Stimulatory effects of the putative metabotropic glutamate receptor antagonist L-AP3 on phosphoinositide turnover in neonatal rat cerebral cortex.

1. The effects of the metabotropic glutamate receptor (mGluR) antagonist, L-2-amino-3-phosphonopropionate (L-AP3) on phosphoinositide turnover in neonatal rat cerebral cortex slices has been investigated. 2. At concentrations of < or = 300 microM, L-AP3 inhibited total [3H]-inositol phosphate ([3H]-InsPx) and Ins(1,4,5)P3 mass responses stimulated by the selective mGluR agonist, 1-amino-cyclopentane-1S, 3R-dicarboxylic acid (1S, 3R-ACPD). Comparison with the competitive mGluR antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG) clearly demonstrated that L-AP3 caused inhibition by a mechanism that was not competitive, as L-AP3 decreased the maximal response to 1S, 3R-ACPD (by approximately 40% at 300 microM L-AP3) without significantly affecting the concentration of 1S, 3R-ACPD required to cause half-maximal stimulation of the [3H]-InsPx response. 3. In contrast, at a higher concentration L-AP3 (1 mM) caused a large increase in [3H]-InsPx accumulation which was similar in magnitude in both the absence and presence of 1S, 3R-ACPD (300 microM). D-AP3 (1 mM) had no stimulatory effect alone and did not affect the response evoked by 1S, 3R-ACPD. L-AP3 (1 mM) also caused a large increase in Ins(1,4,5)P3 accumulation. The magnitude of the response (4-5 fold increase over basal) approached that evoked by a maximally effective concentration of 1S, 3R-ACPD, but differed substantially in the time-course of the response. The stimulatory effects of 1S, 3R-ACPD and L-AP3 on Ins(1,4,5)P3 accumulation were also similarly affected by decreases in extracellular calcium concentration. 4. Detailed analysis of the inositol phospholipid labelling pattern and the inositol (poly)phosphate isomeric species generated following addition of L-AP3 was also performed. In the continued presence of myo-[3H]-inositol, L-AP3 (1 mM) stimulated a significant increase in phosphatidylinositol labelling, but not that of the polyphosphoinositides, and the inositol (poly)phosphate profile suggested that substantial Ins(1,4,5)P3 metabolism occurs via both 5-phosphatase and 3-kinase routes. 5. A significant stimulatory effect of L-AP3 (1 mM) on [3H]-InsPx accumulation was also observed in neonatal rat hippocampus, and cerebral cortex and hippocampus slices prepared from adult rat brain. 6. These data demonstrate that whilst L-AP3 antagonizes mGluR-mediated phosphoinositide responses at concentrations of < or = 300 microM, higher concentrations substantially stimulate this response. The ability of (+/-)-MCPG (1 mM) to attenuate significantly L-AP3-stimulated [3H]-InsPx accumulation, suggests that both the inhibitory and stimulatory effects of L-AP3 may be mediated by mGluRs.     

石杉碱甲对大鼠大脑皮层NMDA受体的调制作用

AIM: To investigate the effects of huperzine A (Hup A) on NMDA receptors in rat cerebral cortex. METHODS: 1) The effect of hup A on NMDA-induced current was studied in acutely dissociated rat hippocampal pyramidal neurons using whole-cell recording. 2) The effect of Hup A on NMDA receptor binding was assessed using [3H] dizocilpine (Diz) binding assay in synaptic membrane preparation of rat cerebral cortex. RESULTS: 1) Hup A reversibly inhibited NMDA-induced current in a concentration-dependent manner with IC50 of 45.4 mumol.L-1. 2) Hup A inhibited the specific binding of [3H]MK-801 to extensively washed synaptic membrane of rat cerebral cortex in a concentration-dependent manner with IC50 of 0.5 (0.1-1.9) mumol.L-1 (n = 4). 3) L-Glutamate 10 mumol.L-1 markedly increased [3H] MK-801 binding. In the presence of L-glutamate, Hup A 0.001-0.1 mumol.L-1 caused a further increase of the binding, whereas Hup A 1-300 mumol.L-1 inhibited the binding in a concentration-dependent manner with IC50 of 12.3 (5.8-26.3) mumol.L-1 (n = 5). CONCLUSION: Hup A acted as an antagonist of NMDA receptor in cerebral cortex in addition to its inhibitory effect on acetylcholinesterase.     

