Organization of connections between the amygdala, medial prefrontal cortex, and lateral hypothalamus: a single and double retrograde tracing study in rats

Abstract

The amygdala and medial prefrontal cortex (mPFC) are highly interconnected telencephalic areas critical for cognitive processes, including associative learning and decision making. Both structures strongly innervate the lateral hypothalamus (LHA), an important component of the networks underlying the control of feeding and other motivated behaviors. The amygdala-prefrontal-lateral hypothalamic system is therefore well positioned to exert cognitive control over behavior. However, the organization of this system is not well defined, particularly the topography of specific circuitries between distinct cell groups within these complex, heterogeneous regions. This study used two retrograde tracers to map the connections from the amygdala (central and basolateral area nuclei) and mPFC to the LHA in detail, and to determine whether amygdalar pathways to the mPFC and to LHA originate from the same or different neurons. One tracer was placed into a distinct mPFC area (dorsal anterior cingulate, prelimbic, infralimbic, or rostromedial orbital), and the other into dorsal or ventral LHA. We report that the central nucleus and basolateral area of the amygdala send projections to distinct LHA regions, dorsal and ventral, respectively. The basolateral area, but not central nucleus, also sends substantial projections to the mPFC, topographically organized rostrocaudal to dorsoventral. The entire mPFC, in turn, projects to the LHA, providing a separate route for potential amygdalar influence following mPFC processing. Nearly all amygdalar projections to the mPFC and to the LHA originated from different neurons suggesting amygdala and amygdala-mPFC processing influence the LHA independently, and the balance of these parallel pathways ultimately controls motivated behaviors.

Keywords: Amygdala; Behavior; Feeding; Hypothalamus; Motivation; Prefrontal cortex.

Fig. 1

Representative images of retrograde tracer labeling in the basolateral area of the amygdala after tracer placement in the ACAd (a′; #32), PL (b′; #25), and ILA (c′; #35). a–c Adjacent Nissl-stained tissue. Scale bars 100 μm

Fig. 2

The distribution of labeling within the amygdala following retrograde tracer injection into the ACAd. a Photomicrograph of the injection center, and illustration of the rostrocaudal extent of the injection spread for case #32. b Illustration of injection center for a comparison injection into ACAd (#52). c Labeled neurons (red dots) were plotted onto rat brain templates derived from Swanson (2004), arranged from rostral to caudal. Numbers denote atlas levels, scale bar 200 μm

Fig. 3

The distribution of labeling within the amygdala following retrograde tracer injection into PL. a Photomicrograph of the injection center, and illustration of the rostrocaudal extent of the injection spread for case #25. b Illustration of the injection centers for comparison injections into PL (#20–22, #33, #46). c Labeled neurons (red dots) were plotted onto rat brain templates derived from Swanson (2004), arranged from rostral to caudal. Numbers denote atlas levels, scale bar 200 μm

Fig. 4

The distribution of labeling within the amygdala following retrograde tracer injection into ILA. a Photomicrograph of the injection center, and illustration of the rostrocaudal extent of the injection spread for case #35. b Illustration of the injection centers for comparison injections into ILA (#27, #36, #45). c Labeled neurons (red dots) were plotted onto rat brain templates derived from Swanson (2004), arranged from rostral to caudal. Numbers denote atlas levels, scale bar 200 μm

Fig. 5

The distribution of labeling within the amygdala following retrograde tracer injection into rostromedial ORB (ORBm, vl). a Photomicrograph of the injection center, and illustration of the rostrocaudal extent of the injection spread for case #40. b Illustration of the injection centers for comparison injections into very rostral mPFC (#43, #48–51). c Labeled neurons (red dots) were plotted onto rat brain templates derived from Swanson (2004), arranged from rostral to caudal. Numbers denote atlas levels, scale bar 200 μm

Fig. 6

Topographical distribution of basolateral area projections to the mPFC. Combined labeling plots in the amygdala following ACAd (orange; from Fig. 2c), PL (blue; from Fig. 3c), and ILA (pink; from Fig. 4c) injections. Labeled neurons were plotted onto rat brain templates derived from Swanson (2004), arranged from rostral to caudal. Numbers denote atlas levels

Fig. 7

Representative images of retrograde tracer labeling in the amygdala; the CEA after tracer placement into dorsal (a′; #23) and ventral (b′; #21) LHA, and the basolateral area of the amygdala after tracer placement into dorsal (c′; #23) and ventral (d′; #21) LHA. a, b Adjacent Nissl-stained tissue. Scale bars 100 μm

Fig. 8

Photomicrographs show retrograde tracer labeling in the mPFC after tracer placement into dorsal (a′; #23) and ventral (b′; #24) LHA, and adjacent Nissl-stained tissue (a, b); scale bars 100 μm. Labeling within layer 5 after dorsal (a″) and ventral (b″) LHA, as indicated by gray boxes on a′ and b′; scale bars 50 μm

Fig. 9

The distribution of labeling within the amygdala (c) and mPFC (d) following retrograde tracer injection into dorsal LHA. a Photomicrograph of the injection center, and illustration of the rostrocaudal extent of the injection spread for case #23. b Illustration of the centers of other injections into dorsal LHA (#26, #28, #37, #40, #41, #55). c, d Labeled neurons (red dots) were plotted onto rat brain templates derived from Swanson (2004), arranged from rostral to caudal. Numbers denote atlas levels, scale bar 200 μm

Fig. 10

The distribution of labeling within the amygdala (c) and mPFC (d) following retrograde tracer injection into ventral LHA. a Photomicrograph of the injection center, and illustration of the rostrocaudal extent of the injection spread for case #21. b Illustration of the centers of other injections into ventral LHA (#24, #29, #51–53). c, d Labeled neurons (red dots) were plotted onto rat brain templates derived from Swanson (2004), arranged from rostral to caudal. Numbers denote atlas levels, scale bar 200 μm

Fig. 11

Distribution of mPFC-projecting (green) and LHA-projecting (red) neurons in the caudal basolateral area of the amygdala. a Illustration of total amygdala projections to all mPFC areas and both LHA regions (combined labeling plots from single-label analyses shown in Figs. 2c, 3c, 4c, 5c, 9c, and 10c). Labeled neurons were plotted onto rat brain templates derived from Swanson (2004); numbers denote atlas levels. b Representative images from a single brain used in the double-label analysis for simultaneous visualization of both tracers (case #20, injection in PL and a large injection in LHA). Arrows indicate double-labeled neurons which project to both the mPFC and LHA (yellow); scale bars 100 μm

Fig. 12

Summary of projections characterized in the present study. Selected other known projections are shown in gray