Beatriz Marcos

Lack of localization of 5‐HT6 receptors on cholinergic neurons: implication of multiple neurotransmitter systems in 5‐HT6 receptor‐mediated acetylcholine release

The involvement of the cholinergic system in learning and memory together with the cognitive enhancing properties of 5‐HT6 receptor antagonists led us to study the relationship between 5‐HT6 receptors and cholinergic neurotransmission. A selective cholinergic lesion, induced by injection of the immunotoxin 192‐IgG‐Saporin into the nucleus basalis magnocellularis, failed to alter the density of 5‐HT6 receptor mRNA or protein expression in the deafferentated frontal cortex, suggesting that 5‐HT6 receptors are not located on cholinergic neurons. The 5‐HT6 receptor antagonist SB‐357134 (0.001–1 µm) induced a concentration‐dependant K+‐evoked [3H]acetylcholine (ACh) release in vitro in rat cortical and striatal slices, which was blocked by tetrodotoxin. SB‐357134, up to 1 µm, stimulated glutamate release in cortical and striatal slices. In the cortex, riluzole (1 µm) blocked the SB‐357134‐induced K+‐stimulated [3H]ACh release, and simultaneous administration of MK‐801 (1 µm) and SB‐357134 (0.05 µm) elicited an increase in K+‐evoked ACh release. In the striatum, SB‐357134, 1 µm, decreased dopamine release, and the increase in K+‐evoked [3H]ACh release induced by 5‐HT6 receptor blockade was reversed by the D1 receptor antagonist, SCH23390 (1 µm). In both the frontal cortex and striatum, bicuculline, 1 µm, showed no effect on SB‐357134‐evoked [3H]ACh. These results are discussed in terms of neurochemical mechanisms involved in 5‐HT6 receptor functions.

Effects of 5-HT6 receptor antagonism and cholinesterase inhibition in models of cognitive impairment in the rat.

The beneficial effect of 5‐HT6 receptor antagonism in cognition remains controversial. This study has been undertaken to reassess the cognition enhancing properties of acute vs subchronic treatment with the selective 5‐HT6 receptor antagonist SB‐271046 in unimpaired rats, as well as against scopolamine (cholinergic‐) or MK‐801 (glutamatergic‐mediated) deficits.

Neonatal stress affects vulnerability of cholinergic neurons and cognition in the rat: Involvement of the HPA axis

Adverse experiences early in life may sensitize specific neurocircuits to subsequent stressors. We have evaluated in maternal separation (MS) rats, an animal paradigm of early-life stress, the effects of a selective cholinergic lesion on cognitive function as well as susceptibility of cholinergic neurons to the lesion. MS rats subjected to a cholinergic lesion by administration of the immunotoxin 192 IgG-saporin, showed significant decreases in both choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity compared to control lesioned rats. Morris water maze results revealed a significant impairment in learning and memory function in MS adult rats and further cognitive deficits after the lesion. The lesion of cholinergic neurons induced a significant decrease in glucocorticoid receptor density in MS rats, accompanied by increases in CRF mRNA expression. Decreases in NGF and increases in NGF-p75NTR expression have also been found in MS rats. Our results suggest that vulnerability of basal forebrain cholinergic nerve cells might be affected by the HPA axis. The present data are discussed not only in terms of conditions that occur during ageing or Alzheimer disease, but also regarding a purported involvement of the cholinergic system in the regulation of HPA axis activity.

Involvement of the GABAergic system in depressive symptoms of Alzheimer's disease

Cognitive and neuropsychiatric (BPSD) symptoms seen in Alzheimers disease (AD) probably result from differential neurotransmitter alterations. The involvement of the glutamatergic and GABAergic system in cognitive and behavioral and psychological symptoms of dementia (BPSD) has been studied in post-mortem frontal and temporal cortex from AD patients who had been prospectively assessed with the Mini-Mental State Examination (MMSE) for cognitive impairment and with the Present Behavioral Examination (PBE) for BPSD. In addition to cholinergic deficits, significant decreases in gamma-amino butyric acid (GABA) content, with no changes in glutamate content, were found in frontal and temporal cortex. Both GABA levels and the glutamate/GABA ratio showed significant correlations with depression in AD. In the temporal cortex, higher densities of GABA(A)/benzodiazepine receptors also correlated with more severe depression. It can be suggested that in a situation of cholinergic deficit, such as AD, an imbalance between the excitatory glutamatergic tone and inhibitory GABAergic tone may be responsible for non-cognitive behavioral disturbances.

