Henrik H. Hansen

Long-term effects on serotonin transporter mRNA expression of chronic neonatal exposure to a serotonin reuptake inhibitor.

Chronic administration of clomipramine or other serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitors to neonatal rats produces behaviours that resemble a depressive state in the adult animal, and this model is therefore regarded as a putative animal model of depression. Alterations in the activity of the central 5-HT system are important in understanding the pathophysiology of depression, and therefore, we examined whether this model was associated with changes in the expression of 5-HT1A receptor, 5-HT1B receptor, and 5-HT transporter mRNA in the dorsal raphe nucleus and the hippocampus. Wistar rats were injected twice daily with the serotonin reuptake inhibitors clomipramine and 5-chloro-1-[3-(dimethylamino)propyl]-1-(4-fluoro-phenyl)-1,3-dihydroi so-benzofurane, hydrochloride (code Lu 10-134-C) at doses of 15 mg kg(-1) or vehicle i.p. from postnatal day 8 for 14 days. Groups of rats (n = 10) were either killed the day after the last injection or left undisturbed for 69 days before they were killed. The expression of 5-HT transporter, 5-HT1A receptor, and 5-HT1B receptor mRNA was examined in the dorsal raphe nucleus and in the CA1 of the hippocampus by means of quantitative in situ hybridisation histochemistry. Both compounds resulted in an increase in 5-HT transporter mRNA expression (40% more than vehicle) in the dorsal raphe nucleus the day after the last injection (postnatal day 22). A small but significant increase in 5-HT1B receptor mRNA expression in the CA1 was seen after clomipramine, but not after Lu 10-134-C, probably reflecting clomipramines affinity for both the 5-HT and noradrenaline transporters as well as for a number of monoamine receptor sites. Levels of 5-HT1A receptor mRNA were unchanged. In contrast, 5-HT transporter mRNA expression in the dorsal raphe nucleus was significantly decreased in the adult after neonatal treatment with either of the two drugs compared to vehicle. No changes in 5-HT1A receptor and 5-HT1B receptor mRNA expression were observed in any of the regions examined in these animals. The results show that the persistent depressive behaviour previously shown in this model is also associated with changes in the expression of 5-HT transporter mRNA. This long-term alteration in gene expression may result from disturbances in 5-HT neurotransmission in the brain of the neonatal animals.

Kv7 channels: interaction with dopaminergic and serotonergic neurotransmission in the CNS.

Neuronal Kv7 channels (also termed KCNQ channels) are the molecular correlate of the M‐current. The Kv7 channels activate at rather negative membrane potentials (≤ 60 mV), thereby ‘fine‐tuning’ the resting membrane potential. The Kv7 channels are widely expressed in the brain with the Kv7.2, Kv7.3 and Kv7.5 channels being the most abundant. The Kv7.4 subunit has the most restricted brain regional expression being present in discrete nuclei of brainstem only. Kv7 channels are expressed at different subcellular locations, being on both somatodendritic, axonal and terminal sites. This complex subcellular distribution of Kv7 channels enables them to participate in both pre‐ and postsynaptic modulation of basal and stimulated excitatory neurotransmission. Activation of neuronal Kv7 channels limits repetitive firing thereby potentially limiting the generation of long bursts, with subsequent inhibition of monoaminergic neurotransmitter release. In this review, we focus on the influence of Kv7 channels on dopaminergic and serotonergic neurotransmission. The data suggest a novel action of Kv7 channel openers which could translate into having therapeutic value in the treatment of disease states characterized by overactivity of dopaminergic (e.g. schizophrenia and drug abuse) and serotonergic neurotransmission (e.g. anxiety).

