Role of accumbens and cortical dopamine receptors in the regulation of cortical acetylcholine release.

Cortical acetylcholine, under resting and stimulated conditions, was measured in frontoparietal and prefrontal cortex using in vivo microdialysis in freely-moving rats. Cortical acetylcholine efflux was stimulated by systemic administration of the benzodiazepine receptor partial inverse agonist FG 7142. Administration of FG 7142 (8.0 mg/kg; i.p.) significantly elevated acetylcholine efflux in both cortical regions (150-250% relative to baseline) for 30 min after drug administration. The ability of endogenous dopamine to regulate cortical acetylcholine efflux under resting or stimulated conditions and the relative contributions of D1- and D2-like dopamine receptor activation was also assessed. In a first series of experiments, systemic administration of the antipsychotic drug haloperidol (0.15, 0.9 mg/kg, i.p.) blocked FG 7142-stimulated acetylcholine efflux in frontoparietal, cortex while the D1-like antagonist, SCH 23390 (0.1, 0.3 mg/kg), was less effective in attenuating stimulated acetylcholine efflux. In a second series of experiments, the effects of infusions of these antagonists and of the D2-like antagonist sulpiride (10, 100 microM) into the nucleus accumbens were assessed. Infusions of haloperidol and sulpiride significantly blocked FG 7142-stimulated acetylcholine efflux while SCH 23390 did not. By contrast, a third series of experiments demonstrated that perfusion of these antagonists (100 microM) locally into the cortex (through the probe) did not affect FG 7142-stimulated acetylcholine efflux. Moreover, none of these dopamine receptor antagonists, whether administered systemically or perfused into the nucleus accumbens or cortex, affected basal cortical acetylcholine efflux. These results reveal similarities in stimulated cortical acetylcholine release across frontal cortical regions and suggest a prominent role for D2-mediated accumbens dopamine transmission in the regulation of cortical acetylcholine release. The findings provide evidence in support of a neural substrate that links dysregulation of mesolimbic dopaminergic transmission to changes in cortical cholinergic transmission. Dysregulation within this circuit is hypothesized to contribute to the etiology of disorders such as schizophrenia, dementia and drug abuse.     

Cholinergic inputs to the rat medial prefrontal cortex mediate potentiation of the cardiovascular defensive response by the anxiogenic benzodiazephine receptor partial inverse agonist FG 7142

Consistent with its putative anxiogenic actions, administration of the benzodiazepine receptor partial inverse agonist FG 7142 has been shown to potentiate defensive-like cardiovascular reactivity to an acoustic stimulus in the rat, an effect that appears to be mediated by the basal forebrain cholinergic system. The present studies tested the hypothesis that the basal forebrain cholinergic projections to the medial prefrontal cortex, an area that has been implicated in both anxiety and autonomic control, may be a relevant pathway underlying this response potentiation. Infusions of the muscarinic receptor agonist carbachol into the medial prefrontal cortex, but not into the lateral prefrontal cortex or the basolateral amygdala, mimicked the effects of systemically administered FG 7142 on the cardioacceleratory response. Infusions of the muscarinic antagonist atropine blocked this effect, as well as the response-potentiating actions of FG 7142. The effects of FG 7142 were also blocked by lesions of the cholinergic inputs to the medial prefrontal cortex produced by local infusions of the immunotoxin 192 immunoglobulin G-saporin into this area. These findings indicate that cholinergic activation of the medial prefrontal cortex is sufficient to enhance the cardioacceleratory defensive response, and that cholinergic inputs to the medial prefrontal cortex are necessary for the response-potentiating effects of FG 7142. These results are consistent with a recent neurobiological model of anxiety and autonomic control that attributes the enhanced processing of anxiety-related stimuli and contexts to increases in activity in cortical cholinergic inputs.     

Effects of the vasopressin (V1b) receptor antagonist, SSR149415, and the corticotropin-releasing factor 1 receptor antagonist, SSR125543, on FG 7142-induced increase in acetylcholine and norepinephrine release in the rat

