Efferent pathways of the mouse lateral habenula

Abstract

The lateral habenula (LHb) is part of the habenula complex of the dorsal thalamus. Recent studies of the LHb have focused on its projections to the ventral tegmental area (VTA) and rostromedial tegmental nucleus (RMTg), which contain γ-aminobutyric acid (GABA)ergic neurons that mediate reward prediction error via inhibition of dopaminergic activity. However, older studies in the rat have also identified LHb outputs to the lateral and posterior hypothalamus, median raphe, dorsal raphe, and dorsal tegmentum. Although these studies have shown that the medial and lateral divisions of the LHb have somewhat distinct projections, the topographic specificity of LHb efferents is not completely understood, and the relative extent of these projections to brainstem targets is unknown. Here we have used anterograde tracing with adeno-associated virus-mediated expression of green fluorescent protein, combined with serial two-photon tomography, to map the efferents of the LHb on a standard coordinate system for the entire mouse brain, and reconstruct the efferent pathways of the LHb in three dimensions. Using automated quantitation of fiber density, we show that in addition to the RMTg, the median raphe, caudal dorsal raphe, and pontine central gray are major recipients of LHb efferents. By using retrograde tract tracing with cholera toxin subunit B, we show that LHb neurons projecting to the hypothalamus, VTA, median raphe, caudal dorsal raphe, and pontine central gray reside in characteristic, but sometimes overlapping regions of the LHb. Together these results provide the anatomical basis for systematic studies of LHb function in neural circuits and behavior in mice. J. Comp. Neurol. 523:32-60, 2015. © 2014 Wiley Periodicals, Inc.

Keywords: Lateral habenula; median raphe; rostromedial tegmental nucleus; ventral tegmental nucleus.

Figure 1. Anterograde tract tracing injection sites and LHb pathway reconstruction

An rAAV expressing an eGFP tract-tracing protein (Methods) was injected into the LHb, and the LHb output pathways were reconstructed using serial two-photon fluorescence tomography. (A–C) rAAV tracing of LHb/a1 (Allen case 147353537). The rAAV injection was positioned in the dorsolateral quadrant of the caudal LHb at bregma -1.9mm. Tracer expression was also evident in the DG, which did not contribute to brainstem projections. (A) Fluorescence signal at injection site. Dashed white line designates the border between MHb and LHb. (B) Anatomical map of the injected area, adapted from a standard atlas (Paxinos and Franklin, 2001). (C) Sagittal reconstruction of fiber tracts. Stippling indicates the area of viral spread. (D–F) rAAV tracing of LHb/a2 (Allen case 120875111). The rAAV injection was positioned in the ventromedial quadrant of the central LHb at bregma -1.6. Tracer expression was also evident in the PV, which did not contribute to brainstem projections. (D) Fluorescence signal at injection site. Dashed white line designates the border between the MHb and LHb; dashed black line indicates border between the LHb and the PV. (E) Anatomical map of the injected area. (F) Sagittal reconstruction of fiber tracts. An extensive projection from PV to the cerebral ganglia and rostral hippocampus is not shown. Nomenclature is derived from Paxinos (2001), see Table.

Figure 2. LHb projections to the hypothalamus

The LHb was injected with a tract-tracing AAV as described in Figure 1. Only case LHb/a2, labeling the ventromedial LHb, showed significant output to the hypothalamus, and is shown here. Views shown are from 2-photon fluorescence imaging (left) and fiber segmentation analysis (right, Methods), and are shown in caudal to rostral order, progressively farther from their source. (A,B) Labeled fibers in the rostral tegmentum, just caudal to the LH, compared to a standard atlas view at bregma -3.06. Ascending fibers that have separated from the FR and turned rostrally are marked with an asterisk. Decussating fibers are marked with arrowheads. Only rare labeled fibers are observed in the VTA at this level. (C,D) Labeled fibers in the PH, compared to a standard atlas view at bregma -2.54. Labeled fibers are also observed in the LH bilaterally. (E,F) Labeled fibers at the level of the median eminence, compared to a standard atlas view at bregma -2.30. Labeled fibers are observed mainly in PH. Scale: 200μm.

