The lateral habenula (LHb) is a well-conserved epithalamic nucleus that participates in value-based decision-making (Hikosaka, 2010). LHb neurons are more active during punishment or following the absence of an expected reward. In this manner, LHb activity signals the negative valence associated with a decision (Hikosaka et al., 2008; Hikosaka, 2010; Metzger et al., 2017). Moreover, increased activity of the LHb is seen in response to chronic stress (Hikosaka, 2010; Nuno-Perez et al., 2018). In humans, positron emission tomography has been used to demonstrate that LHb activity is associated with depressed mood (Morris et al., 1999). In rodents, inhibition of the LHb has been shown to reverse social withdrawal and despair seen in animals subjected to chronic stress (Winter et al., 2011; Sachs et al., 2015; Tchenio et al., 2017). Finally, modulation of the LHb with DBS has been demonstrated to be an effective therapy in a patient with treatment resistant depression (TRD) (Sartorius et al., 2010). For these reasons, the LHb may be an important therapeutic target for depression.

To employ neuromodulation of the LHb for therapeutic purposes, its anatomy in humans must be characterized and targeted. The LHb receives input from a number of brain regions via the stria medullaris (SM). Predominant inputs include the ventral pallidum and the globus pallidus internus (GPi) which is homologous to the rodent entopeduncular nucleus (Knowland et al., 2017; Hu et al., 2020; Liu et al., 2020). Prior studies in the social defeat mouse model of depression have also demonstrated that these inputs mediate depression-like behavior (Knowland et al., 2017; Liu et al., 2020). Furthermore, the LHb serves as a regulator of the dopaminergic system via the fasciculus retroflexus (FR) which connects the LHb to the ventral tegmental area (VTA) (Wise, 2004; Hikosaka et al., 2008). To characterize these fiber tracts in human subjects, we utilized diffusion magnetic resonance imaging (dMRI). Prior studies have reconstructed the SM with dMRI using the LHb as a seed region for probabilistic tractography (Strotmann et al., 2014; Kochanski et al., 2016, 2018; Grodd et al., 2020). While effective, this strategy does not allow for selection of specific input and output tracts that may be associated with depressive symptomatology. Additionally, this strategy requires manual selection of the LHb which can be difficult due to its small size. Here, we selected the pallidum as the seed region and the VTA as the target region to reconstruct the SM and FR. This strategy allowed us to reconstruct these tracts without manual selection of the LHb and enabled characterization of the specific fiber tracts implicated in depression.

In this study, we describe a novel method for reconstruction of the human afferent (SM) and efferent (FR) tracts of the LHb using dMRI data. Prior studies used the LHb itself as a seed region to generate the putative afferent and efferent tracts of this epithalamic nucleus (Strotmann et al., 2014; Kochanski et al., 2016, 2018; Grodd et al., 2020). The LHb is a very small brain region; therefore, manual selection is difficult and may lead to operator dependent variability and inconsistencies in tractography results (Schilling et al., 2021). Our technique employs a seed region automatically generated by FreeSurfer (Fischl, 2012) segmentation, thereby making this method more reliable and operator independent. While the strategy employed here does require manual outlining of the VTA, this region is larger than the LHb and multiple anatomic landmarks exist to identify this region in a consistent manner (Coenen et al., 2012).

Given the LHb’s relevance in affective dysfunction (Hu et al., 2020), targeting this nucleus and/or the SM/FR may be important for therapeutic purposes. The method employed here allows for identification of these tracts for surgical targeting. One limitation of this technique is that the streamlines include both the afferents and efferents, so they are not easily separable. Notably, other previously published techniques have this limitation as well. Furthermore, the LHb itself may be identified as the brain region adjacent to the third ventricle with the highest streamline density (Figure 2). We first used the high quality HCP dMRI data to develop and validate our tractography method and then demonstrated that this method is generalizable to dMRI data that may be acquired in a clinical setting. Hence, this tractography strategy may potentially be used for treatment purposes in the future.

After developing a strategy to generate the SM/FR tracts, we then proceeded to further characterize these tracts. We found hemispheric asymmetry in the number of streamlines generated in a majority of individuals, and this asymmetry was statistically significant in right-handed subjects. The LHb is a well-conserved epithalamic nucleus found in all vertebrates (Bianco and Wilson, 2009). Structural studies in multiple species, including humans, have demonstrated that there are hemispheric differences in the volume of the lateral habenular nucleus (Bianco and Wilson, 2009; Ahumada-Galleguillos et al., 2017). Interestingly in many species of fish, the right LHb is much larger than the left; however, in mammals the hemispheric difference is less pronounced and the left LHb is often larger than the right (Bianco and Wilson, 2009; Ahumada-Galleguillos et al., 2017). To our knowledge, our study is the first to demonstrate in living human subjects that the input and output streamlines of the LHb exhibit hemispheric asymmetry.

