ZFPM1 Necessary for Development of Serotonergic Projections Related to Anxiety and Contextual Fear Learning

The serotonergic system is strongly implicated in anxiety and depression, and it is the first-line target for pharmacological treatment (Tamblyn et al., 2019). Genetic factors predispose people to these disorders, but the anatomic and molecular diversity of the main serotonergic nuclei, the dorsal raphe (DR) and median raphe (MR), and their extensive projections make establishing causal links among genes, physiology, and etiology complex. Nevertheless, genome-wide screening can highlight correlations with genetic or epigenetic markers (Li et al., 2016), whose effects can then be tested through gene knockout in animal models.

A genome-wide screening by Baselmans et al. (2015) found that Zfpm1 methylation was negatively correlated with factors associated with well being. ZFPM1 is a zinc-finger cofactor that interacts with the GATA family of transcription factors and is expressed in certain serotonergic and GABAergic neurons from early development to adulthood. Therefore, Tikker et al. (2020) recently used selective gene knockout to examine the role of ZFPM1 in development and behavior.

Tikker et al. (2020) generated Zfpm1 conditional knockout (Zfpm1^CKO) mice in which Zfpm1 was knocked out selectively in rhombomere 1, an embryonic hindbrain segment in which serotonin neurons are derived. The number of serotonergic neurons was reduced in the ventrolateral DR (DRVL) and increased in the dorsal DR (DRD) in Zfpm1^CKO mice. Notably, a substantial number of DRD serotonergic neurons expressed the DRVL-specific marker BCL11B, and there was a decrease in the number of serotonergic cells expressing this marker in DRVL. No change was detected in ventral DR (DRV) or B8 MR areas, and, importantly, the total number of neurons across DR nuclei was unaffected. Based on these findings, the authors hypothesized that Zfpm1 knockout disrupted cell positioning, rather than generation. The authors speculate that Zfpm1 regulates positioning by repressing the transcriptional processes that cause medial migration of serotonergic precursors, and thus knocking out Zfpm1 causes DRVL neurons to be displaced to DRD.

Notable effects of Zfpm1 knockout were also produced at DR projection sites. The density of serotonergic axons was greatly reduced in the dorsal hippocampus (dHPC), ventral hippocampus (vHPC), dorsal lateral geniculate nucleus, and superior colliculus. In contrast, the density increased in the basolateral amygdala (BLA), although less consistently than the reductions seen elsewhere.

Knockout of Zfpm1 also affected mouse behavior. In an elevated plus maze, Zfpm1^CKO mice made less open arm entries than control littermates, indicating anxiety-like behavior. In a recognition task, Zfpm1^CKO mice spent less time than controls exploring novel objects. Zfpm1^CKO mice also showed more freezing during contextual fear conditioning compared with controls, and this effect was abolished by treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine. Tikker et al. (2020) tested for depression-like behaviors with a forced swim test but found no significant differences between control and Zfpm1^CKO mice, and both groups responded equally positively to fluoxetine in this test. Overall, these results suggest that alterations in specific projections led to enhanced fear and anxiety-like responses in Zfpm1^CKO mice, while behaviors dependent on spared serotonergic projections were unaffected.

The two key findings of Tikker et al. (2020) were that Zfpm1 is critically involved in normal development of the DR serotonergic system and that alterations in Zfpm1 produce anxiety-like and fear-related behaviors. The article also raised two important questions. First, how do the serotonergic axonal density changes produced by Zfpm1 knockout align with our current understanding of DRVL and DRD projections? Second, how does each of the affected areas contribute to the behavioral phenotype seen?

The reduction in hippocampal serotonergic afferents in Zfpm1^CKO mice is surprising if attributed solely to the displacement of DRVL to DRD. In a previous tracing study, Muzerelle et al. (2016) explicitly noted a lack of projections from the DRVL to the hippocampus. This suggests that Zfpm1 knockout either affected a hippocampal-projecting area not examined by Tikker et al. (2020) or altered axonal targeting of neurons in the examined DR regions without affecting cell placement. Serotonergic projections to the hippocampus include dense efferents from the caudal DR (DRC) (B6), MR (B8), and some sparse efferents from DRV and DRD (Soiza-Reilly and Gaspar, 2020). Of these, only the DRC was not specifically examined by Tikker et al. (2020). It may be that Zfpm1 knockout affected the DRC, which is adjacent to the DRD, but this was missed because these neurons were included with other regions during analysis. Substantiating this possibility, it was noted that the DRVL region in which significant change was found was situated caudally. Alternatively, Tikker et al. (2020) may have limited their analysis to B7 cells, which includes the DRD, DRVL, and DRV but excludes DRC, and so did not note changes in the DRC. Complicating the DRC hypothesis, Kast et al. (2017) found that the majority of DRC serotonergic neurons project strongly to the vHPC but not the dHPC, whereas Tikker et al. (2020) found both hippocampal regions were equally affected. Still another possibility is that Zfpm1 knockout altered axonal targeting in the hippocampal projecting MR without affecting cell location. A further experiment using retrograde tracers in the hippocampus of Zfpm1^CKO mice might shed light on the curious decrease in hippocampal serotonergic axon density.

