The inhibitory influence of the lateral habenula on midbrain dopamine cells: ultrastructural evidence for indirect mediation via the rostromedial mesopontine tegmental nucleus

Abstract

The lateral habenula (LHb) provides an important source of negative reinforcement signals to midbrain dopamine (DA) cells in the substantia nigra and ventral tegmental area (VTA). This profound and consistent inhibitory influence involves a disynaptic connection from glutamate neurons in the LHb to some population of γ-aminobutyric acid (GABA) cells that, in turn, innervates DA neurons. Previous studies demonstrated that the GABA cells intrinsic to the VTA receive insufficient synaptic input from the LHb to serve as the primary source of this intermediate connection. In this investigation, we sought ultrastructural evidence supporting the hypothesis that a newly identified region of the brainstem, the rostromedial mesopontine tegmental nucleus (RMTg), is a more likely candidate for inhibiting midbrain DA cells in response to LHb activation. Electron microscopic examination of rat brain sections containing dual immunoreactivity for an anterograde tracing agent and a phenotypic marker revealed that: 1) more than 55% of the synapses formed by LHb axons in the RMTg were onto GABA-labeled dendrites; 2) more than 80% of the synapses formed by RMTg axons in the VTA contacted dendrites immunoreactive for the DA synthetic enzyme tyrosine hydroxylase; and 3) nearly all RMTg axons formed symmetric synapses and contained postembedding immunoreactivity for GABA. These findings indicate that the newly identified RMTg region is an intermediate structure in a disynaptic pathway that connects the LHb to VTA DA neurons. The results have important implications for understanding mental disorders characterized by a dysregulation of reward circuitry involving LHb and DA cell populations.

Figure 1

Light micrographic images of coronal sections through the rat lateral habenula (LHb; A) and rostromedial mesopontine tegmental nucleus (RMTg; B) illustrating immunoperoxidase labeling for PHAL. For both panels, medial is to the left. A: A unilateral injection of PHAL is centered in the medial portion of the caudal LHb (at approximately −3.8 mm posterior to Bregma) but also includes parts of the lateral LHb. It extends to the borders of the stria medullaris (sm) and fasciculus retroflexus (fr) but largely avoids the medial habenula (MHb) adjacent to the lateral ventricle (LV). B: Anterograde transport of PHAL to the RMTg (at approximately −6.8 mm caudal to Bregma) includes clusters of fibers near the crossing of the superior cerebellar peduncle (xscp). The fibers dorsal to the medial lemniscus (ml) and dorsolateral to the interpeduncular nucleus (IP) include branched axons with highly beaded morphology; arrows indicate the same axons at low and high magnification (inset). Scale bar in A = 300 µm in A, B; 60 µm in inset.

Figure 2

A–D: Electron micrographs through the rat RMTg illustrating axons labeled by immunoperoxidase for PHAL transported anterogradely from the lateral habenula (LHb). Some LHb fibers are unmyelinated passing axons containing some vesicles but not forming synapses (LHb-a in A), whereas others exhibit distinct myelination (LHb-ma in B). Other LHb axons form synaptic contacts (black arrows) onto dendrites (LHb-t in C and D). Note the modest postsynaptic densities in both cases. Scale bar = 0.5 µm in D (applies to A–D).

Figure 3

A–D: Electron micrographs through the rat RMTg showing immunoperoxidase reaction product for PHAL in lateral habenula (LHb) axon terminals (LHb-t) in relation to dendrites containing immunogold-silver labeling for γ-aminobutyric acid (GABA) (GABA-d). LHb-ts often synapse (black arrows) onto GABA-ds (A–C). In some cases (A), multiple LHb-ts form convergent contacts onto common immunoreactive dendrites, although synaptic junctions are not always evident at each site. (Insets illustrate points of contact in adjacent serial sections; gray arrows illustrate an apposition that is not clearly synaptic.) In other instances (D), LHb-ts synapse onto unlabeled dendrites (ud) in close proximity to GABA-ds. Most LHb-ts and their postsynaptic targets are surrounded on multiple sides by glial processes (asterisks), some of which contain gold-silver immunolabeling for GABA (A,B,D). In C, a dense-cored vesicle (arrowhead) is evident within the LHb-t; a myelinated LHb axon (LHb-ma) can be seen nearby. Scale bar = 0.5 µm in D (applies to A–D).

Figure 4

Scatter plot showing the diameter of γ-aminobutyric acid (GABA)-immunoreactive or unlabeled (UL) dendrites (Dend) in the rat RMTg that received synaptic input from LHb axons. The group medians are indicated by horizontal lines. The largest diameter dendrite in the GABA-immunolabeled population (7.00 µm) was not included in the plot for ease of presentation, but was factored into the group median.