Figure 3. LHb projections to the rostral brainstem

The LHb was injected with a tract-tracing AAV as described in Figure 1. Labeled fibers are shown for case LHb/a1, injected in the dorsolateral LHb, and LHb/a2, injected in the ventromedial LHb. Images here and in Figure 6 are shown in rostral to caudal order, progressively farther from their source. (A,B) Labeled fibers in the descending FR, compared to a standard atlas view at bregma -3.16. Fibers of passage dominate at this level, but sparse terminal fibers can be seen in the VTA bilaterally (arrows, A). (C,D) Labeled fibers in the RMTg, compared to a standard atlas view at bregma -4.04. The anatomical localization of the RMTg is based on prior studies in the rat (Jhou et al., 2009b; Kaufling et al., 2009). The intensity of the specific ipsilateral projection to the RMTg is much stronger in case LHb/a1, injected in the dorsolateral LHb. Although fibers of passage cannot be definitively distinguished from terminal fibers using anterograde tracing with AAV, the dense labeling of the IP in LHb/a2 is likely to represent fibers of passage projecting to more caudal regions (Figures 6,7). (E, F) Labeled fibers in the MnR and PMnR compared to a standard atlas view at bregma -4.36. In case LHb/a1 there is a strong ipsilateral projection to a region annotated as ATg. However, this appears to be a caudal extension of the RMTg (cRMTg). See text for discussion. Scale: 200μm.

Figure 4. Identification of RMTg neurons in the pontine tegmentum

Retrograde labeling using CTB was used to identify VTA-projecting neurons in the rostral and caudal parts of the RMTg. The injection was performed in Gad1^GFP transgenic mice to facilitate detection of GABAergic neurons. (A) Location of a focal injection of CTB in the VTA. (B) Low-power image of the region containing the rostral RMTg at bregma -3.95. Arrow indicates an area of labeling contiguous with the injection site. Boxed area is enlarged in (D). (C) Low-power image of the region containing the caudal RMTg at bregma -4.4. Boxed area is enlarged in (E). (D) Confocal image of the area boxed in (B), including the rostral RMTg, showing immunofluorescent staining for CTB and GFP. The image is a Z-stack of four adjacent 1μm optical sections and is thus unlikely to include superimposed cells from different levels in the Z-dimension. Gad1^GFP-label is expressed in GABAergic cell somata and diffusely in the neuropil. Gad1-negative cells appear as dark “holes”. Examples of CTB-labeled Gad1^GFP-expressing cells are marked with arrowheads (inset view D′). A single CTB-labeled Gad1^GFP-negative cell is marked with an arrow (inset view D″). (E) Confocal image of the area boxed in (C), including the CTB-labeled cells in the caudal RMTg, an area identified as ATg in standard atlases. Examples of CTB-labeled Gad1^GFP-expressing cells are marked with arrowheads (inset views E′, E″). The image is a Z-stack of six 1μm optical sections. (F) Location of CTB injection in the mammillary body near bregma -2.70. (G) Immunostaining of case shown in (F) sectioned at bregma -4.24 (standard atlas coordinate) shows little retrograde transport of CTB in the area of the caudal RMTg (bracket). (H) Case shown in (F) sectioned at bregma -4.60 (standard atlas coordinate) shows retrograde transport of CTB in the ATg (arrow). Scale: B,C,F, 400μm; D,E, 50μm; G,H, 200μm.

Figure 5. Induction of cFos expression in the mouse RMTg by acute administration of amphetamine

Mice were injected with Amph or saline (N=4 per group), sacrificed after 2 hours, and cFos immunoreactivity was examined throughout the tegmentum and pons. Co-immunostaining for the dopamine transporter (DAT) was performed to localize dopaminergic neurons in the areas examined. (A,B) Expression of cFos at the rostral end of the RMTg. (C,D) Expression of cFos in the central part of the RMTg. (E,F) Expression of cFos in the caudal part of the RMTg, identified as ATg in standard atlases. Scale: A, 200μm.

Figure 6. LHb projections to the dorsal raphe and dorsal tegmental area

The LHb was injected with a tract-tracing AAV as described in Figure 1. Labeled fibers are shown for case LHb/a1, injected in the dorsolateral LHb, and LHb/a2, injected in the ventromedial LHb. (A,B) Labeled fibers in the DRC, DRI and MnR, compared to a standard atlas view at bregma -4.96. Caudal to this level, the extent of labeling in LHb/a2 is much greater than in LHb/a1. (C,D) Labeled fibers in the DRC, DRI, and adjacent dorsal pontine central gray, compared to a standard atlas view at bregma -5.20. (E,F) Labeled fibers in the DRI and pontine central grey, compared to a standard atlas view at bregma -5.34. Note that fibers are observed in the pontine central gray medial and lateral to the DTg nuclei, but not in their core. (G,H) Labeled fibers in the pontine central grey at the most caudal extent of the LHb projection, compared to a standard atlas view at bregma -5.68. Scale: 200μm.

