Paradoxical stimulation of cyclooxygenase-2 expression by glucocorticoids via a cyclic AMP response element in human amnion fibroblasts.

Human amnion fibroblasts produce abundant prostaglandins toward the end of gestation, which is one of the major events leading to parturition. In marked contrast to its well-described antiinflammatory effect, glucocorticoids have been shown to up-regulate cyclooxygenase-2 (COX-2) expression in human amnion fibroblasts. The mechanisms underlying this paradoxical induction of COX-2 by glucocorticoids have not been resolved. Using cultured human amnion fibroblasts, we found that the induction of COX-2 mRNA expression by cortisol was a glucocorticoid receptor (GR)-dependent process requiring ongoing transcription. Upon transfection of a COX-2 promoter-driven reporter gene into the amnion fibroblasts, cortisol stimulated the COX-2 promoter activity. This was abolished by mutagenesis of a cAMP response element (CRE) at -53 to approximately -59bp as well as by cotransfection of a plasmid expressing dominant-negative CRE-binding protein (CREB). The phosphorylation level of CREB-1 was significantly increased by cortisol treatment of the amnion fibroblasts, whereas the effect was attenuated either by the protein kinase A inhibitor H89 or the p38 -MAPK inhibitor SB203580. The induction of the COX-2 promoter activity and the phosphorylation of CREB-1 were also blocked by the GR antagonist RU486. Chromatin immunoprecipitation (ChIP) assay revealed that the binding of CREB-1 to the CRE of the COX-2 promoter was increased by cortisol treatment of the amnion fibroblasts. In conclusion, cortisol, via binding to GR, stimulated COX-2 expression by increasing phosphorylated CREB-1 binding to the CRE of the COX-2 gene. Cortisol may phosphorylate CREB-1 by activating either protein kinase A or p38-MAPK in the amnion fibroblasts.

Expression of progesterone receptor A form and its role in the interaction of progesterone with cortisol on cyclooxygenase-2 expression in amnionic fibroblasts.

CONTEXT: Human amnion fibroblasts produce abundant prostaglandins toward the end of gestation, which is believed to be one of the major events leading to parturition. Glucocorticoids have been shown to up-regulate cyclooxygenase-2 (COX-2) expression, the crucial enzyme catalyzing prostaglandin synthesis, in human amnion fibroblasts. Although a major propregnancy hormone, the effect of progesterone and the associated progesterone receptor subtypes in the regulation of both basal and glucocorticoid-induced COX-2 expression in human amnion fibroblasts have not been resolved. METHODS AND RESULTS: Cultured human amnion fibroblasts prepared from the fetal membranes at term pregnancy without labor mainly expressed the progesterone receptor A form (PRA). Inhibition of endogenous progesterone production with trilostane or knockdown of PRA expression with small interfering RNA significantly enhanced the glucocorticoid receptor (GR)-mediated COX-2 induction by cortisol, whereas overexpression of PRA attenuated the induction by cortisol. Co-immunoprecipitation assay revealed PRA in the GR protein complex. Although exogenous progesterone did not alter COX-2 expression under basal conditions, it attenuated cortisol-induced COX-2 expression at concentrations about 10- to 50-fold higher, which might be achieved by competition with cortisol for GR. CONCLUSIONS: We demonstrated in this study that endogenous progesterone might counteract the induction of prostaglandin synthesis by cortisol via PRA transdominant repression of GR function, whereas high levels of progesterone might further inhibit the induction by cortisol via competitive binding to GR in human amnion fibroblasts. These inhibitory actions of progesterone and PRA on glucocorticoids and GR may partly explain the inconsistent effects of glucocorticoids on parturition in humans.

Benzodiazepine impairment of perirhinal cortical plasticity and recognition memory.

Benzodiazepines, including lorazepam, are widely used in human medicine as anxiolytics or sedatives, and at higher doses can produce amnesia. Here we demonstrate that in rats lorazepam impairs both recognition memory and synaptic plastic processes (long-term depression and long-term potentiation). Both impairments are produced by actions in perirhinal cortex. The findings thus establish a mechanism by means of which benzodiazepines impair recognition memory. The findings also strengthen the hypotheses that the familiarity discrimination component of recognition memory is dependent on reductions in perirhinal neuronal responses when stimuli are repeated and that these response reductions are due to a plastic mechanism also used in long-term depression.