Modulatory effects of NMDA on phosphoinositide responses evoked by the metabotropic glutamate receptor agonist 1S,3R-ACPD in neonatal rat cerebral cortex.

1. The effect of NMDA-receptor stimulation on phosphoinositide signalling in response to the metabotropic glutamate receptor agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) has been examined in neonatal rat cerebral cortex slices. 2. Total [3H]-inositol phosphate ([3H]-InsPx) accumulation, in the presence of 5 mM LiCl, in [3H]-inositol pre-labelled slices was concentration-dependently increased by 1S,3R-ACPD (EC50 16.6 microM) and, at a maximally effective concentration, 1S,3R-ACPD (300 microM) increased [3H]-InsPx accumulation by 12.8 fold over basal values. 3. [3H]-InsPx accumulation stimulated by 1S,1R-ACPD was enhanced by low concentrations of NMDA (3-30 microM), but not by higher concentrations (> 30 microM). [3H]-InsPx accumulations stimulated by 1S,3R-ACPD in the absence or presence of 10 microM NMDA were linear with time, at least over the 15 min period examined; however, in the presence of 100 microM NMDA the initial enhancement of 1S,3R-ACPD-stimulated phosphoinositide hydrolysis progressively decreased with time. 4. In the presence of a maximal enhancing concentration of NMDA (10 microM), the response to 1S,3R-ACPD (300 microM) was increased 1.9 fold and the EC50 for agonist-stimulated [3H]-InsPx accumulation decreased about 4 fold. The enhanced response to the metabotropic agonist was concentration-dependently inhibited by competitive and uncompetitive antagonists of NMDA-receptor activation. 5. 1S,3R-ACPD also stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation with an initial peak response (5-6 fold over basal) at 15 s decaying to a smaller (2 fold), but persistent elevated accumulation (1-10 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of phosphoinositide turnover in neonatal rat cerebral cortex by group I- and II- selective metabotropic glutamate receptor agonists

1. The interactive effects of different metabotropic glutamate (mGlu) receptor subtypes to regulate phosphoinositide turnover have been studied in neonatal rat cerebral cortex and hippocampus by use of agonists and antagonists selective between group I and II mGlu receptors.
2. The group II-selective agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC; 100 μM) had no effect on basal total inositol phosphate ([³H]-InsP_x) accumulation (in the presence of Li⁺) in myo-[³H]-inositol pre-labelled slices, but enhanced the maximal [³H]-InsP_x response to the group I-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) by about 100% in both hippocampus and cerebral cortex. In cerebral cortex the enhancing effect of 2R,4R-APDC occurred with respect to the maximal responsiveness and had no effect on EC₅₀ values for DHPG (-log EC₅₀ (M): control, 5.56±0.05; +2R,4R-APDC, 5.51±0.08). 2R,4R-APDC also caused a significant enhancement of the DHPG-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) mass response over an initial 0–300 s time-course.
3. The enhancing effects of 2R,4R-APDC on DHPG-stimulated [³H]-InsP_x accumulation were observed in both the presence and nominal absence of extracellular Ca²⁺, and irrespective of whether 2R,4R-APDC was added before, simultaneous with, or subsequent to DHPG. Furthermore, increasing the tissue cyclic AMP concentration up to 100 fold had no effect on DHPG-stimulated Ins(1,4,5)P₃ accumulation in the absence or presence of 2R,4R-APDC.
4. 2R,4R-APDC and (2S, 1′R, 2′R, 3′R)-2-(2,3-dicarboxylcyclopropyl)glycine (DCG-IV), the latter agent in the presence of MK-801 to prevent activation of NMDA-receptors, each inhibited forskolin-stimulated cyclic AMP accumulation by about 50%, with respective EC₅₀ values of 1.3 and 0.04 μM (-log EC ₅₀ (M): 2R,4R-APDC, 5.87±0.09; DCG-IV, 7.38±0.05). In the presence of DHPG (30 μM), 2R,4R-APDC and DCG-IV also concentration-dependently increased [³H]-InsP_x accumulation with respective EC₅₀ values of 4.7 and 0.28 μM (-log EC₅₀ (M): 2R,4R-APDC, 5.33±0.04; DCG-IV, 6.55±0.09) which were 3–7 fold rightward-shifted relative to the adenylyl cyclase inhibitory responses.
5. The group II-selective mGlu receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"LY307452","term_id":"1257780057","term_text":"LY307452"}}LY307452 (30 μM) caused parallel rightward shifts in the concentration-effect curves for inhibition of forskolin-stimulated adenylyl cyclase, and enhancement of DHPG-stimulated [³H]-InsP_x accumulation, by 2R,4R-APDC yielding similar equilibrium dissociation constants (K_ds, 3.7±1.1 and 4.1±0.4 μM respectively) for each response.
6. The ability of 2R,4R-APDC to enhance receptor-mediated [³H]-InsP_x accumulation appeared to be agonist-specific; thus although DHPG (100 μM) and the muscarinic cholinoceptor agonist carbachol (10 μM) stimulated similar [³H]-InsP_x accumulations, only the response to the former agonist was enhanced by co-activation of group II mGlu receptors.
7. These data demonstrate that second messenger-generating phosphoinositide responses stimulated by group I mGlu receptors are positively modulated by co-activation of group II mGlu receptors in cerebral cortex and hippocampus. The data presented here are discussed with respect to the possible mechanisms which might mediate the modulatory activity, and the physiological and pathophysiological significance of such crosstalk between mGlu receptors.