The KCNQ Channel Opener Retigabine Inhibits the Activity of Mesencephalic Dopaminergic Systems of the Rat

Homo- and heteromeric complexes of KCNQ channel subunits are the molecular correlate of the M-current, a neuron-specific voltage-dependent K+ current with a well established role in control of neural excitability. We investigated the effect of KCNQ channel modulators on the activity of dopaminergic neurons in vitro and in vivo in the rat ventral mesencephalon. The firing of dopaminergic neurons recorded in mesencephalic slices was robustly inhibited in a concentration-dependent manner by the KCNQ channel opener N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester (retigabine). The effect of retigabine persisted in the presence of tetrodotoxin and simultaneous blockade of GABAA receptors, small-conductance calcium-activated K+ (SK) channels, and hyperpolarization-activated (Ih) channels, and it was potently reversed by the KCNQ channel blocker 4-pyridinylmethyl-9(10H)-anthracenone (XE991), indicating a direct effect on KCNQ channels. Likewise, in vivo single unit recordings from dopaminergic neurons revealed a prominent reduction in spike activity after systemic administration of retigabine. Furthermore, retigabine inhibited dopamine synthesis and c-Fos expression in the striatum under basal conditions. Retigabine completely blocked the excitatory effect of dopamine D2 autoreceptor antagonists. Again, the in vitro and in vivo effects of retigabine were completely reversed by preadministration of XE991. Dual immunocytochemistry revealed that KCNQ4 is the major KCNQ channel subunit expressed in all dopaminergic neurons in the mesolimbic and nigrostriatal pathways. Collectively, these observations indicate that retigabine negatively modulates dopaminergic neurotransmission, likely originating from stimulation of mesencephalic KCNQ4 channels.

Alpha-7 nicotinic acetylcholine receptor agonists selectively activate limbic regions of the rat forebrain: An effect similar to antipsychotics

It is considered that activation of nicotinic α7 receptors (α7 nAChR) is useful for the treatment of cognitive disturbances in schizophrenia and Alzheimers disease. Recently, selective α7 nAChR agonists have been discovered and are used to validate the α7 nAChR as a drug target for the treatment of cognitive disturbances in schizophrenia. One important feature shared by all known antipsychotics is their capacity to induce expression of the neuronal immediate‐early gene c‐fos in the limbic forebrain. Using two novel and selective α7 nAChR agonists, PNU‐282987 and SSR180711, we investigated their ability to induce c‐Fos expression in the limbic forebrain with particular emphasis on the same regions reported to be activated by antipsychotics. Both α7 nAChR agonists increased c‐Fos dose‐dependently in the prefrontal cortex and the shell of nucleus accumbens, while leaving the core of nucleus accumbens and the dorsolateral striatum unaffected. The accumbal and cortical effect of SSR180711 was blocked completely by pre‐administration of the α7 nAChR antagonist methyllycaconitine. Also, SSR180711 displayed no c‐Fos‐inducing effect in α7 nAChR knock‐out mice. In conclusion, these results show that selective pharmacologic stimulation of α7 nAChR function results in activation of forebrain regions similar to conventional antipsychotics.

Regulation of activity-regulated cytoskeleton protein (Arc) mRNA after acute and chronic electroconvulsive stimulation in the rat.

The temporal profile of Arc gene expression after acute and chronic electroconvulsive stimulations (ECS) was studied using semi-quantitative in situ hybridisation in the rat cortex. A single ECS strongly and temporarily increased Arc mRNA levels in dentate granular cells with maximal induction seen up to 4 h after the stimulus, but returned to baseline at 24 h. A single ECS also increased expression of Arc mRNA in the CA1 and the parietal cortex, but the expression peaked within 1 h and returned to baseline levels within 2 h. Repeated or chronic ECS is a model of electroconvulsive therapy and it would be predicted that gene products involved in antidepressant effects accumulate after repeated ECS. However, repeated ECS reduced Arc gene expression in the CA1 24 h after the last stimulus. These results indicate that Arc is an immediate early gene product regulated by an acute excitatory stimulus, but not accumulated by long term repetitive ECS and therefore not a molecular biomarker for antidepressant properties. More likely, Arc is likely a molecular link to the decline in memory consolidation seen in depressive patients subjected to electroconvulsive therapy.