Arginine vasopressin and corticotropin-releasing factor are two neuroactive peptides that regulate hypothalamic-pituitary-axis and associated stress response. While the potential antidepressant and anxiolytic profiles of corticotropin-releasing factor 1 antagonists have been well studied, the concept of blockade of vasopressin system as another approach for the treatment of emotional processes has only been made available recently by the synthesis of the first non-peptide antagonist at the V1b receptor, SSR149415. In the present study SSR149415 has been compared with the corticotropin-releasing factor 1 antagonist SSR125543 and with anxiolytic and antidepressant drugs on the response of hippocampal cholinergic and cortical noradrenergic systems to the anxiogenic benzodiazepine receptor inverse agonist FG 7142. Acute (0.3-10 mg/kg, i.p.) and long-term administration (10 mg/kg, i.p., 21 days) of SSR149415 and SSR125543 reduced the FG 7142-induced increase in extracellular concentrations of acetylcholine in the hippocampus of anesthetized rats measured by microdialysis. By contrast acute and long-term administration of SSR149415 failed to reduce the FG 7142-induced increase in the release of norepinephrine in the cortex of freely moving rats. The present results demonstrate that the two compounds have similar profiles in a model of activation by an anxiogenic drug of the hippocampal cholinergic system and they suggest that SSR149415 and SSR125543 may have anti-stress anxiolytic and antidepressant effects via a mechanism of action different from classical benzodiazepine ligands and noradrenergic antidepressants.     

The cardiovascular startle response: Anxiety and the benzodiazepine receptor complex

Benzodiazepine receptor (BZR) agonists are prototypic anxiolytic agents, whereas BZR inverse agonists exert anxio-genic effects. The effects of these compounds offer a potentially important pharmacological model system to examine the central mechanisms of anxiety. In accord with its putative anxiogenic properties, we previously found that the BZR partial inverse agonist, FG 7142, enhances the cardiovascular defensive response to a nonsignal acoustic stimulus in rats. In contrast, we found in the present study that this agent attenuates both the somatic and cardiovascular components of the acoustic startle response. BZR agonists and inverse agonists are known to modulate the basal forebrain cortical cholinergic system, and we consider the potential involvement of this system in the disparate psychophysiological actions of FG 7142 and in anxiety states in general.     

Decreased sensitivity to diazepam induced by chronic administration of FG 7142

Chronic treatment with the beta-carboline inverse agonist FG 7142 (25 mg/kg i.p. twice a day for 15 consecutive days) enhances in rats the effects of proconvulsant and convulsant beta-carbolines and of the inverse agonist Ro 15-4513 whilst leaving unchanged the response to the benzodiazepine receptor antagonists. Moreover, the same treatment reduces the sedative and the anticonvulsant effects of diazepam. These results are consistent with the view that chronic treatment with FG 7142 may produce a decrease in the pharmacological effects of benzodiazepines, whilst inducing sensitization to the convulsant effect of inverse agonists.     

Bi-directional changes in sham feeding in the rat produced by benzodiazepine receptor ligands

Both the real and sham intake of a 5% sucrose solution were increased by midazolam (3 mg/kg IP), a benzodiazepine agonist. In contrast, both the real and sham intakes of a 20% sucrose solution were reduced by Ro15-3505 (a benzodiazepine antagonist with weak inverse agonist properties) and by FG 7142 (a beta-carboline inverse agonist). These data demonstrate that drugs acting at benzodiazepine receptors can bi-directionally alter ingestional responses, probably by modulation of the oropharyngeal control of consumption.     

The abstinence syndrome in diazepam-dependent cats is precipitated by Ro 15-1788 and Ro 15-4513 but not by the benzodiazepine receptor antagonist ZK 93426

Diazepam-dependent cats were challenged with different benzodiazepine recognition site ligands 24 h after the last dose of chronic treatment with diazepam (7 mg/kg, i.p. at 08.00 and 20.00 h, for 21 consecutive days). The benzodiazepine derivatives Ro 15-4513 (a partial inverse agonist) and Ro 15-1788 (a pure antagonist), precipitated an abstinence syndrome within minutes after i.p. administration. Abstinence signs included tremors, increased muscle tone, irritability, fear, pupillary dilation and vocalizations. On the contrary, the beta-carboline derivatives ZK 93426 (an antagonist) and FG 7142 (a partial inverse agonist) failed to precipitate abstinence signs in diazepam-dependent cats when given at doses that prevented the acute effects of diazepam. Our results demonstrate that the ability to induce withdrawal signs in diazepam-dependent cats strongly depends on the benzodiazepine structure of the challenge drug since beta-carboline antagonist and partial inverse agonists lack this property.     