Figure 7. Anterograde labeling of paraventricular thalamic nucleus

Specific labeling of the PV was achieved using two mouse strains Grm2^Cre (case PV/a1) and Ntrk1^cre (case PV/a2) that express Cre-recombinase in the PV but not in the LHb. No anterograde labeling of fibers was seen in any of the hindbrain areas receiving LHb efferents. (A,B) Injection sites in the PV in case PV/a1 and PV/a2. Grm2^Cre activates fairly widespread expression in the medial thalamus. Ntrk1^Cre activates restricted expression in the PV. (C,D) Sections at the level of the rostral part of the RMTg, at a level matched to Figure 3C,D. (E,F) Sections at the level of the caudal part of the RMTg, at a level matched to Figure 3E,F. (G,H) Sections at the level of the DTg/DRI, at a level matched to Figure 6E,F.

Figure 8. Quantitative analysis of LHb projections to the midbrain and pons

Total fiber volume in each of the defined anatomical areas was determined from the segmentation views of case LHb/a1 and LHb/a2 (Methods). Percentage values were calculated based on the total fiber volume in the midbrain + pons. Only areas receiving greater than 2% of the LHb fibers are shown. Fibers detected in the diencephalon were not included in the analysis. (A,B) Distribution of fiber density in case LHb/a1, injected in the dorsolateral LHb. (C,D) Distribution of fiber density in case LHb/a2, injected in the ventromedial LHb. The RMTg was analyzed as a rostral (rRMTg) and a caudal (cRMTg) component. The caudal component has been assigned as ATg in a standard atlas, but this appears to be a misassignment of this structure (see text). As discussed in Methods, because the FR is not precisely annotated in the Allen Reference Atlas, some fibers of passage within the FR were assigned to adjacent structures in this analysis. Areas which may have a significant contribution from these fibers of passage, always on the side ipsilateral to the injection, are annotated with “fr” on the corresponding bar in the graph. Because the quantitative analysis of fiber density was performed in the 3D Allen Reference Atlas space (Dong, 2008), the anatomical areas used here do not always correspond exactly to other published atlases, or may be differently named. Correspondence between the Allen Reference Atlas terms used here and Paxinos (2001) atlas nomenclature is as follows: Ventral tegmental area, VTA (same); Midbrain reticular nucleus, MRN (deep mesencephalic nucleus, DpMe); periaqueductal gray, PAG (same); red nucleus, RN (R); interpeduncular nucleus, IPN (IP), central linear n raphe, CLI (same); dorsal raphe, DR (same); dorsal tegmental nucleus, DTN (DTg); pontine central gray, PCG (CGPn); superior central nucleus raphe, CS (median raphe, MnR; paramedian raphe, PMnR); laterodorsal tegmental nucleus, LDT (LDTg); nucleus incertus, NI (includes multiple regions including the dorsal raphe, interfascicular, DRI); pontine reticular nucleus, PRNr (Pontine reticular n, caudal part, PnC).

Figure 9. LHb projections to the rostral RMTg mapped onto the Allen Reference Atlas

Case LHb/a1, with a strong ipsilateral projection to the RMTg, was used to assign the location of this nucleus in Figures 9–11. The circled areas on the Allen Reference Atlas show the area of integration for the quantitative analysis of the LHb projections to the RMTg in cases LHb/a1 and LHb/a2 (Figure 8). The designated levels relative to bregma correspond to the nearest levels annotated in a standard mouse brain atlas (Paxinos and Franklin, 2001). The correspondence is not exact because the data are acquired at 100μm intervals corresponding to the Allen Reference Atlas, and the standard atlas intervals are irregular. The most rostral RMTg fiber terminals are identified at bregma -3.64. Beginning at bregma -4.16, the strong ipsilateral projections from the LHb coincide with a region designated anterior tegmental nucleus (“ATg”) in a standard atlas (Paxinos and Franklin, 2001). However, in a standard atlas of the rat brain (Paxinos and Watson, 2005), the ATg is not annotated until a more caudal position relative to other structures (rat bregma -7.64mm, approximately equivalent to mouse bregma -4.40), where the strong ipsilateral projection in case LHb/a1 ends. Thus we infer that regions between bregma -4.16 and bregma -4.48 innervated by the strong ipsilateral projection from the LHb corresponds to the caudal part of the RMTg (cRMTg), not the ATg. See text for discussion. Note also that at the level corresponding to bregma -4.72, the location of the ATg (AT) is Allen Reference Atlas is designated VTg in the standard atlas. A full list of abbreviations used in the Allen Reference Atlas appear in the Atlas documentation at: http://help.brain-map.org/display/mousebrain/Documentation

