Hypothalamic TTF-1 orchestrates the sensitivity of leptin.

OBJECTIVE: Thyroid transcription factor-1 (TTF-1), a homeodomain-containing transcription factor, is predominantly expressed in discrete areas of the hypothalamus, which acts as the central unit for the regulation of whole-body energy homeostasis. Current study designed to identify the roles of TTF-1 on the responsiveness of the hypothalamic circuit activity to circulating leptin and the development of obesity linked to the insensitivity of leptin. METHODS: We generated conditional knock-out mice by crossing TTF-1flox/flox mice with leptin receptor (ObRb)Cre or proopiomelanocortin (POMC)Cre transgenic mice to interrogate the contributions of TTF-1 in leptin signaling and activity. Changes of food intake, body weight and energy expenditure were evaluated in standard or high fat diet-treated transgenic mice by using an indirect calorimetry instrument. Molecular mechanism was elucidated with immunohistochemistry, immunoblotting, quantitative PCR, and promoter assays. RESULTS: The selective deletion of TTF-1 gene expression in cells expressing the ObRb or POMC enhanced the anorexigenic effects of leptin as well as the leptin-induced phosphorylation of STAT3. We further determined that TTF-1 inhibited the transcriptional activity of the ObRb gene. In line with these findings, the selective deletion of the TTF-1 gene in ObRb-positive cells led to protective effects against diet-induced obesity via the amelioration of leptin resistance. CONCLUSIONS: Collectively, these results suggest that hypothalamic TTF-1 participates in the development of obesity as a molecular component involved in the regulation of cellular leptin signaling and activity. Thus, TTF-1 may represent a therapeutic target for the treatment, prevention, and control of obesity.

Suppressive effects of valproic acid on caudal fin regeneration in adult zebrafish.

Zebrafish can regenerate fins following injury through an epimorphic process that includes the formation of new tissues and reconstruction of the original morphology. In this study, the effects of valproic acid (VPA), a widely used anti-epileptic drug, on fin regeneration were studied after the caudal fin amputation of adult zebrafish. In the control group, zebrafish formed new tissues and reconstructed the original rays 14 days after amputation (dpa). Meanwhile, VPA treatments between 20 and 200 µM following amputation suppressed fin regeneration in a dose-dependent manner and altered morphological characteristics, such as bifurcation and segmentation, in the rays. Compared to the control, VPA also delayed blastema formation and decreased cell proliferation in the mesenchymal area of the regenerated fin. The mRNA expression of lef1, a downstream signaling gene in the Wnt pathway, was transiently increased in the regenerated fin of the control at 2 dpa; the same increase was not observed in the VPA-treated zebrafish. Sodium butyrate (SB), an histone deacetylase activity (HDAC) inhibitor, suppressed the fin regeneration without affecting the morphological characteristics of the regenerated ray. Furthermore, the transient increase of lef1 mRNA was not suppressed in the SB-treated zebrafish. These results suggested that VPA's suppressive effects on fin regeneration are partly mediated through decreased cell proliferation and lef1 mRNA expression.

Cell Proliferation in the Isolated Brains of Adult Zebrafish Incubated in Artificial Cerebrospinal Fluid.

The high rate of cell proliferation in the zebrafish brain allows one to perform reliable studies on cellular molecules associated with cell proliferation. In this study, we show that 5-bromo-2'-deoxyuridine (BrdU)-labeled cells were detected in the isolated brains of adult zebrafish that were incubated in oxygenated artificial cerebrospinal fluid (aCSF) in vitro. The number of BrdU-labeled cells shown in the in vitro preparation was close to that in the in vivo study and was modified by treatment with various pharmacological agents, such as valproic acid. In parallel to cell proliferation, protein expression and neural activity could also be measured in the isolated brains maintained in aCSF.

Hypoxia-Induced Neuroinflammation and Learning-Memory Impairments in Adult Zebrafish Are Suppressed by Glucosamine.

