Behavioral and serotonergic consequences of decreasing or increasing hippocampus brain-derived neurotrophic factor protein levels in mice
Introduction
Antidepressants such as Selective Serotonin Reuptake Inhibitors (SSRI) act as indirect agonists of monoamine receptors. Although SSRI drugs produce relatively rapid blockade of serotonin (5-HT) transporters (SERT) in vitro, the onset of clinical benefits usually takes several (4–6) weeks to occur. The paradox between SSRI near-immediate effects on brain neurotransmitter systems and the slow symptomatic recovery in treating major depressive episodes has not been solved yet. Recent years have provided new information about changes in neurogenesis (mainly cell proliferation) and brain-derived neurotrophic factor (BDNF) protein levels in the adult hippocampus following chronic treatment with antidepressant drugs. Indeed, chronic, but not acute, SSRI treatment by increasing 5-HT neurotransmission causes an increase in BDNF expression (mRNA levels) most notably in the dentate gyrus granular cell layer of the hippocampus in adult rats (Nibuya et al., 1995, Nibuya et al., 1996, Malberg et al., 2000) and mice (Santarelli et al., 2003). In addition, animal studies have shown that neurogenesis can be decreased by a variety of stimuli (aging; various stressors; glucocorticoids), while antidepressant drugs are able to reverse the effects (Duman et al., 2001). More recently, it was shown that chronic fluoxetine treatment accelerates the maturation and functional integration of newborn, immature neurons in the dentate gyrus in wild-type SvEv129 adult male mice (Wang et al., 2008).
Thus, a positive regulation of 5-HT on the expression of the gene coding for BDNF may occur in adult hippocampus. At post-synaptic levels, a growth factor, BDNF requires activation of the high-affinity protein kinase receptor family TrkB (Tropomyosine-related kinase B) to exert its biological effects. However, the actual knowledge regarding the relationship between BDNF and serotonin (5-HT) in the hippocampus is limited. For example, is there any reciprocal effect of BDNF on 5-HT neurotransmission? To answer this question, we have developed a dual experimental strategy by inducing either a decrease or an increase in BDNF protein levels in mouse brain.
First, we studied the SSRI response in heterozygous BDNF+/− mice, in which brain BDNF protein levels are decreased by half (Korte et al., 1995). These constitutive mutants develop enhanced inter-male aggressiveness and hyperphagia accompanied by significant weight gain in early adulthood. These behavioral abnormalities are known to correlate with 5-HT dysfunction (Lyons et al., 1999). In the present review, we summarized our data obtained in these mutant mice.
Second, we increased BDNF protein levels by using its local infusion into adult hippocampus in wild-type mice. Indeed, it was already shown that BDNF increases activity of brain monoaminergic systems in rats (Siuciak et al., 1996). Consistent with these preliminary findings, it has been previously reported that intra-hippocampal BDNF injection induces a dose-dependent antidepressant-like effect in rats that was observed 3 days and lasted up to 10 days after its bilateral injection (Shirayama et al., 2002). In awake freely moving mice, an acute intra-hippocampal injection of BDNF decreased basal and KCl-evoked release of 5-HT in adult hippocampus, as measured using microdialysis (Benmansour et al., 2008). Interestingly, these effects of BDNF were blocked by the non-selective antagonist of TrkB receptors, K252a (Deltheil et al., 2008, Benmansour et al., 2008). Furthermore, BDNF potentiated the effects of an acute systemic SSRI administration or that of locally applied citalopram injection on dialysate 5-HT levels in the ventral hippocampus of adult mice.
Here, we extended this approach by studying the behavioral consequences of the intra-hippocampal BDNF injection combined with a systemic SSRI administration.
To study the relationship between BDNF and 5-HT neurotransmission in the hippocampus, we used adult BDNF+/− mice. We assessed the 5-HT reuptake capacity of the selective transporter SERT in vitro and in vivo. We reasoned that, if BDNF reduction plays a pivotal role in depression, a constitutive decrease in hippocampal BDNF in mutant mice would alter the efficacy of SSRI treatment.
The first BDNF mutant mice were generated by Ernfors et al., in 1994 because this member of the neurotrophin family can prevent the death of particular peripheral sensory neurons and of central motor neurons as well as dopaminergic and cholinergic neurons of the basal forebrain during development. Due to the early postnatal lethality of BDNF null mice, constitutive heterozygous BDNF+/− mice (Korte et al., 1995) or mice lacking its main TrkB receptor (Saarelainen et al., 2003) were used. Mice over-expressing the truncated isoform of the TrkB receptor were also generated (Sairanen et al., 2005, Rantamäki et al., 2007) as a supplementary model of blunted BDNF neurotransmission. Results obtained with conditional KO mice with floxed BDNF alleles allowing spatial and temporal regulation of BDNF deletion only appeared later on (Rios et al., 2001).
