Gene regulation in the frontal cortex of rats exposed to the chronic mild stress paradigm, an animal model of human depression.

In the present study, we have coupled the chronic mild stress (CMS) protocol with Affymetrix microarray technology to screen the rat genome for gene changes in the frontal cortex. The aim of our work was to assess whether the CMS protocol could be a useful experimental model to provide insights into the molecular basis of depression. Under our experimental conditions, 59 transcripts changed by more than +/-1.5-fold between naïve and anhedonic rats and showed significantly altered expression levels (P < 0.05). Among these, 18 were upregulated (fold change range +1.509 to +3.161) and 41 were downregulated (fold change range -1.505 to -2.659). To confirm the data obtained with microarrays, we used real-time reverse transcription polymerase chain reaction (RT-PCR). The results confirmed the downregulation of Itga6, Camk2a, Plcb1, Cart, Gad1, Homer1 and Th and the upregulation of Egr2 and Ptgs2 observed in the DNA microarray analysis. Moreover, the fold change data of the nine validated transcripts from microarray analysis and real-time polymerase chain reaction showed a good correlation (r = 0.863, 7 d.f., P < 0.01; slope = 0.976). It is of great interest that prostaglandin-endoperoxide synthase 2, tyrosine hydroxylase, Cart, Homer1 and glutamate decarboxylase have already been implicated in affective disorders by different approaches in previous reports. In conclusion, our findings indicate that the CMS paradigm is a useful preclinical model with which to investigate the molecular basis of anhedonia and to help in the discovery of novel targets for antidepressant drugs.

Histamine H3-receptor blockade in the rat nucleus basalis magnocellularis improves place recognition memory.

RATIONALE: Several lines of evidence have indicated that the central histaminergic system might be involved in learning and memory OBJECTIVES: The aim of the present study was to ascertain the impact on memory processes of putative histaminergic-cholinergic interactions in the nucleus basalis magnocellularis (NBM) of the rat. METHODS: The effects of thioperamide, a histamine H3-receptor antagonist, were studied on the memory performance of rats in a two-trial, delayed, place-recognition task. The drug was injected into the NBM area 2 min prior to the first trial (1.5, 7.5, and 37.5 ng/0.5 microl; pre-acquisition treatment), within 30 s from the end of the first trial (0.3, 1.5, 7.5, and 37.5 ng/0.5 microl; post-acquisition treatment), or 2 min prior to the second trial (1.5, 7.5, and 37.5 ng/0.5 microl; pre-retrieval treatment). RESULTS: Post-acquisition intra-NBM injections of 1.5 ng and 7.5 ng, but not of 0.3 ng and 37.5 ng thioperamide, significantly enhanced memory retention in treated rats. The histamine H(3)-receptor blocker exerted pro-cognitive effects only when administered post-acquisition, since both pre-acquisition and pre-retrieval treatments were ineffective. The post-acquisition effect of the drug was time dependent and disappeared when the drug was injected 90 min after the end of the first trial. The U-shaped dose-response relationship and the time dependency of the effect of thioperamide indicated that the drug acts on mechanisms involved in memory consolidation. CONCLUSIONS: The present findings demonstrate that the pro-cognitive effect of thioperamide is probably due to the modulation of post-acquisition memory processes through an action on the cholinergic basal forebrain. Our results indicate also that H3-antagonists may provide a useful approach for improving spatial recognition memory.

Histamine H(3)-receptor antagonism improves memory retention and reverses the cognitive deficit induced by scopolamine in a two-trial place recognition task.

Several reports have indicated that, under different experimental conditions, the administration of histamine H(3)-receptor antagonists exerts procognitive effects by activating central histaminergic transmission. In the present study the action of thioperamide, a H(3)-receptor blocker, is investigated on consolidation and recall mechanisms of the rat place recognition memory. The animals have been tested on a two-trial delayed comparison paradigm in a Y-maze. Thioperamide enhances the memory retention when administered intraperitoneally (i.p.) post-acquisition (0.7 and 5.0 mg/kg are ineffective, whereas the dose of 2.0 mg/kg improves memory) but does not affect the rat performance when injected 45 min prior to the testing trial. The post-acquisition effect of thioperamide is time-dependent since the administration of the drug 30 min after the end of the training trial has no effect on memory. In addition, thioperamide reverses the amnesia induced by the post-acquisition treatment with 0.02 mg/kg i.p. of scopolamine (SCOP). The procognitive effect of thioperamide is not modified by the contemporary administration of pyrilamine, an histamine H(1)-receptor antagonist. On the contrary, the blockade of H(2)-receptors by zolantidine 10 mg/kg reverses both the effect of thioperamide alone and the drug action on the scopolamine-induced memory deficit. The results indicate that the neuronal histamine released in consequence of the post-acquisition thioperamide treatment improves place recognition memory through the activation of postsynaptic H(2)-receptors.

