Striatal medium spiny neurons terminate in a distinct region in the lateral hypothalamic area and do not directly innervate orexin/hypocretin- or melanin-concentrating hormone-containing neurons

Abstract

Neuronal circuits including medium spiny neurons (MSNs) in the nucleus accumbens (NAc) and melanin-concentrating hormone (MCH)-containing neurons in the lateral hypothalamic area (LHA) are hypothesized to play an important role in hedonic feeding. A reciprocal connection between NAc MSNs and MCH-containing neurons is proposed to form a neuronal circuit that is involved in hedonic feeding. Although NAc MSNs have been shown to receive projection from MCH-containing neurons, it is not known whether MCH-containing neurons in the LHA also receive direct inputs from NAc MSNs. Here, we developed a genetic approach that allows us to visualize almost all striatal MSNs including NAc MSNs. We demonstrate that striatal MSNs terminate in a distinct region within the anterior LHA, and that the terminal area of striatal MSNs in this region contains glutamatergic neurons and is distinctly separate from orexin/hypocretin- or MCH-containing neurons. These observations suggest that NAc MSNs do not directly innervate MCH-containing neurons, but may indirectly signal MCH-containing neurons via glutamatergic neurons in the anterior LHA.

Figure 1.

Genetic visualization of striatal MSNs. a, Schematic illustration of the induced expression of EGFP in striatal MSNs of PDE10A2-tTA/bitetO-GFP mice. b, Illustration of the brain atlas at the similar level of c and f. c, A representative parasagittal section of the PDE10A2-tTA/bitetO-GFP mouse brain visualized with green fluorescence. d, Enlargement of the LHA region in c. e, Schematic illustration of the induced expression of synaptopHluorin (spH) in striatal MSNs of PDE10A2-tTA/tetO-synaptopHluorin mice. f, A representative parasagittal section of the PDE10A2-tTA/tetO-synaptopHluorin mouse brain visualized with green fluorescence. g, Enlargement of the LHA region in f. A distinct green fluorescent dense plexus is seen in a restricted region in the anterior LHA (dotted circle; c, d, f, g). h, A comparison between the anterior LHA region (left) and the posterior LHA region (right) of the PDE10A2-tTA/tetO-synaptopHluorin mouse brain in higher magnification. Green fluorescent plexus is much more clearly observed in the anterior LHA region than in the posterior LHA region. Scale bars: c, f, 300 μm; d, g, h, 100 μm.

Figure 2.

The axons of striatal MSNs, including NAc MSNs, form GABAergic terminals onto neurons in a distinct region in the anterior part of the LHA. a, Immunostainings with anti-VGAT antibody (red) of the green fluorescently labeled terminal regions of striatal MSNs in the anterior LHA of synaptopHluorin (spH)-expressing mice (PDE10A2-tTA/tetO-synaptopHluorin). b, Immunostainings with anti-gephyrin antibody (red) counterstained with TOTO-3 (blue) of the green fluorescently labeled LHA in synaptopHluorin-expressing mice (PDE10A2-tTA/tetO-synaptopHluorin). c, Retrograde tracer FluoSpheres (red) placed within the GFP-labeled region (green) in the LHA of GFP-expressing mice (PDE10A2-tTA/bitetO-GFP) (left) were transported to the GFP-labeled cell bodies (green) of NAc MSNs (middle and right). Scale bars: a, 20 μm; b, c, middle, right, 10 μm; c, left, 100 μm.

Figure 3.

Terminal area of striatal MSNs in the anterior LHA contains many glutamatergic neurons, but few GABAergic neurons. a–c, Serial parasagittal sections of the anterior LHA region in synaptopHluorin-expressing mice (PDE10A2-tTA/tetO-synaptopHluorin) were examined for VGLUT2 mRNA expression (a), green fluorescent synaptopHluorin (spH) in the anterior LHA (b), and Gad67 mRNA expression (c). d, Schematic representation of distribution of glutamatergic and GABAergic neurons in the terminal area of striatal MSNs in the anterior LHA. Yellow dots representing glutamatergic neurons identified in a and red dots representing GABAergic neurons identified in c are placed on b. Scale bars: a–c, 100 μm.

Figure 4.

Terminal area of striatal MSNs in the LHA is distinctly separate from both MCH-containing neurons and orexin/hypocretin-containing neurons. a, b, Immunostainings with anti-MCH (red in a), anti-orexin-A (red in b, left), and anti-orexin-B (red in b, right) of parasagittal brain sections of GFP-expressing mice (PDE10A2-tTA/bitetO-GFP). Scale bars: a, b, 100 μm.

Figure 5.

a, b, Retrograde tracer FluoSpheres (red) placed within the posterior LHA of GFP-expressing mice (PDE10A2-tTA/bitetO-GFP) (a) were transported to cell bodies of neurons that reside in the green fluorescent plexus in the anterior LHA (b). Sections were counterstained with TOTO-3 (blue). Scale bars: a, 100 μm; b, 10 μm.

Figure 6.

A neuronal circuit model composed of NAc MSNs, the anterior LHA glutamatergic neurons, and MCH-containing neurons. MCH-containing neurons send inhibitory (−) efferents to NAc MSNs. GABAergic NAc MSNs send inhibitory (−) efferents to the anterior LHA glutamatergic neurons. The anterior LHA glutamatergic neurons send excitatory (+) efferents to MCH-containing neurons distributed in the tuberal and posterior LHA. This neuronal circuit model allows continuous activation of MCH-containing neurons.