Young adult heterozygous BDNF+/− mice generated on a 129 Sv genetic background exhibit alterations in the expression of several 5-HT receptor mRNA particularly in the cortex, hippocampus, and hypothalamus (Lyons et al., 1999). Thus, from this pioneer study, we have learned that endogenous BDNF is critical for the normal development and function of central 5-HT neurons and for the elaboration of behaviors that depend on these nerve cells. Proliferation of adult progenitors and survival of immature neurons are significantly decreased in BDNF+/− mice (Lee et al., 2002). Therefore, BDNF+/− mice provide a useful model to study human psychiatric disorders related to dysfunction of serotonergic neurons.
We have already shown that constitutive BDNF+/− mice have increased basal extracellular 5-HT levels in the hippocampus associated with a decreased 5-HT reuptake capacity: these data have been published by Guiard et al. (2008). The following main results were obtained.
Conventional intracerebral microdialysis technique suggests that constitutive decreases in BDNF expression produced an elevation in basal dialysate 5-HT levels in the ventral hippocampus (Table 1). In addition, dialysate levels of its major metabolite, 5-hydroxy-indoleacetic acid (5-HIAA) were significantly reduced in the ventral hippocampus in BDNF+/− mice compared to BDNF+/+ mice (p < 0.05).
In vitro [3H]-5-HT uptake by synaptosomes prepared from the hippocampus was decreased in BDNF+/− mice compared to BDNF+/+ mice (Fig. 1). Constitutive reductions in BDNF significantly affected Vmax (528 ± 32 versus 942 ± 59 pmol/mg protein per min in BDNF+/− mice and BDNF+/+ mice, respectively, p = 0.007), but not Km values for [3H]-5-HT uptake.
In the ventral hippocampus, a systemic administration of paroxetine (8 mg/kg, i.p.) increased extracellular 5-HT levels in BDNF+/+ mice, but produced a smaller non-significant effect in BDNF+/− mice (Fig. 2A). Interestingly, we also reported that the neurochemical effects of paroxetine did not differ between BDNF+/− and BDNF+/+ mice in the frontal cortex (Fig. 2B) and dorsal raphe nucleus (Fig. 2C), both regions expressing SERT protein.
Autoradiographic study revealed a significant reduction in the number of [3H]-citalopram binding sites in the ventral hippocampus of BDNF+/− mutants compared to BDNF+/+ mice. In particular, a significant decrease in [3H]-citalopram binding sites was measured in the CA3 (p < 0.01, Table 2), but not in the dentate gyrus and CA1 (p > 0.05) sub-regions of the hippocampus in BDNF+/− mutants compared to BDNF+/+ mice. No differences in the density of the labeling were noted in other brain regions such as frontal cortex, striatum and raphe nuclei with respect to genotype.
Measurements of SERT mRNA transcripts in the brain stem of BDNF+/+ controls and heterozygous BDNF+/− mice revealed that no significant differences in SERT mRNA levels occurred in the brain stem between the two genotypes (Deltheil et al., 2007).
Taken together, these data suggest that an enhancement of basal extracellular 5-HT levels (5-HText) induced a down-regulation of SERT, i.e., a decrease in its capacity to reuptake 5-HT, at serotonergic nerve terminals located in the hippocampus of adult BDNF+/− mice. 5-HT neurotransmission seems to be regulated by BDNF in a region-dependent manner. Results obtained by using chronoamperometry confirmed that 5-HT clearance rate increased markedly with age, and suggests that the profoundly reduced ability of 5- and 10-month-old BDNF+/− mice to clear 5-HT may be due to functional deficits in SERT, e.g., in the machinery/signaling required for insertion of SERTs into the plasma membrane and/or activation of the SERT once it is positioned to take up 5-HT from extracellular fluid (Daws et al., 2007).
In a second part of our study, BDNF protein levels were increased following its local infusion (100 ng) into adult hippocampus. We performed intracerebral microdialysis and behavioral experiments in wild-type mice.
Effects of local intra-hippocampal BDNF injection on paroxetine-induced changes in hippocampal extracellular 5-HT levels in wild-type mice. BDNF potentiated the effects of a systemic administration of paroxetine (4 mg/kg, i.p.) on dialysate 5-HT levels in the adult ventral hippocampus in mice (Fig. 3; from Deltheil et al., 2007).
