Hypothesis-driven structural connectivity analysis supports network over hierarchical model of brain architecture

Abstract

The brain is usually described as hierarchically organized, although an alternative network model has been proposed. To help distinguish between these two fundamentally different structure-function hypotheses, we developed an experimental circuit-tracing strategy that can be applied to any starting point in the nervous system and then systematically expanded, and applied it to a previously obscure dorsomedial corner of the nucleus accumbens identified functionally as a "hedonic hot spot." A highly topographically organized set of connections involving expected and unexpected gray matter regions was identified that prominently features regions associated with appetite, stress, and clinical depression. These connections are arranged as a longitudinal series of circuits (closed loops). Thus, the results do not support a rigidly hierarchical model of nervous system organization but instead indicate a network model of organization. In principle, the double-coinjection circuit tracing strategy can be applied systematically to the rest of the nervous system to establish the architecture of the global structural wiring diagram, and its abstraction, the connectome.

Fig. 1.

Direct comparison of ACB projection specificity and topography. Pathway tracer double COINs were made in different regions entirely within the ACB, allowing the direct comparison of topographic organization of both input and output projections from the two single COINS. (A) Injection site distribution for nine double-COIN experiments (18 COINs), summarized on reference atlas templates (32). PHAL/CTb tracer injections are outlined in purple, and BDA/FG COINs are outlined in orange; filled circles indicate the double-COIN experiment illustrated in B and C. (Top, Right) Each atlas level (AL) and the corresponding distance from the bregma are indicated. (B) Quadruple-labeled confocal image of a double-COIN experiment involving the medial (shell) nucleus accumbens. The background image was derived from a copy of the BDA channel and added to the composite as a separate layer (Fig. S1). For all confocal images (Figs. 1 B–F′′ and 3 B–G), grayscale channels were false-colored according to the tracer imaged; PHAL is always shown in red, CTb in magenta, BDA in green, and FG in cyan. (C) Selective presentation of one tracer from each single COIN shown in B (PHAL, red; BDA, green). (D–F) Comparison of the projections of the medial ACB, labeled by the injections shown in B. (D) Confocal image of CTb and FG neuronal labeling in ILA (AL9) (Inset, AL9, lower left). (E) Confocal image of PHAL and BDA axonal labeling in SI (AL17). (F) Dark-field photomicrograph of ACBdmt projection to LHAa (AL25). In this series of histological sections, tracers were labeled with diaminobenzidine (DAB) (PHAL) and nickel-intensified DAB (BDA) to generate high-contrast, low-power images. Because distinguishing between the brown and black reaction products at this magnification is impossible, a fluorescent companion series was prepared from adjacent sections. Green and red arrows indicate the corresponding groups of BDA- and PHAL-labeled axons illustrated at higher magnification in F′ and F′′. Note that BDA-labeled axons from the ventromedial ACB (F′; green) descend laterally to the projection from the ACBdm in tightly bundled fascicles with few boutons, suggesting virtually no input at this level. In contrast, the PHAL-labeled axons from the ACBdmt (F′′; red) generate a significant input, as indicated by the frequent branches with prominent boutons. aco, anterior commissure olfactory part; CP, caudoputamen; fx, fornix; opt, optic tract; sm, stria medullaris; VL, lateral ventricle. (Scale bars: B–C and F, 100 μm; D, F′, and F′′, 25 μm.)

Fig. 2.

Structural organization of ACBdmt-related neural circuitry. The four nodes of a closed loop (ACBdm > LHAa > PTa/PVTa > ILA > ACBdm; colored circles on left) are emphasized as a test bed for the experimental double-COIN network tracing strategy used to identify the origin, course, and termination of each pathway described in the text. Note a triple descending projection from the cerebral hemisphere (29) to LHAa: excitatory from ILA, inhibitory from ACBdmt, and disinhibitory from SIdmtr. Also note three major LHAa outputs, to (1) BSTamg and ADP, which innervate regions controlling metabolism (including ACTH and glucocorticoid responses) and feeding behavior; (2) parts of the fight-or-flight defensive behavior system, the PMd and PAG (especially the precommissural and commissural nuclei and the dorsomedial and ventrolateral columns); and (3) parts of the behavioral state control system (VTA, IF, and DR). Finally, note (4) feedback from motor output to sensory input (20). Brain part abbreviations are given in the text, and evidence for putative neurotransmitters not documented in the text (5-HT, serotonin; da, dopamine; gaba; glu, glutamate; ne, norepinephrine) is provided in SI Materials and Methods.

Fig. 3.