This study investigated changes in neuroinflammation and cognitive function in adult zebrafish exposed to acute hypoxia and protective effects of glucosamine (GlcN) against hypoxia-induced brain damage. The survival rate of zebrafish following exposure to hypoxia was improved by GlcN pretreatment. Moreover, hypoxia-induced upregulation of neuroglobin, NOS2α, glial fibrillary acidic protein, and S100ß in zebrafish was suppressed by GlcN. Hypoxia stimulated cell proliferation in the telencephalic ventral domain and in cerebellum subregions. GlcN decreased the number of bromodeoxyuridine (BrdU)-positive cells in the telencephalon region, but not in cerebellum regions. Transient motor neuron defects, assessed by measuring the locomotor and exploratory activity of zebrafish exposed to hypoxia recovered quickly. GlcN did not affect hypoxia-induced motor activity changes. In passive avoidance tests, hypoxia impaired learning and memory ability, deficits that were rescued by GlcN. A learning stimulus increased the nuclear translocation of phosphorylated cAMP response element binding protein (p-CREB), an effect that was greatly inhibited by hypoxia. GlcN restored nuclear p-CREB after a learning trial in hypoxia-exposed zebrafish. The neurotransmitters, γ-aminobutyric acid and glutamate, were increased after hypoxia in the zebrafish brain, and GlcN further increased their levels. In contrast, acetylcholine levels were reduced by hypoxia and restored by GlcN. Acetylcholinesterase inhibitor physostigmine partially reversed the impaired learning and memory of hypoxic zebrafish. This study represents the first examination of the molecular mechanisms underlying hypoxia-induced memory and learning defects in a zebrafish model. Our results further suggest that GlcN-associated hexosamine metabolic pathway could be an important therapeutic target for hypoxic brain damage.

Increased cell proliferation and neural activity by physostigmine in the telencephalon of adult zebrafish.

Physostigmine, an acetylcholinesterase inhibitor, is known to affect the brain function in various aspects. This study was conducted to test whether physostigmine affects cell proliferation in the telencephalon of zebrafish. BrdU-labeled cells was prominently observed in the ventral zone of the ventral telencephalon of zebrafish. The increased number of BrdU- and proliferating cell nuclear antigen-labeled cells were shown in zebrafish treated with 200µM physostigmine, which was inhibited by pretreatment with 200µM scopolamine. iNOS mRNA expression was increased in the brain of zebrafish treated with 200µM physostigmine. Consistently, aminoguanidine, an iNOS inhibitor, attenuated the increase in the number of BrdU-labeled cells by physostigmine treatment. Zebrafish also showed seizure-like locomotor activity characterized by a rapid and abrupt movement during a 30min treatment with 200µM physostigmine. Neural activity in response to an electrical stimulus was increased in the isolated telencephalon of zebrafish continuously perfused with 200µM physostigmine. None of the number of BrdU-labeled cells, neural activity, or locomotor activity was affected by treatment with 20µM physostigmine. These results suggest that 200µM physostigmine increased neural activity and induced cell proliferation via nitric oxide production in zebrafish.

Valproic acid decreases the proliferation of telencephalic cells in zebrafish larvae.

Valproic acid (VPA) is used as an antiepileptic drug (AED) or mood stabilizer. Recent studies have shown that exposure to VPA during embryonic development alters neural progenitor cell (NPC) proliferation, and can also lead to behavioral impairment in adult animals. The main goal of this investigation was to evaluate the effects of treatment with 2 mM VPA for 3h on cell proliferation in the telencephalic area of zebrafish larvae of 5 days post-fertilization (dpf) using immunohistochemistry (IHC). It was also determined whether VPA exposure affects the learning ability and anxiety levels of zebrafish during adulthood using bottom-dwelling behavior and passive avoidance tests. Result of the study demonstrated that VPA exposure during development transiently decreased neuronal cell proliferation without inducing apoptosis. Additionally, quantitative real-time PCR (qRT-PCR) data indicated that mRNA expression levels of wnt signaling pathway-related factors such as ß-catenin, lef1, and gsk3ß were altered in the zebrafish treated with VPA. Interestingly, these effects were reversed over time after VPA treatment had ceased. Alterations of passive avoidance learning and bottom dwelling behavior were not observed during adulthood after developmental VPA exposure. These results may be due to the restoration of cell proliferation during the recovery period after VPA treatment.

Exogenous GDNF increases the migration of the neural stem cells with no protection against kainic acid-induced excitotoxic cell death in rats.