The regulation of high-affinity choline uptake in vitro in rat cortical and hippocampal synaptosomes by beta-carbolines administered in vivo

The effects of several beta-carboline derivatives on sodium-dependent high-affinity choline uptake (HACU) were investigated in rat hippocampus and cerebral cortex. HACU was measured in synaptosomal preparations from these areas after in vivo administration of the drugs. The convulsant, picrotoxin (6 mg/kg), stimulated HACU in both hippocampal and cortical synaptosomes. The convulsant inverse agonist benzodiazepine, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) (5 mg/kg) stimulated hippocampal but not cortical HACU. However, other inverse agonists, methyl-beta-carboline-3-carboxylate (beta-CCM) (20 mg/kg) and ethyl-beta-carboline-3-carboxylate (beta-CCE) (20 mg/kg), stimulated HACU in the cortex but not in the hippocampus. The partial inverse agonist, N-methyl-beta-carboline-3-carboxylate (FG-7142) (20 mg/kg), inhibited cortical HACU and had no effect on hippocampal HACU. The antagonist beta-carboline, 3-hydroxymethyl-beta-carboline (3-HMC) (20 mg/kg), had no effect on either cortical or hippocampal HACU. None of these drugs displayed any effect on HACU when they were incubated directly in vitro with synaptosomal preparations at concentrations up to 100 microM, suggesting their activity is not directly on the cholinergic nerve terminal. The results suggest that beta-carbolines regulate hippocampal and cortical cholinergic activity as do other GABAergic drugs. However, unlike diazepam, which depresses cholinergic activity in both the hippocampus and the cortex the beta-carbolines differentiate between the hippocampus and cortex in their action.     

Effects of FG7142 and immobilization stress on the gene expression in the neocortex of mice

Several psychiatric disorders are often precipitated or exacerbated by exposure to stressors. FG7142 (N-methyl-beta-carboline-3-carboxamide), a partial inverse agonist of benzodiazepine receptors, mimics the physiological (an increased release in the adrenal steroid hormone) and neurochemical (an enhanced neurotransmission of monoamines) changes induced by stressful stimuli. We examined the effects of FG7142 and immobilization stress on the gene expression of the mouse neocortex in order to obtain a new insight into the molecular stress-responsive system. The effect of FG7142 (20 mg/kg, i.p.) on the gene expression of the brain area was examined using a DNA microarray method. The genes showing a significant change in expression were investigated in further experiments using the quantitative RT-PCR method. There was an increase in the mRNA of seven genes in the neocortex of mice 1h after treatment with FG7142. In addition, there was an increase in the mRNAs of five of the seven genes (Fos, Cyr61, Btg2, Adamts1, and Gem) in the neocortex of mice exposed to the stress for 1h. The up-regulation of these five genes by both FG7142 and immobilization stress indicates that these genes may be involved in the stress-responsive system. Dysfunctions of the system may be associated with the pathophysiology of psychiatric disorders.     

Recovery of fear memories in rats: role of gamma-amino butyric acid (GABA) in infantile amnesia

Infantile amnesia is a ubiquitous phenomenon, but its neural bases remain largely unknown. The authors identify a role for GABAergic transmission in suppressing retrieval of memories acquired in infancy. Eighteen-day-old rats received pairings of white noise and shock; considerable forgetting of this experience (assessed by freezing) occurred after 10 days. The memory was recovered by pretest administration of the GABAA inverse agonist FG7142 10 days, but not 2 months, after training. This effect of FG7142 generalized when a passive avoidance procedure was used. Also, FG7142 decreased fear of a latently inhibited conditioned stimulus, showing that the observed memory recovery effect was not due to a state-dependent process. It appears that GABA may be involved in infantile amnesia regardless of the emotional content of the memory.     

A developmental dissociation of context and GABA effects on extinguished fear in rats

Although extinction has attracted considerable attention in recent years, there has been very little empirical work on extinction during development. Using Pavlovian fear conditioning, the authors provide evidence for developmental differences in extinction. Specifically, Postnatal Day (PND) 23 rats exhibited recovery of an extinguished freezing response to an auditory conditioned stimulus when tested in a context different from that in which extinction occurred (i.e., renewal) or when injected with the gamma-amino butyric acid (GABA) inverse agonist FG7142 prior to test. In contrast, PND 16 rats failed to exhibit either of these effects, although a subsequent experiment demonstrated that FG7142 alleviated spontaneous forgetting in PND 16 rats. Taken together, it appears that there are fundamental differences in the processes involved in extinction across development.     

Correlation between regional and kinetic heterogeneities of beta-carboline/benzodiazepine receptor binding in rat brain

Kinetic heterogeneity of [3H]methyl beta-carboline-3-carboxylate (CCM) binding was used to distinguish two populations of benzodiazepine binding sites in synaptic membranes of rat cerebral cortex. Curvilinear dissociation plots of CCM binding revealed major high affinity and minor lower affinity populations with slow and rapid dissociation rates, respectively. beta-Carbolines showed some selectivity to displace the higher affinity [3H]CCM binding. The selectivity decreased in the following order: CCM (inverse agonist), FG 7142 (partial inverse agonist), ZK 93426 (antagonist) and ZK 93423 (agonist). Regional selectivity of displacing potencies of these beta-carbolines on [3H]flunitrazepam binding in cerebellar versus hippocampal membranes decreased similarly.     