Effect of Selective Cholinergic Denervation on the Serotonergic System: Implications for Learning and Memory

The cholinergic system has been widely implicated in cognitive processes and cholinergic loss is a classical hallmark in Alzheimer disease. Increasing evidence supports a role of the serotonergic system in cognition, possibly through a modulation of cholinergic activity. We compared selective cholinergic denervation by administration of the immunotoxin 192 IgG-saporin in the nucleus basalis of Meynert (NBM) with intracerebroventricular (ICV) lesions of the basal forebrain in male rats 7 days after lesioning. NBM lesions induced significant changes in cholinergic markers in the frontal cortex, whereas ICV lesions produced significant decreases in cholinergic markers both in the frontal cortex and hippocampus. Only ICV lesions lead to memory impairments in passive avoidance and Morris water maze tasks. Both models lead to reductions of serotonin levels in the frontal cortex. Similar changes in 5-hydroxytriptophan levels were observed, suggesting a downregulation of the rate-limiting enzyme for the synthesis of serotonin along with the cholinergic deficit. Neither 5-HT1A nor 5-HT1B receptors seem to mediate this process. These data imply that the serotonergic system in the frontal cortex can compensate for diminished cholinergic function and support the investigation of the serotonergic system as a therapeutic target to treat Alzheimer disease.

Involvement of an altered 5-HT -{6} receptor function in behavioral symptoms of Alzheimer's disease.

We studied the hypothesis that disturbances in 5-HT_{6} receptor function in the temporal cortex may contribute to clinical symptoms of Alzheimers disease (AD). 5-HT_{6} density and 5-HT levels were significantly decreased in a cohort of AD patients prospectively assessed for cognitive/behavioral symptoms. cAMP formation after stimulation with the selective 5-HT_{6} receptor agonist E-6801 was significantly lower (p<0.01) in AD (170.02 +/- 27.53 pmol/mg prot.) compared to controls (823.33 +/-196.67). In addition, the ratio cAMP formation after stimulation with E-6801/5-HT_{6} receptor density was significantly lower (p< 0.01) in AD (6.67 +/- 0.83) compared to controls (16.67 +/- 3.33). Splitting these results by sex, 5-HT_{6} receptor activation was significantly lower (p< 0.01) in AD females compared to males (121.67 +/- 30.02 vs. 231.67 +/- 34.17 pmol/mg prot). 5-HT_{6} density and 5-HT levels were significantly correlated (p < or = 0.01) in both controls and AD patients, although in AD, this correlation was lost in females. Psychosis factor was the best predictor of reduced 5-HT levels or adenylate cyclase activity after E-6801 stimulation, the former result being due to females. It may be suggested that psychotic symptoms may be related to a dysregulation of 5-HT_{6} activation by 5-HT in the temporal cortex. These results are discussed in terms of purported influence of sex and therapeutical approaches to psychosis in AD.

Functional interaction between 5-HT6 receptors and hypothalamic-pituitary-adrenal axis : Cognitive implications

The serotonin 5-HT(6) receptor has become a promising target for the treatment of neuropsychological diseases, such as affective disorders. Increasing evidence implicates stress and its effector system, the hypothalamic-pituitary-adrenal (HPA) axis, in the neurobiology of depression. In addition, there are important memory disturbances in stress-related psychiatric disorders that have been associated to an impairment of the HPA axis reactivity. The aim of the present work is to study the functional interactions between 5-HT(6) receptors and HPA axis. In a situation of increased HPA axis responsiveness (maternal separation, MS) no differences were found in the expression of 5-HT(6) gene in the hippocampus or frontal cortex, although serotonin levels were higher in the frontal cortex of MS rats. 5-HT(6) receptor mRNA expression increased significantly in the hippocampus in a situation of decreased glucocorticoid levels, such as adrenalectomy. Cognitive deficits associated to HPA dysfunction, such those found in the MS model, were fully reversed by administration of SB271046, a selective 5-HT(6) receptor antagonist. A chronic treatment with SB271046 did not modify CRF mRNA levels in the hypothalamus, but there was a higher glucocorticoid receptor density in the hippocampus compared to control. In contrast, in the frontal cortex, treatment with SB271046 induced a significant decrease in glucocorticoid receptor density. These data suggest that expression of 5-HT(6) receptors might be differentially regulated depending on levels of circulating adrenal corticoids. These results are discussed in terms of therapeutical approaches to the treatment of behavioral (depressive-like) and cognitive disturbances associated to an altered response to stress.

Signalling pathways associated with 5-HT6 receptors: relevance for cognitive effects

A growing body of evidence supports the use of serotonin 5-HT6 receptor antagonists as a promising mechanism for treating cognitive dysfunction. We evaluated 5-HT6 receptor expression and associated biochemical mechanisms in the hippocampus of rats that had been trained in the Morris water maze (MWM), a spatial learning task. Training in the MWM induces a down-regulation of 5-HT6 receptor protein and mRNA receptor expression. The learning procedure or the administration of the selective 5-HT6 receptor antagonist SB-271046 induced an increase in pCREB1 levels while CREB2 levels were significantly reduced. However, although SB-271046 was able to improve retention in the MWM, no further changes in pCREB1 or CREB2 levels were found to be associated with the presence of the 5-HT6 receptor antagonist during the learning procedure. The MWM procedure significantly increased pERK1/2 levels and interestingly, further increases were seen when treating with SB-271046 during the MWM. These results suggest that, in the hippocampus, biochemical pathways associated with pERK1/2 expression, and not with the CREB family of transcription factors, seem to be related to the cognitive-enhancing properties of 5-HT6 receptor antagonists.