Effect of R-(-)-alpha-methylhistamine and thioperamide on in vivo release of norepinephrine in the rat hippocampus.

1. The modifications of hippocampal release of norepinephrine following the administration of R-(-)-alpha-methylhistamine and thioperamide, respectively agonist and antagonist of histamine H3 receptors, were assessed in freely moving rats by microdialysis. 2. Both the systemic (2 mg/kg i.p.) and local (100 microM via the probe) administration of thioperamide caused no modifications of basal release, indicating that the histaminergic system is not tonically involved in regulating the hippocampal noradrenergic activity. 3. R-(-)-alpha-methylhistamine (1 and 100 microM) produced a slight, short-lasting and dose-dependent reduction of norepinephrine release antagonized by local perfusion (100 microM) and prevented by systemic administration of thioperamide 2 mg/kg. 4. The results seem to indicate that the modulation of norepinephrine release through presynaptic H3-receptors in the rat hippocampus plays a minor role in the memory-enhancing effects of thioperamide.

Sex differences in memory performance in the object recognition test. Possible role of histamine receptors.

The mnemonic performances of male and female rats were compared in an object recognition test. Females were still able to recognize a previously identified object after a 90-min between-trial interval, compared with only 60 min in the males. Because histamine (HA) involvement in memory processes has been strongly suggested, the effect of H3-HA autoreceptor antagonist thioperamide was investigated. This drug was found to produce a dose-dependent promnestic effect in both sexes, but it did not influence the time course of memory retrieval. These behavioral data were compared to the density of H1-HA, H2-HA, and H3-HA receptors in cortical membranes. The densities of H1-HA and H2-HA receptors were greater in the females, whereas that of H3-HA was substantially the same in both sexes. The behavioral effect of thioperamide was very similar in both sexes, and this agrees with a similar H3-HA receptor density; however a better memory performance might have been expected in the female after thioperamide treatment (in view of different H1-HA and H2-HA receptor density), but this was not found. Because thioperamide has also been demonstrated to influence the acetylcholine release, its possible role in regulating the cholinergic memory effect was investigated. The scopolamine-reduced visual retrieval was antagonized by thioperamide in a similar way in both sexes. In conclusion, these data have shown a better performance of the female in a visual memory test, but this behavioral difference could not be affected by an H3-HA receptor-dependent manipulation of histaminergic and cholinergic systems.

Behavioral and neurochemical effects of a chronic choline-deficient diet in the rat.

Chronic administration of a choline-deficient diet for 20 days caused no modification in ACh levels in the cortex, hippocampus and olfactory system of the rat and an increase in the density (Bmax) of the muscarinic receptors in the hippocampus. The choline-deficient diet caused no modification in noradrenaline levels and a reduction in the density of alpha1-adrenoceptors in the cortex, hippocampus and olfactory system. This paper discusses the possibility that these neurochemical effects are correlated with the reduction in spontaneous memory decay which was observed in the staircase maze after 20 days of a choline-deficient diet.

Behavioral and neurochemical effects induced by chronic L-DOPA administration.

L-DOPA, in combination with benserazide, in the ratio 4:1 (w/w), was administered orally to rats. In the staircase maze test a low dose of L-DOPA (3 mg/kg/day) reduced the increase in errors caused by 20 days interruption of daily training, while a higher dose (30 mg/kg/day) was ineffective. A decrease in levels of dopamine in the olfactory system and DOPAC in the striatum was seen at all tested doses of L-DOPA, while an increase in 5-HT levels was seen in the hippocampus and in the striatum. 5-HIAA levels did not change. Levels of ACh in the olfactory system were reduced at all doses of L-DOPA, while in the hippocampus this effect was seen only at the dose of 90 mg/kg/day. The density of muscarinic receptors was not altered. All tested doses of L-DOPA caused norepinephrine levels to fall in the hippocampus and increase in the striatum. The density of alpha 1-adrenoceptors was reduced only at the two lower doses of L-DOPA. A comparison of the neurochemical results with the behavioral modifications seen in the staircase maze test suggests that the catecolaminergic systems are implicated in the memory process.

Enhanced acetylcholine release in the hippocampus and cortex during acquisition of an operant behavior.