Effects of alpha 2-drugs and pilocarpine on the high-voltage spindle activity of young and aged control and DSP4-lesioned rats.

The present study investigates the effects of alpha 2-drugs and pilocarpine on the neocortical high-voltage spindle (HVS) activity in young and aged control and DSP4-lesioned rats. DSP4 partially decreased cortical and thalamic noradrenaline levels, but had no effect on HVS activity. The alpha 2-adrenoceptor agonist guanfacine (0.004, 0.02, 0.1 mg/kg) increased HVS activity in young and aged control and DSP4-lesioned rats. Guanfacine produced a significantly smaller increase in HVS activity in aged rats. A combination of pilocarpine (3 mg/kg), a muscarinic agonist, and atipamezole (1 mg/kg), an alpha 2-adrenoceptor antagonist, suppressed HVS activity more effectively than either of the drugs alone in young or aged control and DSP4-lesioned rats. The present results demonstrate that 1) the alpha 2-adrenoceptor antagonist and muscarinic agonist interact in suppressing HVSs in noradrenergically lesioned young and aged rats; 2) alpha 2-adrenoceptor agonists produce a greater increase in HVS activity in young than aged rats; and 3) partial noradrenergic lesions do not affect the HVS-modulating effects of alpha 2-adrenoceptor active drugs in young or aged rats.     

Multiple effects of drugs acting on benzodiazepine receptors

The actions on [³H]noradrenaline release of chlordiazepoxide and FG 7142 were investigated in rat hippocampal synaptosomes. The release evoked by GABA, an action mediated by GABA_A receptors, was enhanced by chlordiazepoxide and depressed by FG 7142. K⁺-evoked release, however, was depressed by both chlordiazepoxide and FG 7142 and occurred in the absence of GABA. The actions on both GABA-evoked and K⁺-evoked release were blocked by Ro 15-1788. The results suggest that the distinction between agonist and inverse agonist applies to the GABA-dependent but not to the GABA-independent action of benzodiazepines.     

Inhibition of synaptically evoked cortical acetylcholine release by adenosine: an in vivo microdialysis study in the rat

The release of cortical acetylcholine from the intracortical axonal terminals of cholinergic basal forebrain neurons is closely associated with electroencephalographic activity. One factor which may act to reduce cortical acetylcholine release and promote sleep is adenosine. Using in vivo microdialysis, we examined the effect of adenosine and selective adenosine receptor agonists and antagonists on cortical acetylcholine release evoked by electrical stimulation of the pedunculopontine tegmental nucleus in urethane anesthetized rats. All drugs were administered locally within the cortex by reverse dialysis. None of the drugs tested altered basal release of acetylcholine in the cortex. Adenosine significantly reduced evoked cortical acetylcholine efflux in a concentration-dependent manner. This was mimicked by the adenosine A(1) receptor selective agonist N(6)-cyclopentyladenosine and blocked by the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The A(2A) receptor agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne hydrochloride (CGS 21680) did not alter evoked cortical acetylcholine release even in the presence of DPCPX. Administered alone, neither DPCPX nor the non-selective adenosine receptor antagonist caffeine affected evoked cortical acetylcholine efflux. Simultaneous delivery of the adenosine uptake inhibitors dipyridamole and S-(4-nitrobenzyl)-6-thioinosine significantly reduced evoked cortical acetylcholine release, and this effect was blocked by the simultaneous administration of caffeine.These data indicate that activation of the A(1) adenosine receptor inhibits acetylcholine release in the cortex in vivo while the A(2A) receptor does not influence acetylcholine efflux. Such inhibition of cortical acetylcholine release by adenosine may contribute to an increased propensity to sleep during prolonged wakefulness.     