Glia cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuron types. In this study, we have evaluated the utility of adenovirus-based vectors (Ad) and hippocampal neural stem cells (NSCs) as genetic tools for the delivery of a therapeutic protein, GDNF, in hippocampus tissues damaged by kainic acid (KA)-induced excitotoxicity. The experimental animals were treated with KA 3 days prior to exposure to Ad-GDNF, NSCs, and NSCs infected with Ad-GDNF (Ad-GDNF-NSCs). Seven days after the treatments with Ad-GDNF, NSCs and Ad-GDNF-NSCs, the effects of the treatments were evaluated. GAD-67 labeled cells originating from the transplanted NSCs were observed at increased levels in the Ad-GDNF-NSCs-treated rats as compared to the NSCs-only rats. In situ apoptosis assays showed that the levels of TUNEL-positive cells were slightly, but not significantly, reduced in the Ad-GDNF and Ad-GDNF-NSCs groups, as compared to the saline and NSCs only groups. GDNF expression by NSCs and Ad-GDNF was upregulated as the consequence of adenoviral gene delivery in the NSCs and Ad-GDNF-treated rats, and the transplanted NSCs were shown to have migrated to the hippocampal regions in Ad-GDNF-NSCs rats to a greater degree than in the NSCs-only rats. Furthermore, in the region in which the NSCs were detected, GDNF and GAD-67 expression were increased. These results indicate that the migration and differentiation of NSCs may be associated with the expression of GDNF. However, cell death consequent to KA administration was not prevented by upregulated GDNF and NSCs transplantation. Collectively, our results indicate that GDNF may exert effects on the migration and differentiation of NSCs, but there are no protective properties with regard to excitotoxically damaged hippocampal tissue.

Improvement of pentylenetetrazol-induced learning deficits by valproic acid in the adult zebrafish.

Pentylenetetrazol (PTZ) has been shown to induce seizure-like behavior, learning deficits in passive avoidance response test, and an increase in hsp70 (heat shock protein 70) mRNA expression in the adult zebrafish; PTZ has been increasingly appreciated as an excellent model system for the study of seizures. In this study, we demonstrate that valproic acid (VPA), an antiepileptic drug, suppresses seizure-like behavior and improves learning ability in adult zebrafish treated with PTZ. Pretreatment with VPA significantly reduces rapid involuntary movement and abrupt changes in moving direction in the PTZ-treated zebrafish. PTZ-induced learning impairments were also improved in the zebrafish pretreated with 200 or 500 microM VPA. However, the scopolamine-induced impairments of learning ability were not improved by VPA pretreatment. It is worth noting that while the zebrafish treated with 500 microM VPA for 1-3 weeks learned the passive avoidance response, those treated with 1 or 2mM VPA for 3h didn't. Furthermore, the increased level of hsp70 expression induced by PTZ, a stress marker protein, was significantly reduced in the VPA-pretreated zebrafish brains. Collectively, our data show the antiepileptic effects of VPA in the adult zebrafish, which coincides with reduced hsp70 mRNA expression, rescued learning impairment under PTZ-treated conditions.

Reduced neuronal proliferation by proconvulsant drugs in the developing zebrafish brain.

Seizures have been reported to modify neural development in the immature brain. In this study, we attempted to determine whether pentylenetetrazol (a GABAergic receptor antagonist, PTZ)-induced seizures influence cell proliferation in zebrafish larvae (5 and 15 days of post-fertilization), using bromodeoxyuridine (BrdU) to label dividing cells. In the brains of 5 dpf larvae, PTZ treatment significantly reduced the number of BrdU-labeled cells in the telencephalic area (pallium and subpallium), diencephalic area (thalamus and preoptic area), medial tectal proliferation zone, and medial cerebellar proliferation zone to 52.4%, 62.9%, 47.2%, and 54.0% of the controls, respectively. In contrast, we noted no reductions in the number of BrdU-labeled cells in the brains of the 15 dpf larvae. The double-label of BrdU and Hu, a neuronal marker, demonstrated that the majority of newborn cells showed the neuronal phenotype. Similarly, kainic acid (200 microM), a glutamatergic receptor agonist, significantly reduced the number of BrdU-labeled cells in the telencephalic area, diencephalic area, and medial tectal proliferation zone to 51.4%, 61.9%, and 40.4% of the controls, respectively. Physostigmine (500 microM), an acetylcholinesterase inhibitor, also reduced the number of BrdU-labeled cells in the telencephalic area, diencephalic area, medial tectal proliferation zone, and medial cerebellar proliferation zone to 52.8%, 35.9%, 30.5%, and 39.8% of the controls, respectively. All of these drugs resulted in electrographic seizures in the larval brain when perfused directly through artificial cerebrospinal fluid. These results indicated that seizures result in a massive reduction in cell proliferation in wide-ranging areas of the developing brain.