Figure 5

Light micrographic images of coronal sections through the rat rostromedial mesopontine tegmental nucleus (RMTg; A,B) and ventral tegmental area (VTA; C) illustrating immunoperoxidase labeling for PHAL. In each panel, medial is to the right. A,B: A unilateral injection of PHAL is centered in the RMTg (at approximately −6.8 mm posterior to Bregma) just above the medial lemniscus (ml) and dorsolateral to the interpeduncular nucleus (IP). Arrows indicate cells that have taken up tracer. At the more caudal level shown in B (about −7.04 mm behind Bregma), a few such cells can be seen at more dorsal positions, consistent with the known caudodorsal shift of the RMTg. C: Anterograde transport of PHAL to the ventral midbrain includes the entire SNc-VTA complex (shown here at roughly −5.3 mm caudal to Bregma), outlining the region known to contain DA neurons. RMTg axons shown at higher magnification in the inset are extensively branched and beaded. (Arrows indicate the same capillary at low and high magnification.) Abbreviations: cp, cerebral peduncle; fr, fasciculus retroflexus; mlf, medial longitudinal fasciculus; SNc, substantia nigra zona compacta. Scale bar in C = 500 µm in A–C; 50 µm in inset.

Figure 6

A,B: Electron micrographic images of the rat VTA showing immunoperoxidase reaction product for PHAL transported anterogradely from the rostromedial mesopontine tegmental nucleus (RMTg). Tracer containing axons are either unmyelinated (RMTg-a), as shown in A in a field of unlabeled axons (ua) or occasionally myelinated as illustrated in B (RMTg-ma). Scale bar = 0.5 µm in B (applies to A,B).

Figure 7

Electron micrographs of the rat VTA showing rostromedial mesopontine tegmental nucleus (RMTg) axon varicosities labeled by immunoperoxidase for PHAL (RMTg-t) synapsing onto dendrites containing gold-silver labeling for tyrosine hydroxylase (TH-d). A,B: The synapses are symmetric (white arrows), the TH-ds receive additional symmetric synapses from unlabeled terminals (ut), and the RMTg-ts are mostly surrounded on nonsynaptic sides by glial processes (asterisks). In B, the small-diameter TH-d is relatively distal and contains fewer gold-silver particles for TH than adjacent dendrites. Nevertheless, the specific labeling of the target dendrite was confirmed in adjacent serial sections. C: The RMTg axon forms a synapse with a thicker postsynaptic density (black arrow) that is more typically asymmetric. Scale bar = 0.5 µm in C (applies to A–C).

Figure 8

Electron micrograph of the rat VTA illustrating an axon varicosity containing immunoreaction product for PHAL transported anterogradely from the rostromedial mesopontine tegmental nucleus (RMTg-t). Although the RMTg-t is in the field of a dendrite containing immunogold-silver labeling for tyrosine hydroxylase (TH-d), it synapses instead (white arrow) onto an unlabeled dendrite (ud). Note the small diameter of the ud. Scale bar = 0.5 µm.

Figure 9

Electron micrographs of the rat VTA showing rostromedial mesopontine tegmental nucleus (RMTg) axon varicosities (RMTg-t) in the vicinity of tyrosine hydroxylase (TH)-immunoreactive perikarya (TH-p; n represents the nucleus) or proximal dendrites (TH-d; ga represents a Golgi apparatus). A: Although the RMTg-t is closely adjacent to the TH-p, a thin glial leaflet (asterisks) separates the two structures. B: The RMTg-t is directly apposed to the proximal dendrite but without evidence of a junctional specialization in this or in adjacent serial sections. Scale bar = 0.5 µm in B (applies to A,B).

Figure 10

Electron micrographic images of the rat VTA illustrating axons (RMTg+GABA-a) or terminal varicosities (RMTg+GABA-t) that contain both pre-embedding immunogold-silver labeling for PHAL (large, irregular particles) transported anterogradely from the RMTg and postembedding immunogold labeling for GABA (small, uniform particles). A: The RMTg+GABA-a is adjacent to an axon terminal that is singly labeled for GABA (GABA-t) and forms a symmetric synapse (white arrow) onto a dendrite. Two nearby unlabeled terminals (ut) forming asymmetric synapses are shown for comparison. B,C: The two RMTg+GABA-ts synapse (white arrows) onto dendrites. In B, a GABA-t and a ut are shown for comparison. Abbreviations: GABA, γ-aminobutyric acid; RMTg, rostromedial mesopontine tegmental nucleus. Scale bar = 0.5 µm in C (applies to A–C).