Microdialysis without acetylcholinesterase inhibition reveals an age-related attenuation in stimulated cortical acetylcholine release

Aging-related differences in the ability of cortical cholinergic inputs to respond to local depolarization was assessed in young (3-6 months) and old (26-33 months) awake rats using in vivo microdialysis in the absence of an inhibitor of acetylcholinesterase. Rats were perfused, using a within-subjects, repeated session design, with vehicle (aCSF) or K(+) (25, 50, 100 mM). Perfusion of K(+) resulted in a dose-dependent increase in cortical ACh efflux with comparable efflux seen between the two ages following 25 mM (50%) and 50 mM (100%) K(+). In contrast, aged rats exhibited a marked attenuation (330%) in ACh efflux relative to young adult rats (650%). These data reveal aging-related decreases in the responsiveness of cortical cholinergic afferents, tested under physiologically relevant conditions, to local depolarization and may provide a neuronal mechanism contributing to the cognitive deficits reported in normal aging- and age-related pathological conditions.     

Effects of pharmacological stressors on c-fos and CRF mRNA in mouse brain: relationship to alcohol seeking

A marked heterogeneity exists among stressors in their ability to reinstate alcohol seeking in rats. We have reported that the pharmacological stressor yohimbine, an alpha-2 adrenoceptor antagonist, potently reinstated alcohol seeking, but FG-7142, a benzodiazepine inverse agonist was ineffective. In rats, we determined that yohimbine elicits patterns of brain expression of the mRNAs for c-fos, a marker of neuronal activation, and corticotropin-releasing factor (CRF) a stress-related peptide, distinct from that produced by FG-7142. The purpose of the present experiment is to determine if these differential effects of yohimbine and FG-7142 on regional c-fos and CRF mRNA expression generalize to another animal commonly used in alcohol research, the C57 BL/6J mouse. In comparing the results of the present study to those of our previous one, we found a number of commonalities in the patterns of activation elicited by yohimbine and FG-7142 between the two species, and some notable differences. As we found in the rat, yohimbine selectively increased c-fos mRNA in the mouse NACs, BLA and CeA. Yohimbine increased CRF mRNA only in the mouse PVN, but was without effect on CRF mRNA in extrahypothalamic sites, the BNST and CeA. This differs from what we saw in the rat, where yohimbine increased CRF mRNA in these extrahypothalamic regions, but not the PVN. The selective induction of c-fos in the NACs, BLA and CeA of mice and rats by yohimbine offers further support for the idea that activation of these structures participates in reinstatement induced by such stressors.     

Presynaptic cholinergic mechanisms in brain of aged rats with memory impairments

Presynaptic cholinergic mechanisms were investigated in various brain regions of aged Fisher 344 rats with documented 24 hr retention deficits measured in a single-trial passive avoidance task. Sodium-dependent high affinity choline uptake was found to be decreased by 22% in hippocampus of 23–26 month old animals as compared to 6 month old controls. Prior depolarization of hippocampal or cortical synaptosomes with K⁺ resulted in stimulation of choline uptake which was similar in aged rats and young controls. No age-related differences were observed either in hippocampal, cortical, striatal acetylcholine or choline concentrations, or in the activity of choline acetyltransferase in hippocampus. Synthesis of acetylcholine in hippocampal and cortical slices under basal conditions, as well as under K⁺-stimulated conditions, did not differ in the two age groups examined. These neurochemical findings are consistent with an age-related decrease in hippocampal cholinergic neuronal activity without an actual loss in cholinergic neuron number. It is further suggested that this reduction in cholinergic neuronal activity may be related to the deficit in cognitive performance observed in aged Fisher rats.     

Cyclopyrrolones, unlike some benzodiazepines, do not induce physical dependence in mice

In a model of physical dependence in mice, treatment with cyclopyrrolones such as zopiclone and suriclone (from 4 to 400 mg/kg/day), did not modify the sensitivity of the gamma-aminobutyric acid (GABA) receptor complex to the partial inverse agonist FG 7142 following their withdrawal, whereas sensitivity changes were observed after treatment and withdrawal from some benzodiazepines (e.g. lorazepam, diazepam, flunitrazepam and triazolam). These data suggest that, in contrast to some benzodiazepines, zopiclone and suriclone may not produce physical dependence.     

The effects of FG7142 on two types of forgetting in 18-day-old rats

The authors studied the role of gamma-aminobutyric acid (GABA) in 2 types of forgetting of fear in the developing rat. One type of forgetting studied was that observed after an intermediate retention interval (the "Kamin effect"); the other type studied was that observed after a longer interval (infantile amnesia). Rats were given pairings of an auditory conditioned stimulus with shock, and learned fear was assessed by freezing. Forgetting at an intermediate retention interval (1 hr) was not alleviated by the GABA-sub(A) receptor partial inverse agonist FG7142 (0, 1, 5, or 10 mg/kg), whereas forgetting at a longer retention interval (48 hr) was alleviated. These results suggest that in the developing rat, forgetting observed at different retention intervals is mediated by different physiological mechanisms.