Scopolamine-induced learning impairment reversed by physostigmine in zebrafish.

In this study, the effects of scopolamine, an acetylcholine muscarinic receptor antagonist, and physostigmine, an acetylcholinesterase inhibitor, on the learning ability and memory of zebrafish were evaluated using a passive avoidance response test. The zebrafish were trained to stay in a dark compartment to avoid a weight dropping into an acryl shuttle chamber with a central sliding door. The crossing time was increased significantly, from 30.7+/-40.8s to 179.3+/-27.3s in the training session and 179.9+/-28.0s in the test session carried out 2h later in the controls. When treatment with 200 microM scopolamine was administered for 1h prior to the training session, the crossing time did not increase. The scopolamine-induced learning deficit was ameliorated by pretreatment with 20 microM physostigmine for 1h prior to scopolamine treatment; the crossing time was similarly increased, as shown with the controls (60.9+/-11.5s, 130.9+/-27.5s, and 183.4+/-26.6s in the training session and 108.1+/-23.9s in the test session). When scopolamine treatment was administered after the training session, the crossing time in the test session was reduced significantly as compared to that noted in the third trial of the training session, which was also ameliorated by physostigmine pretreatment. These results show that scopolamine impairs both the acquisition of passive avoidance response and retention of the learned response, and that physostigmine rescues the amnesic effects of scopolamine in zebrafish.

Cholinergic modulation of neural activity in the telencephalon of the zebrafish.

In this study, we have assessed the effects of muscarinic receptor activation on neural activity in the telencephalon of the zebrafish. Electrically evoked field potentials via the stimulation of the posterior dorsal telencephalon were recorded in the anterior dorsal telencephalon of the ipsilateral hemisphere, with a peak latency of 20-30ms. The application of carbachol (1-25microM) and oxotremorine-M (0.25-60microM) suppressed the amplitude of field potentials in a dose-dependent manner. This suppressive effect was blocked by a muscarinic receptor antagonist, atropine (20microM), and an M1 receptor antagonist, pirenzepine (5microM), but not by the M2 receptor antagonists, methoctramine (5microM) and gallamine (5microM). Oxotremorine-M (1microM) also suppressed the induction of field potentials via the stimulation of the contralateral telencephalon and the medial fiber bundle, which was blocked by atropine (20microM). An acetylcholine esterase inhibitor, physostigmine (20-30microM), reduced the peak amplitude of the evoked field potentials, while increasing late bursting activity. Furthermore, a high dose of physostigmine (100microM) induced spontaneous bursting activity. These results demonstrated the cholinergic influence on the neural activity in the telencephalon of the zebrafish.

Mitochondrial DNA in patients with essential tremor.

Essential tremor (ET) is one of the most common of the movement disorders. However, there has been little agreement in the neurological literature regarding diagnostic criteria for ET. It is not clear to what extent ET is associated with defects of mitochondrial DNA. In this study, we analyzed mitochondrial DNA (mtDNA) from the blood cells of the normal and ET patients using the long and accurate polymerase chain reaction (LA-PCR) and PCR. The large deletions were detected within several regions of mtDNA, but were not detected in the D-loop or CO I regions in ET patients. From our study, it is suggested that ET is a disorder showing a deficiency of mtDNA multicomplexes, and it also appears that mitochondrial dysfunction could be one of the causative factors of ET.

Neuroprotection of adenoviral-vector-mediated GDNF expression against kainic-acid-induced excitotoxicity in the rat hippocampus.