Figure 10. LHb projections to the central RMTg mapped onto the Allen Reference Atlas

See Figure 9 for legend.

Figure 11. LHb projections to the caudal RMTg mapped onto the Allen Reference Atlas

See Figure 9 for legend.

Figure 12. LHb efferent terminal fibers in the hypothalamus and rostral tegmentum

The LHb was injected with an AAV virus encoding a YFP tracer (Methods, case LHb/a3), and YFP-labeled fibers were examined in the tegmentum and pons. (A) Extent of YFP expression in the central LHb, encompassing both the medial and lateral divisions. (B) Extent of YFP expression in the caudal LHb. (C,D) LHb projections to the hypothalamus. Fibers showing a beaded morphology, indicative of terminal fibers, can be seen in the enlargement of the boxed area shown in D (40x, confocal image). (E,F) LHb projections to the central part of the RMTg. Short fibers with punctate areas of intense fluorescence suggestive of fiber terminals characterize this area (F). (G,H) LHb projections to the caudal part of the RMTg. Fibers in this area frequently show a beaded morphology indicative of terminals. Scale: A, C 100μm; D, 25μm.

Figure 13. LHb efferent terminal fibers in the MnR and DTg

Continuation of case LHb/a3 shown in Figure 12. (A,B) LHb projections to the MnR/PMnR. Fibers showing a beaded morphology, indicative of terminal fibers, can be seen in the enlargement of the boxed area shown in B (40x, confocal image). (C,D) LHb projections to the DRC. (E,F) LHb projections to the pontine raphe. (G,H) LHb projections to the area surrounding the PDTg. Scale: A, 100μm; B, 25μm.

Figure 14. Retrograde tracing of the LHb-hypothalamic pathway

LHb projections to the tegmentum were traced by the focal injection of CTB. Sections are counterstained for Pou4f1(Brn3a) which is expressed in the nuclei of neurons distributed through the caudal 2/3 of the LHb. In the rostral LHb, where Pou4f1-expressing neurons are sparse, the lateral border of the LHb is traced with a dashed line. (A,B) Locations of small focal injections of CTB in the PH (standard atlas bregma -2.54) and LH (standard atlas bregma -2-3), respectively. (C–E) Location of neurons labeled from the PH in the rostral, central and caudal LHb, respectively. In the central LHb, labeled neurons were largely confined to the medial division of the nucleus. (F,G) Location of neurons labeled from the LH in the rostral and central LHb, respectively. The area labeled ATg in a standard atlas corresponds to the caudal RMTg (cRMTg, see text). (H,I) Confocal imaging of CTB-labeled neurons in (G). Rare examples of neurons that show co-localization of CTB and Pou4f1 are indicated by arrowheads. (J) Location of neurons labeled from the LH in the caudal LHb. Scale: C, F 100μm; H, 50μm.

Figure 15. Retrograde tracing of LHb projections to the VTA and RMTg

LHb projections to the tegmentum were traced by the focal injection of CTB and counterstained for Pou4f1 as described in Figure 14. (A,B) Location of a small focal injection of CTB in the VTA, compared to a standard atlas view at bregma -3.64. DA neurons in the injected area are labeled for tyrosine hydroxylase (TH) immunoreactivity. (C,D) Location of neurons labeled from the VTA in the central and caudal LHb; few labeled cells were seen in rostral part of the LHb (not shown). (E) Confocal image of CTB and Pou4f1 expression in the area boxed in (D). In the central LHb bilaterally, 15/32 nuclei of CTB-labeled neurons were Pou4f1⁺. (F,G) Location of a large injection of CTB in the RMTg, overlapping the adjacent MnR/PMnR, compared to a standard atlas view at bregma -4.04. DA neurons are not present in the area of injection. (H–J) Location of neurons labeled from the RMTg in the rostral, central, and caudal LHb. Scale: B, G 400μm; C, H 100μm; E, 50μm.