Epigenetic regulation of Arc and c-Fos in the hippocampus after acute electroconvulsive stimulation in the rat

Electroconvulsive stimulation (ECS) remains one of the most effective treatments of major depression. However, the underlying molecular changes still remain to be elucidated. Since ECS causes rapid and significant changes in gene expression we have looked at epigenetic regulation of two important immediate early genes that are both induced after ECS: c-Fos and Arc. We examined Arc and c-Fos protein expression and found Arc present over 4 h, in contrast to c-Fos presence lasting only 1 h. Both genes had returned to baseline expression at 24 h post-ECS. Histone H4 acetylation (H4Ac) is one of the important epigenetic marks associated with gene activation. We show increased H4Ac at the c-Fos promoter at 1 h post-ECS. Surprisingly, we also observed a significant increase in DNA methylation of the Arc gene promoter at 24 h post-ECS. DNA methylation, which is responsible for gene silencing, is a rather stable covalent modification. This suggests that Arc expression has been repressed and may consequently remain inhibited for a prolonged period post-ECS. Arc plays a critical role in the maintenance phase of long-term potentiation (LTP) and consolidation of memory in the rat brain. Thus, this study is one of the first to demonstrate DNA methylation as a regulator of ECS-induced gene expression and it provides a molecular link to the memory deficits observed after ECS.

The GLP-1 receptor agonist liraglutide reduces pathology-specific tau phosphorylation and improves motor function in a transgenic hTauP301L mouse model of tauopathy

In addition to a prominent role in glycemic control, glucagon-like peptide 1 (GLP-1) receptor agonists exhibit neuroprotective properties. There is mounting experimental evidence that GLP-1 receptor agonists, including liraglutide, may enhance synaptic plasticity, counteract cognitive deficits and ameliorate neurodegenerative features in preclinical models of Alzheimers disease (AD), predominantly in the context of β-amyloid toxicity. Here we characterized the effects of liraglutide in a transgenic mutant tau (hTauP301L) mouse tauopathy model, which develops age-dependent pathology-specific neuronal tau phosphorylation and neurofibrillary tangle formation with progressively compromised motor function (limb clasping). Liraglutide (500 µg/kg/day, s.c., q.d., n=18) or vehicle (n=18) was administered to hTauP301L mice for 6 months from the age of three months. Vehicle-dosed wild-type FVB/N mice served as normal control (n=17). The onset and severity of hind limb clasping was markedly different in liraglutide and vehicle-dosed transgenic mice. Clasping behavior was observed in 61% of vehicle-dosed hTauP301L mice with a 55% survival rate in 9-month old transgenic mice. In contrast, liraglutide treatment reduced the clasping rate to 39% of hTauP301L mice, and fully prevented clasping-associated lethality resulting in a survival rate of 89%. Stereological analyses demonstrated that hTauP301L mice exhibited hindbrain-dominant neuronal accumulation of phosphorylated tau closely correlated to the severity of clasping behavior. In correspondence, liraglutide treatment significantly reduced neuronal phospho-tau load by 61.9±10.2% (p<0.001) in hTauP301L mice, as compared to vehicle-dosed controls. In conclusion, liraglutide significantly reduced tau pathology in a transgenic mouse tauopathy model.

GABAA Receptor α Subunits Differentially Contribute to Diazepam Tolerance after Chronic Treatment