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for several types of neurons. In the present study, we examined the protective roles of adenoviral-vector-delivered GDNF (Ad-GDNF) in the hippocampus damaged by kainic-acid (KA)-induced excitotoxicity using GAD-67 immunoreactivity, immunoblot analysis, behavioral test, 5-bromo-2-deoxyuridine (BrdU) and TUNEL assay. Ad-GDNF was pre-inoculated into the KA-treated rat hippocampus 7 days before KA injection. Ad-GDNF resulted in the suppression of KA-induced tonic-clonic convulsions. In situ apoptosis assay demonstrated a significant reduction in apoptotic cells in the CA3 and dentate hilus regions of the Ad-GDNF-pre-inoculated rats (Ad-GDNF-KA), compared to the KA rats. Striking reductions in the density of GAD-67 neurons were also observed in the CA3 and dentate hilus regions of the KA rats. On the other hand, the number of GAD-67-positive cells was recovered to the control levels in the Ad-GDNF-KA rats. Immunoblot analysis further confirmed that GAD-67 and Bcl-2 expression increased in the Ad-GDNF-KA rats compared to KA rats. Taken together, these results suggest that Ad-GDNF may serve to control KA-induced hippocampal cell loss and behavioral seizure.

Maternal stress produces learning deficits associated with impairment of NMDA receptor-mediated synaptic plasticity.

Stress in adulthood can have a profound effect on physiology and behavior, but the extent to which prolonged maternal stress affects the brain function of offspring when they are adult remains primarily unknown. In the present work, chronic immobilization stress to pregnant mice affected fetal growth and development. When pups born from stressed mice were reared to adulthood in an environment identical to that of nonstressed controls, several physiological parameters were essentially unaltered. However, spatial learning and memory was significantly impaired in the maternally stressed offspring in adulthood. Furthermore, electrophysiological examination revealed a significant reduction in NMDA receptor-mediated long-term potentiation in the CA1 area of hippocampal slices. Subsequent biochemical analysis demonstrated a substantial decrease in NR1 and NR2B subunits of the NMDA receptor in synapses of the hippocampus, and the interaction between these two subunits appeared to be reduced. These results suggest that prolonged maternal stress leads to long-lasting malfunction of the hippocampus, which extends to and is manifested in adulthood.

Tamoxifen-induced cell death and expression of neurotrophic factors in cultured C6 glioma cells.

The effects of ( Z)-2[ p -(1,2-diphenyl-1-butenyl)phenoxy]-N ,N -dimethylamine citrate (tamoxifen) on cell survival and the expression of neurotrophic factors (NTF) were investigated in rat C6 glioma cells (C6). C6 cells do not express the estrogen receptor. Cytotoxic effect was detected from 24 h after the treatment with 10 microM tamoxifen and increased with time in a dose-dependent manner. C6 cells treated with tamoxifen also displayed various morphological types such as elliptical, round and aggregated form. As the treatment time increased, the proliferation of C6 cells was reduced remarkably and most of them became the round or aggregated form. To examine the relationship of the expression of NTF and the cytotoxicity of tamoxifen, the mRNA level of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and basic fibroblast growth factor (bFGF) was measured after 24 h treatment with tamoxifen by RT-PCR. The expression of mRNA of BDNF or GDNF in C6 cells treated with various concentrations of tamoxifen was comparable to controls. The expression of bFGF mRNA was significantly reduced in C6 cells treated with 10 or 15 microM tamoxifen. The results suggest that tamoxifen exerts cytotoxic effect on estrogen receptor-negative C6 cells through the inhibition of the transcription of bFGF.

Calcium-independent CaMKII activity is involved in ginsenoside Rb1-mediated neuronal recovery after hypoxic damage.

Recent studies have indicated that Ginsenoside Rb1, one of the major components of ginseng root, may play an important role in protecting cells from damage. Here, we investigated the neuroprotective activity of Rb1 after hypoxic injury in young rats. About 50% animals were dead by exposing hypoxic condition three times in three consecutive days. Then, the pretreatment with Rb1 prior to hypoxic stimulation reduced animal death to 12%, and also significantly reduced the recovery time from hypoxia-related, compromised symptoms in survived animals. Rb1 also significantly reduced levels of lactate dehydrogenase (LDH) release from primary hippocampal neurons which were maintained at low oxygen concentration, indicating increased neuronal survival by Rb1. Ca(2+)/calmodulin-dependent kinase II (CaMKII) activity in the hippocampal tissues of hypoxia-induced rats was decreased to about 50% of the control animal. Then Rb1-treatment prior to hypoxic stimulation significantly elevated Ca(2+)-independent kinase II activity when measured 48 hr after hypoxic stimulation. Thus, the present data suggest that calcium independent CaMKII activity may be involved in the process of ginsenoside Rb1-mediated recovery of neuronal cells after hypoxic damage.

