Paradoxical effects of lithium on serotonergic receptor function: an immunocytochemical, behavioural and autoradiographic study.

Lithium is the preferred treatment for bipolar affective disorder, yet its mechanism of action is poorly understood. Our study was designed to investigate the effect of lithium on the 5-HT2A or 5-HT2C (5-HT2A/2C) receptor subtypes, by comparing the consequences of chronic pre-treatment of rats with lithium on 5-HT2A/2C receptor-mediated behavioural responses, Fos expression, and the density of these receptors in the brain. In addition, the time-course and persistence of the effect of chronic lithium on 5-HT2A/2C receptor-mediated Fos expression was examined. Furthermore, the acute action of lithium on Fos expression was also examined. In an investigation of the dose response of Fos to the 5-HT2A/2C agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), rats received saline or 1, 2, 4, 8, 12, 16, 24 or 32 mg/kg DOI, then were sacrificed 3 h later for immunocytochemical localisation of Fos. In a chronic lithium study, rats received either control or lithium-containing (0.1% LiCO3) chow for 3 weeks prior to challenge with 8 mg/kg DOI. DOI-induced locomotor activity was measured for 30 min immediately following the drug challenge, then 150 min later, the animals were sacrificed for Fos immunocytochemistry. The brains of another group of rats, also receiving either control or lithium-containing diet for 3 weeks, were analysed for the distribution and density of 5-HT2A receptor binding sites by quantitative [3H]ketanserin autoradiography. One group of chronic lithium treated rats received ritanserin (0.4 mg/kg), a 5-HT2A/2C receptor antagonist, 40 min before DOI challenge and were sacrificed 3 h later for Fos localisation. In the time-course experiment, rats received lithium-containing diet for 3 weeks followed by normal, control diet for 48 h, 1, 2 or 4 weeks prior to DOI or saline challenge. A further group of animals received an injection of LiCl (3 mM/kg) before being challenged with DOI or saline 12, 24, 36 or 48 h later. The dose-response experiment revealed that little Fos-like immunoreactivity was evident above basal levels following administration of 1 mg/kg DOI. However, at all other doses examined, Fos-like immunoreactivity was elevated in a number of brain areas, particularly in cerebral cortex, olfactory tubercle and amygdala. Following 24 mg/kg DOI, the number of Fos-positive nuclei appeared to have reached a plateau level. Treatment of rats with chronic lithium significantly enhanced DOI-induced locomotor activity and Fos-like immunoreactivity throughout the cerebral cortex. This elevation in Fos-like immunoreactivity was completely abolished by prior treatment with ritanserin. In contrast, chronic lithium treatment had no effect on the density of [3H]ketanserin binding to 5-HT2A receptors in any brain region examined. The results of the time-course experiment demonstrated that the enhancing effect of lithium on 5-HT2A/2C receptor-mediated Fos expression was short-lived such that Fos-like immunoreactivity returned to untreated levels within 48 h. In the acute lithium experiment, administration of lithium to rats 12 or 24 h before DOI resulted in a similar elevation of Fos-like immunoreactivity to that seen in chronically treated animals. Administration of acute lithium 36 or 48 h before DOI had no effect. The effects of lithium on 5-HT2A/2C receptor function thus appear to be complex. In particular, the results of this study indicate that the enhancing effects of lithium on DOI-induced locomotor activity and Fos-like immunoreactivity are not accompanied by any alteration in the density of 5-HT2A receptor binding sites. If changes in receptor numbers therefore do not account for the physiological effect of chronic lithium, other explanations must be sought. The study also suggests that the inositol depletion hypothesis of lithium's therapeutic action does not adequately explain the mechanism of action of lithium in man.

Differential expression of DCC mRNA in adult rat forebrain.

DCC is a member of the immunoglobulin superfamily of cell adhesion molecules, whose role in the function of the adult nervous system is unknown. DCC mRNA expression was studied in adult rat dorsal hippocampal sections using in situ hybridization histochemistry. High levels of DCC transcript were detected in hippocampus and medial habenula, whereas lower mRNA expression was found in cerebral cortex, hypothalamus and thalamus. The higher relative expression of DCC mRNA in hippocampus, compared with the remainder of the brain was confirmed using RT-PCR analysis. These data confirm the presence of DCC mRNA in adult rat brain and indicate that DCC mRNA is differentially expressed between forebrain regions, suggesting a role for DCC in the function of the adult rat central nervous system.

BDNF enhances neuronal growth and synaptic activity in hippocampal cell cultures.