Cholinergic depletion by IgG192-saporin retards development of rat barrel cortex.

This study examines the role of cholinergic projections from the basal forebrain on development of the rodent barrel cortex. Pups were administered the immunotoxin IgG192-saporin (0.1 microg) intraventricularly at postnatal day (P) 0 and sacrificed at P1-P7. One ventricle was injected with saporin while the other side received saline, allowing comparison between the two sides of the same animal, as well as with controls receiving saline only. Compared to control animals, neuronal loss in the basal forebrain was present on both sides of saporin-treated pups but was significantly greater on the toxin-treated side, in all age groups and regions sampled. Depletion of acetylcholine did not prevent the formation of the barrel pattern, however it delayed its emergence by approximately 1 day. At P4, the thickness of layer IV barrel cortex was also significantly reduced; this reduction was undetectable by P7. From P3 to P5, the ratios of intensity of staining for acetylcholinesterase between the barrel centres and septa on the toxin-treated side were significantly lower than those on the saline side, although normal densities were present by P7. Thus, the depletion of cholinergic innervation at birth causes a transient delay in the development of the barrel pattern during the first postnatal week.

IP3-gated channels and their occurrence relative to CNG channels in the soma and dendritic knob of rat olfactory receptor neurons.

Olfactory receptor neurons respond to odorants with G protein-mediated increases in the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) and/or inositol-1,4,5-trisphosphate (IP3). This study provides evidence that both second messengers can directly activate distinct ion channels in excised inside-out patches from the dendritic knob and soma membrane of rat olfactory receptor neurons (ORNs). The IP3-gated channels in the dendritic knob and soma membranes could be classified into two types, with conductances of 40 +/- 7 pS (n = 5) and 14 +/- 3 pS (n = 4), with the former having longer open dwell times. Estimated values of the densities of both channels from the same inside-out membrane patches were very much smaller for IP3-gated than for CNG channels. For example, in the dendritic knob membrane there were about 1000 CNG channels x microm(-2) compared to about 85 IP3-gated channels x microm(-2). Furthermore, only about 36% of the dendritic knob patches responded to IP3, whereas 83% of the same patches responded to cAMP. In the soma, both channel densities were lower, with the CNG channel density again being larger ( approximately 57 channels x microm(-2)) than that of the IP3-gated channels ( approximately 13 channels x microm(-2)), with again a much smaller fraction of patches responding to IP3 than to cAMP. These results were consistent with other evidence suggesting that the cAMP-pathway dominates the IP3 pathway in mammalian olfactory transduction.

Ion permeation and selectivity of wild-type recombinant rat CNG (rOCNC1) channels expressed in HEK293 cells.

The permeation properties of adenosine 3', 5'-cyclic monophosphate (cAMP)-activated recombinant rat olfactory cyclic nucleotide-gated channels (rOCNC1) in human embryonic kidney (HEK 293) cells were investigated using inside-out excised membrane patches. The relative permeability of these rOCNC1 channels to monovalent alkali cations and organic cations was determined from measurements of the changes in reversal potential upon replacing sodium in the bathing solution with different test cations. The permeability ratio of Cl(-) relative to Na(+) (P(Cl)/P(Na)) was about 0.14, confirming that these channels are mainly permeable to cations. The sequence of relative permeabilities of monovalent alkali metal ions in these channels was P(Na) > or = P(K) > P(Li) > P(Cs) > or = P(Rb), which closely corresponds to a high-strength field sequence as previously determined for native rat olfactory receptor neurons (ORNs). The permeability sequence for organic cations relative to sodium was P(NH3OH) > P(NH4) > P(Na) > P(Tris) > P(Choline) > P(TEA), again in good agreement with previous permeability ratios obtained in native rat ORNs. Single-channel conductance sequences agreed surprisingly well with permeability sequences. These conductance measurements also indicated that, even in asymmetric bi-ionic cation solutions, the conductance was somewhat independent of current direction and dependent on the composition of both solutions. These results indicate that the permeability properties of rOCNC1 channels are similar to those of native rat CNG channels, and provide a suitable reference point for exploring the molecular basis of ion selectivity in recombinant rOCNC1 channels using site-directed mutagenesis.

The role of heat shock proteins in mammalian differentiation and development.