Figure 16. Retrograde tracing of LHb projections to the median raphe and anterior tegmental nucleus

LHb projections to the tegmentum were traced by the focal injection of CTB and counterstained for Pou4f1 as described in Figure 14. (A,B) Location of a large injection of CTB in the MnR, compared to a standard atlas view at bregma -4.36. (C–E) Location of neurons labeled from the MnR in the rostral, central, and caudal LHb. (F,G) Location of a small focal injection of CTB in the MnR plus the area identified as ATg in a standard atlas, here designated the “caudal RMTg” (Figure 3E, 10, see text), compared to a standard atlas view at bregma -4.16. (H–J) Location of neurons labeled from the caudal RMTg/ATg in the rostral, central and caudal LHb. Arrowheads in (I,J) indicate labeled neurons in a dorsomedial position that are not evident in VTA/RMTg-injected cases and may be attributable to spread of the label into the dorsal MnR. Scale: B, 400μm; C,H 100μm; G, 200μm.

Figure 17. Retrograde tracing of LHb projections to the paramedian raphe and rostral dorsal tegmentum

LHb projections to the tegmentum were traced by the focal injection of CTB and counterstained for Pou4f1 as described in Figure 14. (A) Location of a small CTB injection in the PMnR, corresponding to bregma -4.96 in a standard atlas. (B) Relationship of the injected area to 5HT neurons of the MnR, marked by immunofluorescence for Tph2. (C) Retrograde labeling of LHb neurons in the central LHb. Labeled cells are seen mainly in the medial division of the LHb. Few labeled neurons were seen at rostral levels in this case (not shown). (D) Retrograde labeling of LHb neurons in the caudal LHb. (E) Location of a large CTB injection in the rostral part of the dorsal tegmentum (primarily LDTg), corresponding to bregma -5.02 in a standard atlas. (F–H) Retrograde labeling of neurons in the rostral, central, and caudal LHb, respectively. Scale: B,C,F 100μm.

Figure 18. Retrograde tracing of LHb projections to the dorsal tegmentum and caudal dorsal raphe

The fiber inputs to this area can be seen in Figure 6E–H. (A) Location of a CTB injection in the dorsal pontine gray/DRI at bregma -5.3. (B) Relationship of the injection in (A) to 5HT neurons in the DRI. (C,D) Location of neurons labeled from the DRI in the central and caudal LHb. Nearly all labeled neurons are in the medial division of the LHb. Few labeled neurons were seen at rostral levels in this case (not shown). (E) Confocal image of inset area shown in (D). Some fibers of passage labeled in this section (examples indicated by arrowheads) are not counted as CTB-labeled neurons. (F) Location of a CTB injection in the dorsal pontine gray, near the most caudal extent of LHb projections, at bregma -5.6. (G) Relationship of the CTB injection in (F) to cholinergic neurons in the LDTg at bregma -5.3, slightly rostral to the center of the injection shown in (F). (H-J) Location of neurons labeled from the LDTg in the rostral, central and caudal LHb. Scale: B,C,E,G 100μm.

Figure 19. Topography of LHb projections to brainstem targets

The location of LHb neurons retrogradely labeled from each of the stated structures is shown. Only cell bodies showing a partial or complete nuclear outline were recorded. All maps are taken from the corresponding views of the central LHb (bregma -1.6 to -1.7) in Figures 15–18. Maps for midline injections show only a composite of both hemispheres. (A) Location of LHb neurons projecting to the LH. (B) LHb neurons projecting to VTA (derived from Figure 15C). (C) LHb neurons projecting to RMTg (Figure 15I). (D) LHb neurons projecting to the MnR (Figure 16D). (E) LHb neurons projecting to the caudal RMTg (Figure 16I). (F) LHb neurons projecting to the caudal PMnR (Figure 17C). (G) LHb neurons projecting to the CGPn/DRI (Figure 18C). (H) LHb neurons projecting to the LDTg (Figure 18I). (I) Subdomains of the central LHb defined by projections to specific targets. (J–M) Overlay of LHb neurons projecting to RMTg with those projecting to other areas.