     

Modulation of NMDA receptor by huperzine A in rat cerebral cortex 1

目的：研究石杉碱甲（ＨｕｐＡ）对大脑皮层ＮＭＤＡ受体的影响．方法：１）用急性分离海马锥细胞全细胞记录研究ＨｕｐＡ对ＮＭＤＡ诱发电流的影响．２）用大脑皮层突触膜标本研究ＨｕｐＡ对［３Ｈ］Ｄｉｚ特异性结合的影响．结果：１）ＨｕｐＡ可逆地抑制ＮＭＤＡ诱发的电流反应（ＩＣ５０＝４５４μｍｏｌ·Ｌ－１）．２）在突触膜标本,ＨｕｐＡ抑制［３Ｈ］Ｄｉｚ的结合量（ＩＣ５０＝０５（０１－１９）μｍｏｌ·Ｌ－１,ｎ＝４）．３）Ｌ谷氨酸１０μｍｏｌ·Ｌ－１增加［３Ｈ］Ｄｉｚ结合量．加入Ｌ谷氨酸后,ＨｕｐＡ０００１－０１μｍｏｌ·Ｌ－１进一步增加结合量;ＨｕｐＡ１－３００μｍｏｌ·Ｌ－１则抑制结合量（ＩＣ５０＝１２３（５８－２６３）μｍｏｌ·Ｌ－１,ｎ＝５）．结论：ＨｕｐＡ在大脑皮层除了抑制乙酰胆碱酯酶外,还是ＮＭＤＡ受体拮抗剂     

Neuroprotection by the NMDA receptor-associated open-channel blocker memantine in a photothrombotic model of cerebral focal ischemia in neonatal rat.

Excessive accumulation of glutamate or other excitatory amino acids and the subsequent overactivity of NMDA receptors is currently thought to lead to neuronal injury in cerebral ischemia. Therefore, antagonists of the NMDA receptor may offer an approach for the treatment of ischemic brain injury. Dizocilpine (MK-801), an NMDA receptor-associated channel blocker, protects neurons in several rodent stroke models. However, this drug has numerous side effects and causes apoptosis of neonatal neurons. Recently, another NMDA receptor-associated channel blocker, memantine, has been shown to ameliorate NMDA-receptor mediated neurotoxicity in neuronal cell cultures and in focal cerebral ischemia models in adult rats without substantial side effects. Memantine has been used clinically in the treatment of Parkinson's disease and spasticity for a number of years. Here we tested the effects of memantine on focal stroke caused by photochemical thrombosis in neonatal rats and demonstrated a neuroprotective effect of memantine in this model. We also found excellent correlation between infarct size determined by magnetic resonance imaging (MRI) and histopathological analysis in the same animals. A single pre-ischemic dose of memantine (20 mg/kg) given 15 min prior to induction of stroke reduced the infarct size by 36.3% when compared to control animals treated with normal saline (P < 0.0001). At this dosage, memantine manifests few, if any, neurobehavioral side effects. Thus memantine appears to be both safe and effective in neonatal as well as adult animal models of stroke.     