The activity of the septo-hippocampal and nucleus basalis-cortical cholinergic pathways was investigated by measuring changes in the extracellular acetylcholine levels in the hippocampus and parietal cortex, by means of transversal microdialysis, during the acquisition and recall of a positively reinforced operant behavior. Adult male Wistar rats were trained in a sound-isolated operant chamber equipped with a single lever. The positive reinforcement was represented by food pellets and the number of cumulative reinforced responses was recorded every 30 min. Five groups of rats were used. Unoperated animals were used as controls. In two groups of untrained animals, the microdialysis tubes were transversally implanted in the parietal cortex, and hippocampus and the training in the operant behavior chamber began 24 h after surgery. In two further groups the microdialysis tubes were implanted in the parietal cortex, and hippocampus after training for 15 days in the operant chamber. Food was removed 12 h before training. The time needed by the control rats to reach a stable baseline of reinforced responses was 83 +/- 12 min, while in the untrained rats implanted with dialysis probes in the cerebral cortex and in the hippocampus was 621 +/- 129 and 521 +/- 126 min, respectively, and in those pretrained and implanted in cerebral cortex and in the hippocampus was 116 +/- 38 and 217 +/- 59 min, respectively. In the untrained operated rats, both cortical and hippocampal extracellular acetylcholine levels remained constant until the number of reinforced responses was low but increased significantly (+156% in the cortex and +183% in the hippocampus) in the first 30 min period in which there was a sharp rise in the reinforced responses. In the pretrained operated rats, neither in the cortex nor in the hippocampus was the increase in response rate accompanied by a statistically significant increase in extracellular acetylcholine levels. Our findings demonstrate that activation of the forebrain cholinergic pathways occurs during the acquisition of a rewarded operant responses, while recall of the same behavior is not associated with the activation of the cholinergic system.

Habituation of exploratory activity in rats: action of N6phenylisopropyladenosine, caffeine and their combination.

1. L-PIA (0.2 mg/kg), caffeine (15 mg/kg) and their combination were given subcutaneously to rats tested in open field. 2. The acute administration of L-PIA reduced ambulation. Caffeine alone was ineffective but increased ambulation in combination with L-PIA. These effects may have been determined by an interaction of L-PIA and caffeine on adenosine receptors. 3. Tolerance to L-PIA was observed after a chronic administration (20 days). The chronic administration of caffeine alone or in combination with L-PIA increased ambulation. 4. In the habituation test the reduction of ambulation in the 3 successive trials was increased by L-PIA. Caffeine did not antagonize the L-PIA effect and, in part, had the same effect. These results suggest that L-PIA and, in part, caffeine improve memory consolidation in open field, and do not support the notion that L-PIA and caffeine action on memory processes is due to an interference on adenosine receptors.

The action of arecoline on retrieval and memory storage evaluated in the staircase maze.

Rats were trained to run on a staircase stopping on the 3rd, 6th, 9th, and 12th steps (correct responses). Stopping on any other step was considered an error. Acute administration of 0.5 mg/kg of arecoline 15 min before the trial improved behavior and 3.5 mg/kg of arecoline caused a reduction of correct responses. An interruption of the daily training for 20 days caused a 15% reduction of correct responses in control animals. Chronic administration of arecoline during the first 15 days of a no-training period caused an increase of errors only at 3.5 and 8 mg/kg/day. The interpretation of these results is that arecoline improves the retrieval process and accelerates the spontaneous decay of memory. The increase of correct responses after the acute administration of 0.5 mg/kg of arecoline was not evident in rats treated for 15 days with arecoline 6.5 mg/kg/day. These results suggest that arecoline improves retrieval only in the absence of tolerance development.

The cyano-NNO-azoxy function in the design of an irreversible label for alpha 1 adrenoreceptors.

A potential alpha 1-adrenergic irreversible antagonist 6, containing the cyano-NNO-azoxy function was synthesized and tested. The effects of norepinephrine on rat thoracic aorta were irreversibly blocked by this compound at the concentration of 1 x 10(-5) M after 60 minutes. Binding studies showed that 6, at 1 x 10(-6) M, did not modify the KD of Prazosin and caused a 30% decrease of the Bmax. Substitution in 6 of the bis (2-chloroethyl)amino moiety for the cyano-NNO-azoxy function afforded 7 which behaves as an irreversible antagonist able to change KD of Prazosin without influencing Bmax.

Action of chronic choline administration on behavior and on cholinergic and noradrenergic systems.

Chronic administration of low doses (0.2-0.8 g/kg/day) of choline caused in the rat an increase of errors evaluated in the staircase maze after 20 days of interruption of daily training. An analogous pharmacological treatment caused no modification of the acetylcholine (ACh) and norepinephrine (NE) levels and no consistent modification of the density of muscarinic and alpha 1-adrenergic receptors. Only at higher doses (2.5 g/kg/day) did chronic administration (20 days) of choline cause in several sections of the CNS, an increase of ACh and NE levels and of the muscarinic receptor density. These observations indicate that only at high doses of choline are there consistent modifications of the central cholinergic systems, suggesting that the behavioral modifications observed at low doses of choline are not determined by an upregulation of the central cholinergic system.