Heat shock proteins (HSPs) have been identified in all cells, prokaryotic and eukaryotic, to protect the cells from harmful insults and stress. Increased HSP synthesis can also result during normal cellular functions and also respond to exposure from environmental stress and infection. Although the molecular mechanisms responsible for HSP cellular protection are still not fully understood, their expression is critical for cellular survival and can be modified by cell signal transducers such as intracellular pH, cyclic AMP, Ca2+ Na+, inositol [correction of inostitol] triphosphate, protein kinase C, and protein phosphates. Most HSPs interact, assemble, fold, unfold, bind, transport, translocate and 'chaperone' other proteins in the cell and alter their function.

Cholinergic depletion reduces plasticity of barrel field cortex.

This experiment examines the impact of the cholinergic input from the basal forebrain on the plasticity of the vibrissa-related somatosensory cortex. Newborn rat pups received intraventricular injections of the cholinergic immunotoxin IgG192-saporin, after bilateral removal of the C-line whisker follicles. Compared with saline-injected control animals, unilateral injections of 0.1 microg IgG192-saporin decreased the number of cholinergic neurons on the toxin injected side by 78% in the basal nucleus of Meynert and the vertical limb of the diagonal band of Broca, 80% in the magnocellular preoptic nucleus and the horizontal limb of the diagonal band of Broca, and 54% in the medial septal nucleus. Neuronal loss contralateral to the toxin was approximately half that on the ipsilateral side. The size of the C and D row barrels were compared from tangential sections through the barrel field. In control animals, D row barrels expanded into C row territory, giving a ratio of areas for D/C barrels of 2.03. Depletion of the cholinergic neurons reduced the expansion of D row barrels and hence decreased the D/C ratio, with a greater reduction on the toxin-treated side (1.43, P <0.005) compared with the contralateral side (1.64, P <0.05). This study implicates the basal forebrain cholinergic projection in somatosensory cortical plasticity.

Differential activation of the rat hippocampus and perirhinal cortex by novel visual stimuli and a novel environment.

Two groups of rats were shown individual novel visual objects. One group had been familiarised to the environmental context within which the objects were shown, the other experienced the situation for the first time. The activation of neurones in perirhinal cortex and the hippocampal formation was determined by counts of nuclei stained for products of the immediate early gene c-fos. The ratio of counts in the hippocampal formation to that in perirhinal cortex was compared for the two groups: the ratio was significantly higher (4.2:1) in the group experiencing the environment for the first time. Thus, whereas perirhinal neurones are activated by novel rather than familiar objects, hippocampal neurones are preferentially activated by a novel rather than a familiar environment.

Mapping visual recognition memory through expression of the immediate early gene c-fos.

To study brain regions involved in familiarity discrimination, rats were shown sets of novel and familiar objects. On each trial two objects were shown simultaneously to a rat so that one eye saw a novel object while the other saw a familiar object. Thus novel and familiar objects were seen with the same conditions of alertness and eye movements. Activated neurones were revealed by staining for products of the immediate early gene c-fos. Familiar stimuli activated significantly fewer neurones than novel stimuli in perirhinal cortex and area TE of temporal cortex, and the ventral lateral geniculate nucleus of the thalamus, but not in the hippocampus or other areas sampled. These findings are discussed in relation to recognition memory.

Effects of the novelty or familiarity of visual stimuli on the expression of the immediate early gene c-fos in rat brain.

To investigate substrates of recognition memory, the cellular expression of Fos protein in rat brain has been studied after groups of rats were either shown sets of novel or highly familiar objects, or were exposed to the same pattern of illumination without objects being shown. Counts of stained nuclei were made in eight brain regions, where information about novel or familiar visual stimuli is likely to be processed or stored. The counts were relatively high in occipital visual association cortex and area TE of temporal cortex, intermediate in perirhinal cortex, entorhinal cortex, anterior cingulate cortex and the diagonal band of Broca, and low in the hippocampal formation and mediodorsal nucleus of the thalamus. The number of Fos-stained cells was significantly higher for the rats shown novel objects than for those shown familiar objects in perirhinal cortex, area TE, occipital cortex and anterior cingulate cortex. Arguments are advanced that these differences in counts indicate areas involved in the processing and/or storage of information about the novelty or familiarity of visual stimuli, information important to recognition memory.

Changes in neuronal activity related to the repetition and relative familiarity of visual stimuli in rhinal and adjacent cortex of the anaesthetised rat.

