf tubes containing 85 microL of a physiological salt solution equilibrated with 40% O2/5% CO2 at 37 degrees C (hyperoxia). At the end of the incubation period the medium was drawn off, and measured for ACh, DA, and NE using HPLC-ECD methods. Next 85 microL of the medium and the tubes were equilibrated with a hypoxic gas mixture (4% O2/5% CO2) and the carotid bodies were incubated for 10 (DA, NE) or 15 (ACh) min, at the end of which the medium was drawn off and measured for ACh, DA, and NE. In the ACh studies there followed a post-hypoxic hyperoxic exposure (40% O2/5% CO2). ACh tubes were then made 100 microM with respect to adenosine, and the hyperoxic, hypoxic, and post-hypoxic hyperoxic challenges were repeated. One of the two DA, NE tubes had the 100 microM adenosine from the start. Adenosine significantly increased the release of ACh, but significantly decreased the hypoxia-induced release of DA. Potential mechanisms for these changes are reviewed.     

A novel choline-sensitive nicotinic receptor subtype that mediates enhanced GABA release in the chick ventral lateral geniculate nucleus

Nicotinic acetylcholine receptors modulate the release of GABA, glutamate, acetylcholine and dopamine in the brain. Here we describe a novel choline-sensitive nicotinic acetylcholine receptor that mediates enhanced GABA release in the chick ventral lateral geniculate nucleus. Whole-cell recordings in slices demonstrated that choline (0.03-10 mM), generally considered an alpha7-selective agonist, and carbachol (3-300 microM), a non-selective cholinergic agonist, both increased the frequency of spontaneous GABAergic events in ventral lateral geniculate nucleus neurons. Tetrodotoxin (0.5 microM) partially reduced responses to carbachol, but eliminated responses to choline. During long-term (5 min) exposure to choline the GABA enhancement was maintained until choline was washed out. Choline (300 microM) enhanced the frequency of spontaneous GABAergic events by 4.28-fold in control artificial cerebrospinal fluid. This choline-mediated enhancement was significantly reduced by the following nicotinic acetylcholine receptor antagonists: 1 microM dihydro-beta-erythroidine (1.49-fold increase, P<0.001), 1 microM methyllycaconitine (1.53-fold, P<0.001) and 0.2 microM alpha-conotoxin ImI (1.84-fold, P<0.001). In contrast, no significant change was seen in the presence of 0.1 microM dihydro-beta-erythroidine, 0.1 microM methyllycaconitine, 0.1 microM alpha-bungarotoxin, 0.1 microM alpha-conotoxin MII, 0.1 microM kappa-bungarotoxin, or 1 microM alpha-conotoxin AuIB.These results indicate that choline, at concentrations as low as 100 microM, activates a nicotinic acetylcholine receptor that is distinct from the classical alpha7 nicotinic acetylcholine receptors previously known to be activated by choline.     

Involvement of cholinergic and gabaergic systems in the fragile X knockout mice

Fragile X syndrome is an inherited cause of mental retardation. We used extra- and intracellular recordings in brain slices obtained from wild type and fragile X knockout mice to establish whether bath application of the cholinergic agent carbachol (5 microM) induces different responses in neurons of the subiculum, a limbic structure involved in learning and memory. We found that carbachol diminished excitatory post-synaptic responses induced by CA1 stratum radiatum stimulation in wild type mice, but caused an unexpected increase in knockout animals. Moreover, these responses augmented in knockout mice after carbachol washout, a phenomenon that resembled the muscarinic long-term potentiation seen in wild type mice during application of carbachol and GABA(A) receptor antagonists. We also used paired-pulse stimulation to determine whether the changes in synaptic excitability induced by carbachol were caused by pre- or post-synaptic mechanism. Under control conditions, this protocol induced facilitation in both wild type and knockout mice; in contrast, during carbachol application, this facilitatory effect was seen in wild type mice only.In conclusion, our data highlight for the first time differences in cholinergic and GABA-ergic mechanisms that may contribute to the phenotype of fragile X patients.