In addition to structural asymmetry, several groups have reported differences in LHb function between the two hemispheres (Lawson et al., 2014; Hennigan et al., 2015; Amiri et al., 2021). One group used resting state functional MRI (fMRI) to show that patients with TRD had increased functional connectivity of the left LHb compared to controls (Amiri et al., 2021), while another group demonstrated decreased node degree of the right LHb in individuals with subclinical depression relative to controls (Zhu et al., 2019). Other groups have explored the role of the LHb using fMRI in healthy subjects and have demonstrated that LHb activity is associated with an aversive stimulus (shock) (Lawson et al., 2014; Hennigan et al., 2015). While both studies demonstrated asymmetric LHb activation, Hennigan et al. (2015) reported increased activity of the left LHb compared to the right, but Lawson et al. (2014) reported increased activity of the right LHb, not the left. Lawson and colleagues suggested that they observed right LHb activation because the shock was delivered to the subjects’ left hand (Lawson et al., 2014). In contrast, the Hennigan et al. study reported left LHb activation with shocks delivered to the subjects’ left foot (Hennigan et al., 2015). These authors did not speculate regarding the cause of asymmetric LHb activation, but their results do not support the hypothesis that the location of the noxious stimulus dictates which LHb nucleus is activated. Considered together, the structural and functional data suggest that the left LHb is dominant in most individuals. Neither of the fMRI studies reported the handedness of their subjects. Our structural data suggest that handedness may potentially explain the divergent findings of these studies. Although the streamline count was greater on the left in 80% of right-handed subjects, there were some subjects with a greater streamline count on the right. Asymmetric LHb volume or afferent/efferent streamline number may correlate with functional LHb asymmetry, and future studies could explore the correlation between LHb structure and function within individuals.

Psychological studies have demonstrated that individuals associate positive valence to objects/individuals in the space in which their dominant hand acts more easily (i.e., right-handed individuals associate positive valence to objects in rightward space) (Casasanto, 2009). It is intriguing to speculate that LHb functional/structural asymmetry may contribute to this behavior. While a recent meta-analysis did not find handedness to be a risk factor for depression (Packheiser et al., 2021), hemispheric differences in the treatment of depression using transcranial magnetic stimulation (TMS) have been observed (Chung et al., 2015) suggesting that LHb streamline asymmetry may be of therapeutic importance as well.

After exploring hemispheric asymmetry, we next investigated sex differences in LHb streamline number. We found that women have a greater number of SM/FR streamlines on average compared to men. Our group has previously demonstrated that significant structural and functional differences exist between the male and female human brain (Ingalhalikar et al., 2014; Tunç et al., 2016). The current study supports and extends these findings by revealing increased LHb afferent/efferent streamline count in females compared to males. Female sex is a well-known risk factor for depression and TRD (Taipale et al., 2020). Given the LHb’s role in processing stimuli of negative valence, it is intriguing to speculate that increased LHb streamline number may contribute to susceptibility to depression. One limitation of this study is that the streamlines were not reconstructed in individuals with a diagnosis of major depressive disorder. Future studies could more thoroughly explore the relationship between sex, LHb streamline count, and depression.

Using the input and output brain regions of the LHb, we have developed a method to reconstruct the afferent and efferent tracts of this epithalamic nucleus without having to identify it manually, which is challenging and error prone due to its small size. This technique enables surgical targeting of the SM and FR tracts as well as indirect LHb targeting. Furthermore, we have demonstrated that these streamlines are more prominent in the female human brain and that most right-handed individuals have a greater number of streamlines in the left hemisphere. These findings may have therapeutic implications for treating affective disorders such as TRD and warrant further investigation in larger studies that have clinical measures for correlation.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

FH: conceptualization, methodology, formal analysis, writing – original draft, and visualization. DP: resources and data curation. AY: writing – review and editing and conceptualization. SB: writing – review and editing, supervision, and funding acquisition. RV: conceptualization, writing – review and editing, supervision, and funding acquisition. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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