Effects of lithium and desipramine administration on agonist-stimulated inositol phosphate accumulation in rat cerebral cortex

The effects of acute lithium and chronic desipramine administration on the inositol phosphate responses of rat cerebral cortical slices to noradrenaline, carbachol and 5-hydroxytryptamine, were determined. Acute injection of lithium (4 meq/kg s.c. 24 hr before sacrifice) significantly increased the inositol-1-phosphate response to noradrenaline, while chronic administration of desipramine (10 mg/kg i.p. for 3 weeks) produced a smaller increase in this parameter. Chronic desipramine also decreased the serotonin responses in terms of all three inositol phosphates measured. No potentiative or additive interactions between the lithium and desipramine effects were observed.     

Characterization of ouabain high-affinity binding to rat cerebral cortex. Modulation by melatonin.

High-affinity [3H]ouabain binding to membrane preparations of rat cerebral cortex was examined using a rapid filtration procedure. At 37 degrees C, binding reached equilibrium in about 60 min. Scatchard analyses of the data at equilibrium revealed a single population of binding sites with a dissociation constant of KD = 3.1 +/- 0.36 nM and a binding site concentration of Bmax = 246.4 +/- 18.4 fmol/mg protein. Kinetic analyses of the association and dissociation curves indicated a kinetic KD = 4.6 nM, which is in good agreement with the value obtained at equilibrium. When various digitalis compounds were tested for their ability to inhibit [3H]ouabain binding, the following Ki values (nM) were obtained: ouabain (3.9); digoxin (18); acetyl-digitoxin (66); k-strophanthin (95); digitoxin (236). When melatonin was added to the incubation medium, the ability of ouabain to inhibit [3H]ouabain binding increased in a dose-related manner to yield the following Ki values (nM): melatonin 10 nM (2); melatonin 20 nM (1.2); melatonin 40 nM (0.8). These data suggest the existence in the rat cerebral cortex of high-affinity ouabain binding sites which may be a locus for the molecular action of melatonin.     

Effects of memantine on recombinant rat NMDA receptors expressed in HEK 293 cells.

1. The actions of the uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists, memantine (1-amino-3,5-dimethyladamantane) and (+)-MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate, dizocilpine), on recombinant NMDA receptors has been studied by use of the whole-cell patch clamp technique. 2. Human embryonic kidney (HEK) 293 cells were transiently transfected with different NMDA receptor subunit combinations (NR1a/NR2A, NR1a/NR2B and NR1a/NR2D). A mutant form of the green fluorescent protein (GFP) cotransfected with the NMDA receptor subunits to enable the visualization of transfected cells. 3. Memantine (0.3-30 microM) blocked L-glutamate (100 microM)-mediated currents in a concentration-dependent manner in NR1a/NR2A, NR1a/NR2B and NR1a/NR2D transfected cells with IC50 values (at -70 mV) of 0.93 +/- 0.15 microM, 0.82 +/- 0.12 microM and 0.47 +/- 0.06 microM (mean +/- s.c. mean), respectively. 4. The memantine-induced block was strongly voltage-dependent. Alteration of the holding potential from -70 mV to +60 mV resulted in an e-fold increase in the IC50 values per 30-33 mV change in membrane potential, for all 3 subunit combinations investigated. 5. The kinetics of the actions of memantine (30 microM) were investigated for the NR1a/2A combination, in 6 cells (13-15 determinations). At -70 mV, the block and recovery from block were both best described by two exponentials with time-constants of 201 +/- 23 ms (81 +/- 2%) and 3.9 +/- 0.6 s and 597 +/- 94 ms (18 +/- 1%) and 18.6 +/- 2.4 s, respectively. The predominant effect of depolarization was to increase the weight of the faster recovery time-constant. Kinetic analysis suggests that these results are consistent with previously proposed Markov models. 6. (+)-MK-801 was studied briefly for comparative purposes. (+)-MK-801 (200 nM) preferentially blocked NMDA receptor currents (at -70 mV) in NR1a/NR2A and NR1a/NR2B (82 +/- 10% and 93 +/- 2% depressions) compared to NR1a/NR2D (38 +/- 7%) transfected cells. (+)-MK-801 appeared to be less voltage-dependent than memantine on all three receptor combinations. 7. In conclusion, memantine was a voltage-dependent antagonist of recombinant rat NMDA receptors expressed in HEK 293 cells but showed little selectivity between the subunits investigated. Its actions on these recombinant receptor combinations are similar to its actions on native NMDA receptors.     