NMDA receptor-dependent long-term potentiation in the telencephalon of the zebrafish.

In Mg2+ -free aCSF, bursting discharges were induced in the posterior telencephalon of zebrafish following an electrical stimulation of the anterior telencephalon. The bursting discharges were partially reduced by CNQX (10 microM), an AMPA receptor antagonist, and the remaining activity was completely blocked by an additional treatment of APV (50 microM), an NMDA receptor antagonist. Long-term potentiation that lasted more than 1 h was also induced after 20 min of perfusion with KCl (10 mM). The degree of KCl-induced long-term potentiation (K-LTP) was reduced when a concomitant electrical stimulation was not delivered during a KCl perfusion. K-LTP was blocked by APV (50 microM) but not by nifedipine (1 microM), an L-type Ca2+ channel blocker. Furthermore, K-LTP was not induced in the presence of a broad spectrum inhibitor for protein kinases, H-7 (10 microM). These results suggest that NMDA receptors and protein kinases play important roles in the synaptic plasticity of the zebrafish brain.

Carbachol induces a form of long-term potentiation in lateral amygdala.

We examined the effects of carbachol, a muscarinic acetylcholine receptor agonist, on excitatory synaptic transmission at thalamo-amygdala synapses in rat brain slices. The application of a low concentration of carbachol (0.25 microM) produced a form of long-term potentiation (cLTP) and a transient suppression of synaptic responses, which was blocked by a muscarinic receptor antagonist, atropine (10 microM). M2 receptor agonist produced only a transient suppression, whereas M1 receptor agonist induced both a transient suppression and a long-term potentiation. Induction of cLTP required simultaneous low-frequency afferent stimulation, and was also dependent upon the activation of NMDA receptors. SQ22536 (50 microM), an adenylyl cyclase inhibitor completely blocked cLTP. Consistently, pretreatment with a maximal concentration of forskolin (10 microM) reduced cLTP.

Identification and functional evidence of GABAergic neurons in parts of the brain of adult zebrafish (Danio rerio).

The distribution of GABA-containing neurons was studied in the brain of the adult zebrafish by Nissl staining and immunohistochemistry. GABA immunoreactivity (GABA-IR) was demonstrated in parts of the brain such as olfactory bulb (OB), telencephalon, tectum stratum, and in the hypothalamus. GABA-IR appeared in the area where Nissl-stained cell bodies were abundant. The internal cellular layer of the OB was most densely stained by Nissl staining, and also showed a high level of GABA-IR. The telencephalon and the hypothalamus revealed a similar pattern to the OB in terms of Nissl staining and GABA-IR. However, the distribution and shape of stained cells of the tectum stratum were distinct from those in other regions: Nissl-stained neurons were ubiquitously present throughout all cellular layers including the stratum griseum centrale, the stratum album centrale (SAC), and the stratum periventriculare (SP). However, GABA-IR was weakly expressed in a limited number of neurons only in the SAC and SP. Whether GABA serves as an inhibitory neurotransmitter was also tested in the isolated telencephalon preparation by using extracellular field potential recordings. The synaptic activity recorded in the posterior dorsal telencephalon in response to the electrical stimulation of the anterior dorsal telencephalon was increased in the presence of the GABAA receptor antagonist, BMI, suggesting an inhibitory role for GABA-immunoreactive neurons in the adult brain of the zebrafish.

Long-lasting facilitation by dehydroevodiamine. HClof synaptic responses evoked in the CA1 region of rat hippocampal slices.

We tested the effects of dehydroevodiamine.Cl (DHED) on field excitatory postsynaptic potentials (fEPSPs) evoked by the electrical stimulation of Schaffer collaterals-commissural fibres in the CA1 region of rat hippocampal slices. Bath applications of 10 microM DHED for 20 or 40 min induced long-lasting facilitation of fEPSPs, which outlasted the presence of DHED. A 10 min treatment with a higher concentration (100 microM) also induced long-lasting facilitation. The long-lasting facilitation was blocked either by 10 microM atropine, the muscarinic receptor antagonist, or by 50 microM D-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. These results show that DHED produces long-lasting facilitation of synaptic transmission, and that this facilitation depends upon the activation of both the muscarinic and NMDA receptors.