Involvement of the Serotonergic System in Cognitive and Behavioral Symptoms of Alzheimers Disease

Alzheimers disease (AD) is a chronic progressive disorder characterized by dementia, but often featuring behavioral and psychological syndromes (BPSD), such as depression, overactivity, psychosis or aggressive behavior. Traditional treatments for BPSD are neuroleptics and sedatives, which are not devoid of serious adverse effects. Neurochemically, the classical hallmark of AD is the disruption of basal forebrain cholinergic pathways and consequent cortical cholinergic denervation of the neocortex and hippocampus. However, it is conceivable that, according to the complexity and diversity of BPSD, more than one transmitter system may contribute to a particular behavioral syndrome. The serotonergic system has been implicated not only in cognitive processes, but also in depression, psychosis or aggression. In AD, extensive serotonergic denervation has been reported. In particular, there is growing interest in the pathological functions and implication in BPSD of 5-HT6 receptors, due to its high affinity for antipsychotic drugs and its distribution in the brain. In this study we will review the present knowledge of the involvement of the serotonergic system and its receptors in cognitive deficits and BPSD. From the currently available data, it is possible to conclude that pharmacological manipulation of serotonergic system may improve not only cognitive function but behavioral disturbances in dementia.

Effects of chronic blockade of 5-HT(6) receptors on NMDA receptor subunits expression.

Several studies with structurally different compounds have shown that 5-HT6 receptor antagonists improve learning and memory in animal models (see reviews by King et al., 2008; Woolley et al., 2004). As a result, the 5-HT6 receptor has become an increasingly promising target for improving cognition in both normal and disease states (Mitchell and Neumaier, 2005). Reductions in 5-HT6 receptor density have been found in cortical areas of Alzheimer’s disease patients (Garcia-Alloza et al., 2004). In addition, the affinity of some atypical antipsychotics for 5-HT6 receptors has prompted interest on the involvement of 5-HT6 receptors in psychiatric disorders. 5-HT6 receptor-modulation of glutamate may contribute to the effect of 5-HT6 receptor antagonists (Dawson et al., 2000, 2001). Previous studies have shown that atypical antipsychotics with high affinities for 5-HT6 receptors, such as clozapine, increased the striatum levels of the NR2 subunit of the glutamate NMDA receptor (Meshul et al., 1996). Based on these facts, we checked the effects of chronic treatment with a selective 5-HT6 receptor antagonist on the mRNA and protein expression of the main NMDA receptor subunits: NR1, NR2A, and NR2B. Male Wistar rats, weighing 230–250 g were used. Animals were kept at constant room temperature (21 6 18C) and relative humidity (55 6 5%) with a 12h light/dark cycle (dark from 8 p.m.) and free access to food and water. All the experiments were performed in strict compliance with the recommendations of the EU (DOCE L 358/1/18/2/1986) for the care and use of laboratory animals. SB-271046 (provided by GlaxoSmithKline, Harlow, UK) is a selective 5-HT6 receptor antagonist with a pKi of 8.9 at the human 5-HT6 receptor, with greater than 200-fold selectivity over more than 51 receptors, ion channels and enzymes (Routledge et al., 2000). SB-271046 was administered p.o., in 1% methylcellulose solution, b.i.d. for 7 days, at a dose of 10 mg/kg (Dawson et al., 2000, 2001; Routledge et al., 2000). Under this administration regimen, in our hands, SB-271046 has been shown to have beneficial effects on cognition (Marcos et al., 2008). For in situ hybridization studies, the oligonucleotides used were: 50-TTC CTC CTC CTC CTC ACT GTT CAC CTT GAA TCG GCC AAA GGG ACT-30 for NR1 mRNA, 50-CCC TTT GCA TTC AGT GCT GAC AGC CAC CTC CAC TGT GTT GGG GTT GGA CTC-30 for NR2A mRNA and 50-GGC CTC CTG GCT CGC TGC CAT CGG CTA GGC ACC TGT GGT AAC CC-30 for NR2B mRNA (all from Sigma Genosis, UK). Negative controls including sense oligonucleotides showed minimal background signals. The sections were incubated overnight in humid chambers at 36.98C for NR1, 41.18C for NR2A, and 43.68C for NR2B. The exposition times to Biomax MR film (Kodak) was 5 weeks (NMDA subunits) at 48C. The relative abundance mRNA in each region was determined by densitometric quantification of autoradiograms using an image analysis system (Scion Image, Scion Corporation, US) correcting for nonspecific signals. Densitometric values from three sections of each animal were averaged and expressed as nCi/g tissue. In Western blot studies, total NR2A and NR2B receptor proteins were detected with primary antibody (1:1000 TBST; Chemicon, Temecula, CA) and immunopositive bands were visualized by a chemilumi-