Differential projections from the medial ACB to the lateral hypothalamic area and the ventral midbrain. (A–D) Fluorescence photomicrographs of retrograde neuronal labeling in the medial ACB (A, Inset; B) resulting from multiple tracer injections in the LHAa (C, Inset, AL25) and ventral midbrain (D; AL37) in the same animal. To specifically label terminating axons in the LHA, the retrograde tracer Fluorogold (FG) was used for injections, because it is largely resistant to uptake by undamaged fibers of passage. To maximally label all projections in the VTA and medially adjacent parts of the substantia nigra, the retrograde tracer True Blue (TB) was used, because it is avidly taken up by terminating and passing fibers. In addition, TB was delivered as desiccated “crystals” of tracer mechanically ejected from glass pipettes with tip diameters of up to 500 μm (D). The typical pattern of retrograde neuronal labeling produced by these combined injections is shown in A, where the bright-yellow FG-filled neurons are clustered in the ACBdmt, whereas all other parts of the striatum contain numerous intensely blue TB-filled neurons projecting to the ventral midbrain. The approximate location of the labeling is shown schematically in the insets in A and also in a low-power (2.5×) dark-field photograph of the same section in B. The arrows in A and B indicate features recognizable in both images. ACBdmt, nucleus accumbens dorsomedial tip; aco, anterior commissure olfactory part; ccr, rostrum of corpus collosum; CP, caudoputamen; cpd, cerebral peduncle; fr, fasciculus retroflexus; fx, fornix; MB, mammillary body; opt, optic tract; PVHpm, paraventricular nucleus of hypothalamus, posterior magnocellular part; SNr/c, substantia nigra, reticular and compact pars; V3, third ventricle. (Scale bars: A, 100 μm; B–D, 200 μm.)

Fig. 4.

Axonal projections from the ACBdmt-recipient part of the LHAa. Single COINs were stereotaxically placed in this region (A, Inset, AL25; compare with Fig. 1F) and confirmed by the distribution of retrograde neuronal labeling in the ACB (B; AL13). Large injections like the BDA-FG experiment shown in A were acceptable, because the ACBdmt projection has a discrete origin and circumscribed terminal region in the LHA. All injections showing this pattern also generated dense axonal projections to the thalamic paratenial nucleus (C; AL23), lateral habenula (D; AL32), and bilateral dorsal premammillary nucleus (E; AL33), and lighter projections listed in the text. The arrows in B indicate the medial border of the ACB. The arrows in C and E indicate the midline. aco, anterior commissure olfactory part; CP, caudoputamen; fx, fornix; isl, island of Calleja; LH, lateral habenula; MH, medial habenula; opt, optic tract; PMd, dorsal premammillary nucleus; PT, paratenial nucleus; PVT, thalamic paraventricular nucleus; sm, stria medullaris; SO, supraoptic nucleus; V3m, third ventricle mammillary recess; V3t, third ventricle thalamic part; VL, lateral ventricle. (Scale bars: 100 μm.)

Fig. 5.

All major ACBdmt axonal projections contribute to a single thalamo > cortico > striatal closed chain (circuit). Shown are analyses of circuit connections based on placement of matched pairs of COINs (A and B) (Fig. S2 A and B) and the resulting interactions between labeled tracers (C–F) (Fig. S2C). The general strategy for circuit tracing involves hypothesizing specific structural relationships between a limited number of known projections. This four-node loop (G) can then be tested with COIN pairs (red circles) in each of the two possible sets of indirectly connected, or “opposite,” nodes (X/Y or A/B) to detect the presence or absence of interactions (blue circles), indicated by overlapping anterograde and retrograde labeling in both “adjacent” nodes. If opposite nodes are connected, then both X/Y COINs will contribute to interactions in each of the A/B nodes, whereas the complementary COIN pair in A/B will label corresponding interactions in nodes X/Y. Because an ILA > ACBdm > LHAa > PTa/PVTa > ILA circuit was hypothesized (H), the X/Y double-COIN experiment targeted PTa/PVTa (A, Inset, AL23) and ACBdm (B; AL13), predicting contacts in the LHAa (C; AL25) and ILA (D; AL9). The complementary A/B pair corresponds to COINs in ILA and LHAa (Fig. S2 A and B) and shows robust interactions not only in the PTa (Fig. S2C) and ACBdmt (E; AL13), but also in SI/ventral pallidum (F; AL16). In A–F, the yellow letters in the upper right refer to corresponding positions in the generic circuit (G). Panels showing tracer interactions also include the relationship to adjacent nodes and, by extension, the direction of tracer transport, indicated by arrows. In all panels, PHAL is shown in red, CTb in magenta, BDA in green, and FG in cyan. CP, caudoputamen; VL, lateral ventricle; sm, stria medullaris; V3t, third ventricle thalamic part. (Scale bars: A and B, 100 μm; C, 5 μm; D–F, 25 μm.)