Chronic lithium treatment inhibits basal and agonist-stimulated responses in rat cerebral cortex and GH3 pituitary cells.

Li+ is used clinically in the management of bipolar-disordered (manic-depressive) illness, but the mechanism of its clinical efficacy remains unclear. Li+ inhibits the metabolism of certain inositol phosphates, leading to a decreased cycling of inositol that may be sufficient to reduce phosphoinositide metabolism. We have tested this hypothesis in slices of rat cerebral cortex and in rat pituitary GH3 cells grown in the presence of low extracellular inositol. We show that basal and stimulated mass levels of inositol-1,4,5-trisphosphate were reduced in rat cerebral cortex and in GH3 cells after chronic, but not acute, treatment with a therapeutic concentration of Li+. In GH3 cells chronic treatment with Li+ also decreased basal levels of intracellular Ca2+ and secretion of prolactin, effects that were prevented by the presence of myo-inositol. Agonist-stimulated mobilization of Ca2+ and prolactin release were also reduced in Li(+)-treated cells. These findings show that chronic perturbation of the phosphoinositide pathway by Li+ is sufficient to reduce basal and agonist-stimulated cellular responses, an action that may underlie its effectiveness in the alleviation of affective disorders.     

Age-dependent changes in receptor-stimulated phosphoinositide turnover in the rat hippocampus.

To study the changes in the hippocampal cholinergic system of chronologically old and behaviorally impaired animals, old (21 months of age) and young (3 months of age) male, Fischer-344 rats were used. The aged animals were tested on a reference memory task (Morris water maze) and found to be functionally impaired as compared to the young controls. Carbachol-stimulated phosphoinositide metabolism was measured in hippocampal slices from young and old rats. Slices were prelabeled with 3H-inositol for 120 min and subjected to muscarinic stimulation in the presence of lithium. Following extraction of the slices with acidified solvent mixture, the inositolphosphates present in the aqueous fraction were isolated by ion exchange chromatography. Receptor-stimulated release of inositolphosphates (IPs) was found to be increased in the hippocampus of older animals. This age-related enhancement of IP release was in contrast to the decrease in choline acetyltransferase (CHAT) activity in the hippocampus. We postulate that alterations in the G-protein coupling with the muscarinic receptor leads to an increase in the phosphoinositide turnover in part as a compensatory mechanism for neuronal cell death and reduced transmitter levels.     

Effects of histamine on phosphoinositide metabolism in chick cerebral cortex

Effects of histamine (HA) and agonists of HA receptors on phosphoinositide metabolism in chick cerebral cortex have been studied using two approaches - measurement of inositol 1,4,5-trisphosphate (IP3) level by a specific and sensitive IP3 receptor radioassay, and analysis of [3H]inositol phosphates accumulation in cortical slices prelabeled with myo-[3H]inositol. HA concentration-dependently elevated IP3 levels in slices of chick cerebral cortex. The effect of HA was mimicked by 2-methylHA, a selective agonist of H1-HA receptors, and blocked by mepyramine, an H1 receptor antagonist. 4-MethylHA and Ralpha-methylHA, selective agonists of H2- and H3-HA receptors, respectively, did not affect IP3 level in the chick cerebrum. In cerebral cortical slices prelabeled with myo-[3H]inositol, 2-methylHA significantly stimulated [3H]inositol phosphates accumulation, whereas HA only slightly and non-significantly increased phosphoinositide metabolism. It is suggested that phospholipase C-coupled H1-HA receptors are present in the chick cerebral cortex, yet their number seems to be a small one.     