Synthesis and H2-antagonist properties of some 1,2,5-thiadiazole-1-oxide derivatives.

A series of 1,2,5-thiadiazole-1-oxide derivatives has been synthesized and studied for its H2-antagonist properties. These derivatives can be considered derived from classical H2-antagonists in which the structure was deeply modified in order to evidence the minimal structural requirements for the activity. It was found that it is sufficient to have the 1,2,5-thiadiazole-1-oxide ring substituted with an alkylamino moiety and with an aliphatic chain linked to the hydroxy or ether group to achieve compounds as active as cimetidine. A few considerations on the binding on guinea-pig cerebral cortex of a series of H2-antagonists with more and more simplified structures are also reported.

Potential histamine H2-receptor antagonists: synthesis and pharmacological activity of derivatives containing acylamino-furazan moieties.

Analogues of 3-amino-4-[2-[(5-dimethylaminomethyl-2-furyl)methylthio]ethylamino] furazan (1) containing carbonyl groups joined to the amino functions linked to the furazan system have been synthetized and investigated for their H2-antagonist properties on the isolated guinea pig right atrium. The presence of the carbonyl group lowers the activity in respect to the corresponding leads. The decrease in activity is only by 1-2 orders of magnitude in the 3-acylamino-furazan series versus inactivity in the 4-acylamino isomers and in the diacylated series.

Characterization of an accessory binding area in the histamine H2 receptor.

The histamine H2 receptor-blocking activity of ranitidine and lamtidine analogues has been investigated to gain information on the structure of the receptor area adjacent to the site fitted by the polar group. The introduction of differently shaped alkyl moieties on the polar group is always accompanied by maintenance or by an increase of H2 receptor antagonism with respect to the starting lead compound (KB on guinea-pig isolated atria ranging from 49 nM to 1.5 nM). The results seem to indicate the presence in the histamine H2 receptor of an area of a predominantly hydrophobic nature located at the boundary of the site fitted by the polar group.

Action of a chronic disulfiram administration on memory decay and on central cholinergic and adrenergic systems.

The chronic administration (15 days) of disulfiram reduced the levels of noradrenaline (NA) in the olfactory system and in the subcortex at all the tested doses (50, 200 and 400 mg/kg/day). No modification of the density (Bmax) and of the dissociation constant (Kd) for alpha 1-adrenergic receptors was observed (radioligand [3H]prazosin). Only in the hippocampus the acetylcholine (ACh) levels and the Bmax for muscarinic receptors (radioligand [3H]QNB) were increased at all doses tested. Modifications of the cholinergic system were observed in the subcortex and in the cortex only at the higher doses of disulfiram. After 20 days interruption of the daily training in the staircase maze, 50, 100 and 200 mg/kg/day of disulfiram accelerated spontaneous decay of memory. It is hypothesized that the modifications of the hippocampal cholinergic system (increase of the ACh levels and of the density of the muscarinic receptors) may be the condition determining the acceleration of the decay of memory caused by disulfiram.

Behavioral and neurochemical modifications caused by chronic alpha-methylparatyrosine administration.

The chronic administration of alpha-methylparatyrosine (AMT) caused a reduction of the noradrenaline levels in the hippocampus (at 150 and 300 mg/kg/day) and in the subcortex (at 30, 150 and 300 mg/kg/day). The acetylcholine levels were reduced in the hippocampus and in the olfactory brain at all the tested doses of AMT. An increase of the Bmax of muscarinic and alpha 1-adrenoceptors was observed at 30 mg/kg/day of AMT; only in the subcortex AMT caused no modification of the density of muscarinic receptors. The degree of increase of the receptors density at 30 mg/kg/day was reduced at the higher doses of AMT. AMT 30 mg/kg/day caused a reduction of the errors in the staircase maze after 20 days of interruption of the daily training. These results might suggest a correlation between the behavioral effect and the increase of density, not only of the adrenoceptors, but also of the muscarinic receptors. It is proposed that the behavioral effects caused by chronic AMT are the consequence of complex neurochemical interactions.

Effects of combinations of arecoline and atropine on mouse motor activity.

1. Effects on motor activity were studied after acute administration of arecoline, atropine alone and in combination in the mouse. 2. Atropine from 15 to 45 mg/kg increased motor activity. 3. A reduction in activity was observed at doses of arecoline above 0.2 mg/kg. 4. An antagonism between arecoline and atropine was observed only at low doses of arecoline, while higher doses of arecoline in association with atropine increased activity.