Brain-derived neurotrophic factor (BDNF) (20 ng/ml) significantly enhanced the growth of the somata of GABA-immunoreactive neurones in primary cultures of hippocampal neurones from postnatal rats after only 24h. Whole-cell patch-clamp experiments showed an increase in spontaneous synaptic activity between neurones with time in culture. After 10 days in culture, 90% of neurones sampled in control cultures showed spontaneous synaptic activity, whereas in cultures treated with BDNF, 100% of neurones had synaptic inputs after only 6 days. This difference in spontaneous activity was not due to the lack of synaptic inputs as KCl-induced synaptic activity was equally effective in BDNF and control cultures. These experiments demonstrate the rapid rate at which BDNF can promote neuronal growth and also show that BDNF can promote long term synaptic activity.

P-chloroamphetamine induces c-fos in rat brain: a study of serotonin2A/2C receptor function.

Recent studies suggest that a class of substituted amphetamines, which includes p-chloroamphetamine, causes an acute release of serotonin (5-hydroxytryptamine) and appears to act preferentially on axons arising from the dorsal raphe nucleus. The postsynaptic targets of these axons are not well characterized, but they have been localized in close proximity to the distribution of serotonin2A receptor binding sites. In the present study, c-fos immunocytochemistry has been used to investigate this anatomical relationship further. Administration of p-chloroamphetamine or the serotonin2A/2C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane to rats resulted in similar patterns of Fos-like immunoreactivity in some, but not all, forebrain areas. Areas which expressed Fos following either treatment included cerebral cortex, claustrum, amygdala and nucleus accumbens. A particularly close match was seen in layer Va of the somatosensory cortex. No specificity of p-chloroamphetamine for dorsal raphe nucleus-innervated areas was noted. Prior treatment of animals with p-chloroamphetamine two weeks before a second challenge with the same drug, or with the serotonin2A/2C receptor antagonist ritanserin 30 min before p-chloroamphetamine challenge, resulted in an attenuation of p-chloroamphetamine-induced Fos-like immunoreactivity in the olfactory tubercle, the islands of Calleja and the caudate-putamen. The reduction was most noticeable in layer Va of the somatosensory cortex. The results of this study indicate that a close anatomical correlation may exist between the fine serotonin axon terminals that show vulnerability to the neurotoxic effects of p-chloroamphetamine and serotonin2A receptors in some brain regions. This association may prove to be important in explaining the actions of certain psychotropic drugs, for example in the control of affective states.

Chronic electroconvulsive shock enhances 5-HT2 receptor-mediated head shakes but not brain C-fos induction.

Chronic electroconvulsive shock (ECS), a widely used treatment for intractable depression, increases the density of 5-HT2A receptor binding sites and mRNA in rat frontal cortex. In contrast, this treatment appears to have no significant effect on 5-HT-stimulated phosphatidyl inositol turnover in rat brain. To investigate the effect of chronic ECS on the 5-HT2 receptor family further, we determined its effects on head shakes and c-fos expression in the rat in response to the 5-HT2A/2C receptor agonist DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane]. Chronic ECS (5 electroconvulsive shocks over 10 days, via earclips under halothane anaesthesia) caused a significant enhancement in the number of head shakes counted in a 30 min period after administration of 2 or 8 mg/kg DOI. In contrast, this treatment had no effect on Fos expression, induced by either dose of DOI, in any region of rat forebrain examined. Fos expression was low-to-undetectable in the brains of animals treated with chronic ECS followed by saline and sham ECS animals that had been treated identically, but with no administration of electrocurrent. Thus the lack of any change in PI turnover, following chronic ECS administration, appears to be mirrored by the failure of this treatment to alter 5-HT2 receptor-mediated Fos expression.

Induction of c-fos in rat forebrain by pharmacological manipulation of 5-hydroxytryptamine levels.

The immunocytochemical localization of the immediate-early gene c-fos has been used to map sites of neuronal activity in the rat brain associated with 5-hydroxytryptamine function. Behavioural studies have shown that brain 5-hydroxytryptamine function is increased by treatment of animals with a combination of the 5-hydroxytryptamine precursor L-tryptophan (100 mg/kg) and the monoamine inhibitor tranylcypromine (20 mg/kg). We now report that such treatment induces a specific anatomical pattern of expression of c-fos in rat forebrain in many limbic, striatal and cortical areas which corresponds well with the distribution of 5-hydroxytryptamine-immunoreactive terminals. To investigate further the involvement of 5-hydroxytryptamine in this response, we pretreated animals with the tryptophan hydroxylase inhibitor p-chlorophenylalanine and observed the effects on Fos-like immunoreactivity after L-tryptophan and tranylcypromine challenge. Two-day pretreatment with p-chlorophenylalanine (300 mg/kg) prior to tranylcypromine and L-tryptophan resulted in a significant attenuation of Fos-like immunoreactivity in specific brain areas, including the piriform and frontal cortices, nucleus accumbens, caudate-putamen, paraventricular hypothalamus and paraventricular thalamic nucleus. A marked reduction of the hyperactivity syndrome was also seen, as has been reported in earlier studies. The results of this study suggest that the elevation in Fos-like immunoreactivity following treatment with tryptophan and a monoamine oxidase inhibitor is mainly due to increased 5-hydroxytryptamine synthesis and release. It is well known that 5-hydroxytryptamine mediates mood and affect, and this study indicates potential brain loci of action of serotonergic drugs.