Depolarisation-evoked release of acetylcholine can mediate phosphoinositide hydrolysis in slices of rat cerebral cortex

Depolarisation of [3H]inositol prelabelled slices of cerebral cortex of the rat, with elevated extracellular K+ or the alkaloid veratrine, induced a marked accumulation of [3H]inositol monophosphate in the presence of 5 mM Li+. The effects of these stimuli were concentration-related with maximal responses obtained at 30 mM K+ and 30 microM veratrine. Larger concentrations produced submaximal responses but also markedly suppressed the incorporation of [3H]inositol into phospholipid. The responses to K+ or veratrine were not sensitive to atropine, prazosin, mepyramine, ketanserin or the peptidase inhibitor bacitracin. However, in the presence of the cholinesterase inhibitor physostigmine, the responses to these stimuli were greatly enhanced and this could be blocked by atropine. Both veratrine and K+ markedly stimulate release of endogenous acetylcholine from the slices. Release appears to be linear with time over the 45 min period of continuous stimulation. Reduction of extracellular calcium severely suppressed both the release of acetylcholine and the atropine-sensitive component of the phosphoinositide response to K+. The results suggest that endogenous acetylcholine can stimulate phosphoinositide metabolism by interacting with muscarinic receptors. The atropine-insensitive component, at least in part, represents entry of Ca2+ through voltage-sensitive channels and perhaps a direct effect on phosphoinositide metabolism.     

Angiotensin II stimulates phosphoinositide turnover in the rat myometrium

Angiotensin II (AII) activates rat myometrium phospholipase C resulting in the hydrolysis of phosphoinositides. The response to AII is concentration- and time-dependent, and it is blocked by saralasin. During pregnancy there is a loss of responsiveness to AII at the site of placental implantation that appears to return during parturition.     

Inhibition of carbachol-stimulated phosphoinositide turnover by U-50,488H in rat hippocampus--involvement of GTP-binding protein.

The effect of U-50,488H, a selective kappa-opioid agonist, on carbachol-stimulated phosphoinositide (PI) turnover response in rat hippocampal slices was examined. U-50,488H which stimulates PI turnover response in this preparation (Periyasamy and Hoss, 1990, Life Sci. 47, 219), inhibited carbachol-stimulated PI turnover in a concentration-dependent manner with an IC50 value of 33 +/- 9.0 microM. The inhibitory effect of U-50,488H was not blocked by the kappa-selective antagonists, e.g., nor-binaltorphimine (10 microM), and MR2266 (10 microM), or tetrodotoxin (1 microM) suggesting that the effect of U-50,488H was mediated neither through the kappa-receptors nor through the release of an endogenous neurotransmitter(s). A Lineweaver-Burke plot of the stimulation of PI turnover by carbachol in the presence and absence of U-50,488H showed that the Km was not changed (11.4 +/- 3.4 and 11.5 +/- 2.6 microM) whereas the Vmax was reduced from 3849 +/- 460 to 1534 +/- 31 cpm indicating that the inhibition was non-competitive. U-50,488H also inhibited guanosine 5'-[beta, gamma-imido]triphosphate (Gpp[NH]p)-stimulated PI turnover in rat hippocampal membranes in a concentration-dependent manner with an IC50 value of 33 +/- 12 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of antidepressants on GABAB and beta-adrenergic receptors in rat cerebral cortex.

The effects of chronic administration of antidepressant drugs on beta-adrenergic and gamma-amino-butyric acid (GABA)B receptors have been assessed with radioligand binding. Tricyclics [imipramine (IMI), 30 mg/kg/day, and desmethylimipramine (DMI), 10 mg/kg day] or monoamine oxidase inhibitors [(+/-)-tranylcypromine (TCP), 1 mg/kg/day, and phenelzine (PLZ), 10 mg/kg/day] were administered to male Sprague-Dawley rats by constant infusion via Alzet 2ML4 osmotic minipumps for 28 days. Pumps were implanted s.c. in the interscapular region. On day 28 the animals were killed and their brains removed; [3H]GABA binding to GABAB receptors was measured in frontal cortex and the remaining cortical tissue was used to measure [3H]dihydroalprenolol ([3H]DHA) binding to beta-adrenoceptors. All drugs tested induced a significant decrease in density (Bmax) of [3H]DHA binding, although no significant changes in affinity (Kd) were observed. [3H]GABA binding was not altered significantly by chronic antidepressant treatment. TCP-treated animals showed a tendency towards increased [3H]-GABA binding, but the differences did not reach statistical significance. No effects on Kd were observed. These data do not support the proposal that an increase in the total population of cortical GABAB receptors is a common effect of chronic antidepressant treatment.