Employing the same techniques as have been used with conscious rats, this study describes neuronal responses signalling information concerning the prior occurrence of visual stimuli in unconscious rats. Recordings of the activity of 387 neurons were made while anaesthetised rats were shown objects. Changes in neuronal responses related to stimulus repetition and the relative familiarity of visual stimuli were sought. The areas sampled were lateral occipital cortex, area TE of temporal cortex, perirhinal cortex and the hippocampal formation. The response to the first presentations of unfamiliar objects was significantly different from that to their second presentations for 30 (35%) of 86 visually responsive neurones; for 23 of the neurones the response was smaller when the stimulus was repeated, whereas for 7 it was larger. For all of these neurones the response change was maintained across intervening trials on which other stimuli were shown. For 4 (25%) of 16 neurones so tested, the response decrement persisted across at least 10 intervening trials. The activity of 63 neurones was recorded while rats were shown highly familiar as well as unfamiliar objects. The response to unfamiliar objects was significantly different from that to highly familiar objects for 3 (23%) of 13 visually responsive neurones. The types of neuronal response and their incidence expressed as a proportion of the number of visually responsive neurones were similar to those found in unanaesthetised rats (though the proportion of visually responsive neurones encountered in the anaesthetised rat was lower). The results indicate that information concerning the prior occurrence of stimuli is processed even under anaesthesia.

Neuronal signalling of information important to visual recognition memory in rat rhinal and neighbouring cortices.

This study was conducted to discover whether the rat cortex contains neurons that signal information concerning the previous occurrence of stimuli, as has been found in the primate. Recordings of the activity of 396 single neurons were made while unanaesthetized rats were shown objects. The effects on neuronal responsiveness of stimulus repetition and of the relative familiarity of the stimuli were sought. The areas sampled were the rhinal (entorhinal and perirhinal) cortex, area TE of the temporal cortex, the lateral occipital cortex and the hippocampal formation. The response to the first presentations of objects was significantly different from that to their second presentations for 63 (34%) of the 185 responsive neurons; for 39 of the neurons the response was smaller when the stimulus was repeated, whereas for 24 it was larger. The incidence of decremental responses was higher in the non-hippocampal cortex than in the hippocampal formation, while the incidence of incremental responses was higher in the hippocampal formation than other cortical areas. The response to unfamiliar objects was significantly different from that to highly familiar objects for 15 (22%) of 67 responsive neurons so tested; for 12 of the neurons the response was smaller when the stimulus was repeated, and for three it was larger; most of these neurons were found in area TE. The responses of ten familiarity neurons varied significantly with the relative familiarity of the stimuli but not with stimulus repetition; the responses of seven recency neurons varied significantly upon stimulus repetition but not with the relative familiarity of the stimuli. Thus information concerning stimulus repetition and familiarity is separably encoded at the single neuron level in the rat cortex. The results demonstrate that in the rat cortex as in the monkey cortex there are neurons that signal information concerning the prior occurrence of stimuli; such information is of importance to recognition memory, working memory and priming memory.

Minimal changes with long-term administration of anxiolytics on septal driving of hippocampal rhythmical slow activity.

In free-moving male rats, the function relating frequency to the threshold current required to drive hippocampal rhythmical slow activity (RSA; "theta") with septal stimulation has a minimum at 7.7 Hz. Classical anxiolytics all increase thresholds in the region of 7.7 Hz, and so does the novel anxiolytic buspirone. However, unlike classical anxiolytics, 2 or 3 weeks are normally required for the onset of the clinical effects of buspirone. This study tested the effects of long-term administration of chlordiazepoxide and buspirone on septal driving of RSA. Separate groups of naive rats received three IP injections per day of chlordiazepoxide (0.4 mg/kg), buspirone (0.1 mg/kg) or saline for 50 days. Both chlordiazepoxide and buspirone increased thresholds at 7.7 Hz, as expected. These acute effects were not significantly changed with chronic administration. Buspirone and chlordiazepoxide produced similar, statistically significant, but small cumulative reductions in thresholds at 6.9 Hz. The present experiments suggest that if the effects of anxiolytic drugs on septally driven RSA provide any basis for their clinical action, then classical anxiolytics may have two actions: an immediate effect on euphoria and tension and a delayed effect on anxiety proper--with buspirone sharing only the latter effect.

Effects of long-term administration of phenelzine on reticular-elicited hippocampal rhythmical slow activity.