Background Within the GABAA-receptor field, two important questions are what molecular mechanisms underlie benzodiazepine tolerance, and whether tolerance can be ascribed to certain GABAA-receptor subtypes. Methods We investigated tolerance to acute anxiolytic, hypothermic and sedative effects of diazepam in mice exposed for 28-days to non-selective/selective GABAA-receptor positive allosteric modulators: diazepam (non-selective), bretazenil (partial non-selective), zolpidem (α1 selective) and TPA023 (α2/3 selective). In-vivo binding studies with [3H]flumazenil confirmed compounds occupied CNS GABAA receptors. Results Chronic diazepam treatment resulted in tolerance to diazepams acute anxiolytic, hypothermic and sedative effects. In mice treated chronically with bretazenil, tolerance to diazepams anxiolytic and hypothermic, but not sedative, effects was seen. Chronic zolpidem treatment resulted in tolerance to diazepams hypothermic effect, but partial anxiolytic tolerance and no sedative tolerance. Chronic TPA023 treatment did not result in tolerance to diazepams hypothermic, anxiolytic or sedative effects. Conclusions Our data indicate that: (i) GABAA-α2/α3 subtype selective drugs might not induce tolerance; (ii) in rodents quantitative and temporal variations in tolerance development occur dependent on the endpoint assessed, consistent with clinical experience with benzodiazepines (e.g., differential tolerance to antiepileptic and anxiolytic actions); (iii) tolerance to diazepams sedative actions needs concomitant activation of GABAA-α1/GABAA-α5 receptors. Regarding mechanism, in-situ hybridization studies indicated no gross changes in expression levels of GABAA α1, α2 or α5 subunit mRNA in hippocampus or cortex. Since selective chronic activation of either GABAA α2, or α3 receptors does not engender tolerance development, subtype-selective GABAA drugs might constitute a promising class of novel drugs.

The nicotinic α7 acetylcholine receptor agonist ssr180711 is unable to activate limbic neurons in mice overexpressing human amyloid-β1-42

Recent studies have demonstrated that amyloid-beta1-42 (Abeta1-42) binds to the nicotinergic alpha7 acetylcholine receptor (alpha7 nAChR) and that the application of Abeta1-42 to cells inhibits the function of the alpha7 nAChR. The in vivo consequences of the pharmacological activation of the alpha7 nAChR have not been examined. The aim of this study has been to evaluate the efficacy of alpha7 nAChR modulators in transgene mice that overexpress human amyloid precursor protein and accumulate Abeta1-40 and Abeta1-42. In accordance with observations in human Alzheimer tissues, we show here through the use of co-immunoprecipitation that human Abeta-immunoreactive peptides bind to mice alpha7 nAChR in vivo. Agonists of the alpha7 nAChR improve memory and attentional properties and increase immediate early gene expression in the prefrontal cortex and the nucleus accumbens. We show that acute systemic administration of the alpha7 nAChR agonist SSR180711 (10 mg/kg) result in a significant increase in Fos protein levels in the shell of nucleus accumbens in wild-type mice, but has no effect in the transgene mice. There were fewer cell bodies expressing Fos in the prefrontal cortex of transgene mice, and in this region no induction was achieved after administration with SSR180711 in either of the two groups. These results suggest that overexpression of human Abeta peptides perhaps via direct interaction with alpha7 nAChR, inhibit alpha7 nAChR-dependent neurotransmission in vivo and emphasize that clinical trials testing alpha7 nAChR agonists should be related to the content of Abeta peptides in the patients nervous system.

GABA Regulates the Rat Hypothalamic‐Pituitary‐Adrenocortical Axis via Different GABA‐A Receptor α‐Subtypes

The control of the corticotropin‐releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) is balanced by excitatory and inhibitory inputs. The GABA‐A receptor, which is a major target for the inhibitory control, is composed of five subunits. The presence of an α1‐, α2‐, α3‐, or α5‐subunit in the GABA‐A receptor protein complex is necessary for benzodiazepines to exert their potentiating effect on the receptor. We postulate that the effect of nonselective benzodiazepines on the hypothalamo‐pituitary adrenocortical (HPA) axis is critically dependent on the composition of the GABA‐A receptor subunits through which they act. We show here that positive modulators of α1‐subtype containing GABA‐A receptors with zolpidem (10 mg/kg) increase HPA activity in terms of increase in plasma corticosterone and induction of Fos in the PVN, whereas activation of non‐α1‐subtype GABA‐A receptors using L‐818,417 (10 mg/kg) likely inhibits the activity. We also show that the α2‐subunit gene is highly expressed in the PVN, but its expression is not affected by chronic mild stress. These results show that the stimulatory effect on HPA activity after positive modulation of GABA‐A receptors composed of α1‐subunit(s) affects a selective afferent system than the PVN, which is distinct from another afferent system(s) activated by non α1‐containing GABA‐A receptors.