Paroxetine, and also BDNF, increased the swimming duration in the FST in adult male Swiss mice (*p < 0.05 for both drugs). Co-administration of [BDNF + paroxetine] potentiated this effect (***p < 0.001 when compared to the effects of paroxetine alone; Fig. 4).
Mice treated acutely with paroxetine (4 mg/kg) or vehicle followed by an intra-hippocampal BDNF (100 ng) injection were tested in the open field (OF) to assess their anxiety-like state. BDNF induced an anxiogenic-like profile since a significant decrease in the time spent in the center was measured (p < 0.001; Fig. 5). The anxiogenic-like effect after an intra-hippocampal BDNF injection did not affect the locomotor activity (data not shown). By contrast to paroxetine, the benzodiazepine diazepam (1 mg/kg, i.p. used here as a reference drug), displayed an anxiolytic-like activity since it induced an increase in the time spent in the center (p < 0.05). Finally, the anxiogenic-like effect of BDNF was prevented by a paroxetine pre-treatment since co-administration of paroxetine with BDNF did not affect the time spent in the center (p > 0.05).
Section snippets
Discussion and conclusion
The present study assessed whether a decrease (i.e., a constitutive deletion of one copy of the BDNF gene) or an increase (i.e., a local intra-hippocampal BDNF injection) in BDNF protein levels in the mouse brain can affect hippocampal 5-HT neurotransmission in adulthood. In both cases, decreasing or increasing BDNF protein levels led to selective alterations of 5-HT neurotransmission in the mouse adult ventral hippocampus.
First, heterozygous BDNF+/− mice (generated by Korte et al., 1995)
Acknowledgments
D.T., C.J. were recipient of a fellowship from “Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche” (MENESR, Paris, France) during the performance of this work.
This work has been supported by the technical assistance of the Animal care facility of the “Institut Fédératif de recherche-IFR141” of the Paris XI University.
References (30)
- et al.
Conditional mutant mice using tetracycline-controlled gene expression system in the brain
Neurosci. Res.
(2007) - et al.
Influence of brain-derived neurotrophic factor (BDNF) on serotonin neurotransmission in the hippocampus of adult rodents
Eur. J. Pharmacol.
(2008) - et al.
Postsynaptic 5-HT1A receptors mediate 5-hydroxytryptamine release in the amygdala through a feedback to the caudal linear raphe
Eur. J. Pharmacol.
(1997) - et al.
Control of serotonergic neurons in the dorsal raphe nucleus by the lateral hypothalamus
Brain Res.
(2002) - et al.
Consequences of altered brain-derived neurotrophic factor protein levels on hippocampal serotonin neurotransmission and behavior
Pharmacol. Biochem. Behav.
(2008) - et al.
In vivo inhibition of neuronal activity in the rat ventromedial prefrontal cortex by midbrain-raphe nuclei: role of 5-HT1A receptors
Neuropharmacology
(2003) - et al.
The in vivo microdialysis recovery of dopamine is altered independently of basal level by 6-hydroxydopamine lesions to the nucleus accumbens
J. Neurosci. Methods
(1991) - et al.
BDNF increases monoaminergic activity in rat brain following intracerebroventricular or intraparenchymal administration
Brain Res.
(1996) - et al.
Exploring the relationship between serotonin and brain-derived neurotrophic factor: analysis of BDNF protein and extraneuronal 5-HT in mice with reduced serotonin transporter or BDNF expression
J. Neurosci. Methods
(2004) - et al.
Control of dorsal raphe serotonergic neurons by the medial prefrontal cortex: involvement of serotonin-1A, GABA(A), and glutamate receptors
J. Neurosci.
(2001)
Serotonin transporter function, but not expression, is dependent on brain-derived neurotrophic factor (BDNF): in vivo studies in BDNF-deficient mice
J. Neurochem.
Regulation of adult neurogenesis by psychotropic drugs and stress
J. Pharmacol. Exp. Ther.
Effects of chronic paroxetine treatment on dialysate serotonin in 5-HT1B receptor knockout mice
J. Neurochem.
Transgenic brain-derived neurotrophic factor expression causes both anxiogenic and antidepressant effects
Proc. Natl. Acad. Sci. U.S.A.
Brain-derived neurotrophic factor-deficient mice exhibit a hippocampal hyperserotonergic phenotype
Int. J. Neuropsychopharmacol.
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The first two authors contributed equally to this work.