All anxiolytics so far tested show a common reduction in the frequency of reticular-elicited hippocampal rhythmical slow activity (RSA). Acute administration of the tricyclic antidepressant imipramine shares this effect with anxiolytics. The present experiment tested whether the MAO inhibitor antidepressant phenelzine shares this common effect of anxiolytics and imipramine on hippocampal RSA. Rats, implanted with reticular stimulating electrodes and subicular recording electrodes, received four acute doses (0.2, 2.0, 6.0 and 18 mg/kg) or one chronic dose (2 mg/kg/day for 35 days) of phenelzine. Acute administration of phenelzine failed to systematically affect RSA frequency. Chronic injections of phenelzine eventually produced a reduction in RSA frequency combined with a gradual increase in baseline RSA frequency. The absence of immediate action and the production of a chronic reduction in RSA frequency are distinct from the documented effects of anxiolytics and imipramine, whereas the increase in baseline RSA frequency is similar to imipramine. The different influence of phenelzine on RSA frequency compared with anxiolytics (including imipramine) is consistent with the different clinical profiles of these drugs.

Similar effects of buspirone and chlordiazepoxide on a fixed interval schedule with long-term, low-dose administration.

Buspirone is a novel anxiolytic which does not share the muscle relaxant, anticonvulsant and sedative properties of classical anxiolytics such as the benzodiazepines. Its effects on behavioural tests of anxiolytic action generally match those of classical anxiolytics provided a low dose is used. However, in a previous experiment, buspirone appeared to affect fixed interval responding in a way which differed qualitatively as well as quantitatively from the classical anxiolytic chlordiazepoxide. It takes as much as 2 weeks for the clinical effects of anxiolytics to develop, during which time the side effects of benzodiazepines undergo tolerance. We, therefore, decided to compare long-term pre-administration (60 days, three injections/day) of buspirone and chlordiazepoxide on learning of a fixed interval 60-s schedule. The doses were based on previous acute dose-response tests of hippocampal theta rhythm in freely moving animals. Buspirone (0.1 mg/ kg i.p.) and chlordiazepoxide (0.4 mg/kg i.p.) produced similar increases in responding, especially in the middle of acquisition of the fixed interval schedule. Consistent with our acute electrophysiological tests, the effects of 0.4 mg/kg chlordiazepoxide were somewhat larger than those of 0.1 mg/kg buspirone. These results suggest that the acute effects of buspirone, but probably not chlordiazepoxide, on fixed interval responding are contaminated by side effects which do not seriously affect the results with long-term administration. The effects of both novel and classical anxiolytics on control of hippocampal theta rhythm appear to predict the magnitude of their common anxiolytic effects and to be unrelated to their different side effects.

The interaction of serotonin depletion with anxiolytics and antidepressants on reticular-elicited hippocampal RSA.

Hippocampal rhythmical slow activity (RSA) can be elicited by stimulation of the midbrain reticular formation. Buspirone, chlordiazepoxide and imipramine are all anxiolytic and have all been shown to decrease the frequency of RSA. All these compounds have been suggested to affect, directly or indirectly, 5-HT metabolism and function. The present experiments tested the possibility that buspirone, chlordiazepoxide and imipramine reduce RSA frequency via 5-HT1A autoreceptors. Rats received buspirone (10 mg/kg), chlordiazepoxide (5 mg/kg) and imipramine (30 mg/kg) after 5-HT depletion with p-chlorophenylalanine (pCPA, 100 mg/kg/day for 3 days or 350 mg/kg/day for 2 days) or after pretreatment with 5-HTP (40 mg/kg, to replete 5-HT) as well as pCPA. The frequency-reducing effects produced by buspirone and chlordiazepoxide were unchanged by either dose of pCPA, whereas the frequency-reducing effect of imipramine was completely eliminated by the high dose of pCPA. Pindolol, but not beta-blockers (a combination of metoprolol and ICI118,551), was able to block the effect of imipramine on RSA frequency. Pindolol has been reported to block the effects of buspirone but not chlordiazepoxide. These data suggest that: (1) buspirone obtains its frequency-reducing effects via pre- or post-synaptic 5-HT1A receptors rather than 5-HT1A autoreceptors; (2) chlordiazepoxide obtains its frequency-reducing effect via benzodiazepine receptors and GABA with no direct or indirect involvement of 5-HT systems; and (3) imipramine obtains its frequency-reducing effect by increasing the availability of 5-HT at 5-HT1A receptors which are not autoreceptors.