Evidence for cholinergic vagal afferents and vagal presynaptic M1 receptors in the ferret.

The distribution of muscarinic receptor binding was examined in the ferret brainstem vagal nuclei using the non-selective ligand [3H]quinuclidinyl benzilate and the relatively M1 receptor-selective ligand [3H]pirenzepine. The highest density of receptor sites are found in the subnucleus gelatinosus and lower levels in the other subnuclei of the nucleus of the tractus solitarius and in the area postrema and dorsal motor nucleus of the vagus nerve. Dense binding was also seen in the adjacent hypoglossal nucleus. Following unilateral cervical nodose ganglion excision binding in the subnucleus gelatinosus was attenuated ipsilateral to the lesion compared with the contralateral side. In contrast, [3H]pirenzepine binding was only seen in the subnucleus gelatinosus and in no other region at this level of the brainstem. This binding was reduced in the subnucleus as a whole by 52% ipsilateral to a cervical vagotomy. In the more rostral parts of the subnucleus gelatinosus, binding was undetectable ipsilateral to the lesion but more caudally, appreciable levels of binding persisted. This distribution parallels the known rostro-caudal variation in cross-over of vagal afferent fibres in the ferret dorsal vagal complex and indicates a presynaptic localization of [3H]pirenzepine binding sites on vagal afferent terminals. The distribution of binding of the high affinity choline uptake site blocker, [3H]hemicholinium-3, was also examined in the ferret brainstem using autoradiography. High densities of [3H]hemicholinium-3 binding were seen in the hypoglossal nucleus, the subnucleus gelatinosus and in the area postrema, with lower levels in the dorsal motor nucleus of the vagus, the trigeminal nucleus and other subnuclei of the nucleus of the tractus solitarius.(ABSTRACT TRUNCATED AT 250 WORDS)

Lithium enhances 5-HT2A receptor-mediated c-fos expression in rat cerebral cortex.

The role of lithium in treating bipolar affective disorder is poorly understood; however, it may involve effects on brain 5-HT function. We have shown that the 5-HT2A/2C receptor agonist DOI (2,5-dimethoxy-4-iodophenylisopropylamine) induces the expression of c-fos in rat brain which correlates with the distribution of 5-HT2A receptors. We now report on the effect of lithium on 5-HT receptor activation. Rats were treated chronically with dietary lithium before being given either DOI or the 5-HT1A receptor agonist 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino) tetralin), and their brains were processed for c-fos immunohistochemistry. Lithium treatment greatly enhanced levels of Fos seen after DOI, but not after 8-OH-DPAT; layer II of caudal piriform cortex, previously devoid of staining, exhibited the most marked labelling. This suggests that chronic lithium selectively alters immediate-early gene expression in brain. Such alteration may underlie the action of lithium in treating bipolar affective disorder.

Serotonin2/1C receptor activation causes a localized expression of the immediate-early gene c-fos in rat brain: evidence for involvement of dorsal raphe nucleus projection fibres.

Immunocytochemistry has been used to monitor the expression of the immediate-early gene c-fos in rat brain following administration of the serotonin2/1C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane. At parenteral doses of 2 or 8 mg/kg the drug caused a highly localized expression of the Fos protein in frontal, parietal, cingulate and piriform cortex as well as in claustrum, mamillary bodies, globus pallidus, amygdala, nucleus accumbens and dorsomedial striatum. In particular, the location of heavy Fos immunoreactivity in the primary somatosensory cortex corresponds precisely to that region (layer Va) shown in other reports to receive a dense input of fine, non-varicose fibres which may arise from the dorsal raphe nucleus. All of the Fos-positive brain regions in the present study have been previously demonstrated to contain serotonin2 receptor ligand binding sites. Interestingly, no Fos-positive cells were found in the hippocampus, another brain region known to contain serotonin2 receptors. Pretreatment of animals with the serotonin2/1C receptor antagonist ritanserin (0.4 mg/kg) markedly attenuated Fos expression in all reactive areas of the brain. Counts of reactive cells indicated that this antagonism of the Fos response was statistically significant in these brain regions. Spiperone (1 mg/kg), a mixed serotonin2 and dopamine D2 antagonist, also attenuated the Fos response in the same regions, but had the effect of inducing Fos expression on its own in other extrapyramidal brain regions. Double labelling of reactive cells with different antisera recognizing Fos and neuron-specific enolase, and lack of double labelling with a glial fibrillary acidic protein antiserum, indicated that the Fos expression was in neurons within the brain nuclei examined.(ABSTRACT TRUNCATED AT 250 WORDS)