The enhanced anxiety-related behavior and contextual fear conditioning reported by Tikker et al. (2020) are consistent with the known functions of the regions affected by Zfpm1 knockout. In particular, the DRVL is strongly linked to anxiety-like behaviors. The intrinsic excitability properties of DRVL neurons appear to mediate greater activation by stressful stimuli (Crawford et al., 2010). For example, one previous study showed that an elevated T-maze task increased c-fos expression specifically in the DRVL (Vilela-Costa et al., 2019). That same study also showed that chronic, but not acute, fluoxetine treatment increased time spent in the open arm, decreased c-fos expression in nonserotonergic cells, and increased c-fos expression in serotonergic cells. 5-HT1A heteroreceptors on nonserotonergic cells were speculated to be a mechanism for this shift. The effect of fluoxetine suggests DRVL serotonergic output is anxiolytic, while DRVL nonserotonergic activity may be anxiogenic. Consequently, anxiety-like behavior may arise in Zfpm1^CKO mice through both decreased serotonergic output in DRVL projection sites and increased nonserotonergic activity in the DRVL.

Reduction in the number of serotonergic neurons in DRVL was mirrored by an increase in DRD. This increase may also be instrumental in the anxiety-like behavioral phenotype. Ren et al. (2018) found that activation of DR projections to the central amygdala (CeA) promoted anxiety-like behaviors. In addition, activity of serotonergic neurons in the CeA strongly increased after footshock (Ren et al., 2018). And inhibition of CeA neurons that express 5-HT2A receptors downregulates learned fear responses (Isosaka et al., 2015). Accordingly, increased DRD serotonin output in the CeA in response to footshock may contribute to the enhanced contextual fear conditioning.

As the hippocampus is associated with contextual fear learning and anxiety-like behaviors, reduced serotonergic afferents could play a key role. In contextual fear conditioning, hippocampal neurons have been proposed to represent the contextual cues that come to elicit a conditioned response by exciting the BLA, which in turn activates the CeA to produce fear responses (Maren et al., 2013). Waider et al. (2019) showed that knockout of Tph2, which is necessary for serotonin production, increased activity in the dHPC along with fear responses. Conversely, Mlinar and Corradetti (2018) found that application of serotonin in the dHPC decreased activity. Therefore, Zfpm1 knockout may enhance fear responses by reducing serotonergic inhibition in the dHPC, thus enhancing activation of BLA. Notably, Zfpm1 knockout also slightly increased the number of serotonergic axons in the BLA itself. Furthermore, the BLA has been linked to neophobia (Sarowar et al., 2017), another behavioral phenotype reported by Tikker et al. (2020).

Intriguingly, chronic serotonin depletion in the hippocampus, as presumably occurred in Zfpm1^CKO mice, appears to paradoxically produce anxiogenic effects similar to those of acute serotonin activation. For example, Ohmura et al. (2020) found that optogenetic activation of serotonergic terminals in vHPC was anxiogenic. This seeming contradiction may mirror the effect of SSRIs in acutely increasing synaptic serotonin, worsening anxiety and depression, but chronically rebalancing release and uptake to alleviate symptoms. One long-term effect of serotonin in the hippocampus that may contribute to improving symptoms is the upregulation of neurogenesis. Mahar et al. (2014) describe how chronic unpredictable stress decreases both DR output and hippocampal neurogenesis, producing depression-like behavior, all of which are alleviated by chronic SSRIs. Tikker et al. (2020) performed a test for depression, the forced swim test, and they found fluoxetine-induced immobility reduction was unaffected by Zfpm1^CKO mice. However, Mahar et al. (2014) discuss how fluoxetine decreases immobility independently of hippocampal neurogenesis. Therefore, the reduction in serotonergic afferents to the hippocampus in Zfpm1^CKO mice may have reduced neurogenesis without influencing the forced swim test. Further investigation of depression-like behavior and hippocampal neurogenesis may be fruitful in Zfpm1^CKO mice.

As a final point of interest, the role of Zfpm1 in hippocampal function may not be limited to the developmental effects discussed here. Li et al. (2016) identified Zfpm1 as a novel gene that is methylated in the hippocampus following acute stressful restraint. This suggests a dynamic role for Zfpm1 in the adult brain, which warrants future examination.

In conclusion, Tikker et al. (2020) found that Zfpm1 is required for the normal development of DRVL serotonergic neurons that project to brain regions involved in anxiety and fear learning. The behavioral phenotype found in Zfpm1^CKO mice suggests that Zfpm1 may be a useful genetic or epigenetic etiologic marker for investigating anxiety-related disorders.

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