Effects of long-term administration of antidepressants on septal driving of hippocampal RSA.

All classical anxiolytics raise the thresholds of septal-elicited hippocampal RSA overall, but do so mostly at 7.7 Hz (130 ms). The novel anxiolytic/antidepressant buspirone shows partial similarity with classical anxiolytics on septal driving thresholds. These effects of both the classical and novel anxiolytics are unchanged by long-term administration. The present experiment tested whether the tricyclic antidepressant imipramine and the monoamine oxidase inhibitor antidepressant phenelzine share these common effects of classical and novel anxiolytics with long-term administration. Rats, implanted with septal stimulating electrodes and subicular recording electrodes, received 15 mg/kg imipramine (twice per day) and 2 mg/kg phenelzine (once per day) for 28 days. Chronic administration of imipramine mimicked the documented effects of anxiolytics while chronic administration of phenelzine produced essentially opposite effects to the effects of anxiolytics on septal driving of RSA. Since both acute and chronic administration of imipramine but not phenelzine also produce similar effects to anxiolytics on the frequency of reticular-elicited hippocampal RSA, we suggest that (1) imipramine has a separate anxiolytic action, in addition to its antidepressant action; and (2) phenelzine may have no central anxiolytic action despite its capacity to relieve somatic symptoms in atypical depression.

A comparison of the acute effects of a tricyclic and a MAOI antidepressant on septal driving of hippocampal rhythmical slow activity.

In free-moving male rats, the function relating frequency to the threshold current required to drive hippocampal rhythmical slow activity (RSA) with septal stimulation has a minimum at 130 ms. Both classical anxiolytics (e.g. benzodiazepines) and the novel anxiolytic buspirone show similar effects on septal driving of RSA. The tricyclic antidepressant imipramine may be as effective as anxiolytic drugs in treatment of generalized anxiety disorder. The antidepressant monoamine oxidase inhibitor phenelzine has also been reported to be effective in treating anxiety, but this may reflect an action on "atypical depression" rather than "anxiety". The present study therefore compared the effects of acute administration of imipramine and phenelzine on septal driving of RSA to determine whether either would mimic anxiolytics in this test. Rats were chronically implanted with septal stimulating electrodes and subicular recording electrodes. Three groups of rats received IP injection of either imipramine (5.9-13.3 mg/kg or 13.3-30 mg/kg) or phenelzine (0.2-5.4 mg/kg). The effects produced by imipramine were very similar to the effects produced by anxiolytic drugs. In contrast, the effects produced by phenelzine were essentially opposite to those of both anxiolytic drugs and imipramine. The present experiment suggests that imipramine may act as a true anxiolytic, in addition to its conventional antidepressant properties. In contrast, phenelzine may be effective in cases where the etiology is essentially that of depression even when the symptomatology appears to be that of anxiety.

Effects of long-term administration of anxiolytics on reticular-elicited hippocampal rhythmical slow activity.

Buspirone is effective in treating clinical anxiety but, unlike classical anxiolytics, does not have anti-convulsant, sedative or muscle relaxant side-effects and does not interact with GABA. Buspirone may also differ from classical anxiolytics in requiring a period of 2 weeks or more to achieve its full therapeutic action. It has previously been shown that all anxiolytic drugs, including buspirone, reduce the frequency of reticular-elicited hippocampal rhythmical slow activity (RSA). The present experiments tested whether the time course of the effect of buspirone on rhythmical slow activity differed from that of the anxiolytic benzodiazepine chlordiazepoxide. Rats, implanted with reticular stimulation electrodes and subicular recording electrodes, received three intraperitoneal injections per day of buspirone (2.5 mg/kg), chlordizepoxide (5 mg/kg) or saline for 45 days. Both buspirone and chlordiazepoxide reduced the frequency of rhythmical slow activity on the first day of testing and Ro15-1788 (10 mg/kg) blocked the effects of chlordiazepoxide but not buspirone. There was no increase in the effect of buspirone with time. These results showed that, if the effect of anxiolytic drugs on rhythmical slow activity provides any basis for their clinical action, then some additional factors are required to explain both the delayed action of buspirone and the immediate action of classical anxiolytic drugs.