Hypothesis-Driven Structural Connectivity Analysis Supports Network Over Hierarchical Model of Brain Architecture

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

Richard H. Thompson and Larry W. Swanson1

Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089

Contributed by Larry W. Swanson, June 30, 2010 (sent for review May 14, 2010) The brain is usually described as hierarchically organized, although (9) and a massive input from the SUBv (10). Only two major an alternative network model has been proposed. To help distin- brainstem inputs were identified: the dorsal raphé (DR), the guish between these two fundamentally different structure-function presumptive source of ACBdmt serotonin (11), and the inter- hypotheses, we developed an experimental circuit-tracing strategy fascicular nucleus (IF), the presumptive source of ACBdmt do- that can be applied to any starting point in the nervous system and pamine (12) that is medial to the expected ventral tegmental area then systematically expanded, and applied it to a previously obscure (VTA) itself (13). Thus, the ACBdmt receives direct inputs from dorsomedial corner of the nucleus accumbens identified functionally three massively interconnected cerebral regions implicated in as a “hedonic hot spot.” A highly topographically organized set of stress and depression, as well as restricted brainstem sites pro- connections involving expected and unexpected gray matter regions viding dopaminergic and serotonergic terminals. was identified that prominently features regions associated with Only one ACBdmt axonal output was labeled with PHAL (Fig. appetite, stress, and clinical depression. These connections are 1A; the filled purple area is a representative injection site): a arranged as a longitudinal series of circuits (closed loops). Thus, the descending pathway through the medial forebrain bundle with two results do not support a rigidly hierarchical model of nervous system clear terminal fields. As expected (4), one field was highly re- organization but instead indicate a network model of organization. stricted to a small oval patch in a rostrolateral sector of the sub- In principle, the double-coinjection circuit tracing strategy can be stantia innominata (SI; also called the ventral pallidum) (Fig. 1E, NEUROSCIENCE applied systematically to the rest of the nervous system to establish red axons), referred to here as the ACBdmt-recipient zone the architecture of the global structural wiring diagram, and its ab- (Sidmtr) (Fig. 2). Surprisingly, virtually no input was labeled to the straction, the connectome. VTA (Fig. 3), the other major ACB target. Instead, a large, clear, circumscribed terminal field was labeled in, and restricted to, brain systems | connectome | appetite | depression | stress a rostromedial sector of the anterior lateral hypothalamic area (LHAa) (Fig. 1 F and F′). This result was confirmed in two ways. he starting point that we chose for neural systems analysis is First, all ACB COINs ventral to the ACBdmt labeled a clear pro- Ta 1-mm3 region of brain tissue in which injected μ-opioids injection to VTA (Fig. 1 A, C,andF′); second, very large retrograde crease ingestive behavior, possibly by increasing the hedonic im- pathway tracer implants centered in the VTA extensively labeled pact of sweetness (1). The identified region, which has been called the ACB except in the ACBdmt, whereas another retrograde a hedonic hot spot, lies dorsomedially in the nucleus accumbens tracer placed in the LHAa of the same animal labeled the ACBdmt (ACB), a part of the basal ganglia’s ventral striatum long thought (Fig. 3). to play a role in controlling foraging behavior and the rewarding We next established the global axonal outputs of the newly properties of food or other natural rewards and substances of identified LHAa region targeted by the ACBdmt. Single COINs of abuse (2, 3). This functional heterogeneity of the ACB suggested each type were used for this, because retrograde labeling concen- to us that the ACB’s extrinsic axonal connectivity is also topo- trated in ACBdmt confirmed accurate LHAa injection placement graphically organized and specialized to a greater extent than (Figs. 2 and 4 A and B). Three long projections were labeled with demonstrated previously (4). Thus, we used the dorsomedial ACB PHAL and confirmed with BDA (Fig. 2). Ascending LHAa axons (ACBdm) as a circumscribed, readily identifiable anchor point for innervate various parts of the septal region, including the BST our hypothesis-driven structural connectivity analysis. anteromedial group (BSTamg), with outputs to regions controlling feeding behavior and metabolism, including the release of ACTH Results and glucocorticoids (14); the SIdmtr (a bidirectional connection We first established differential extrinsic axonal inputs and outputs due to the presence of intermixed retrograde labeling); and adja- of the ACBdm dorsal tip (ACBdmt) with a new double-coinjection cent hypothalamic neuron populations, including the anterodorsal (COIN) network tracing strategy. In the same animal, two tiny, preoptic nucleus (ADP), a critical node in the hypothalamic vis- nonoverlapping anterograde/retrograde tracer COINs—mixtures ceromotor pattern generator network (15). A dorsally directed of biotinylated dextran amine/Fluorogold (BDA/FG) and Pha- hypothalamo > thalamic pathway innervates restricted adjacent seolus vulgaris leucoagglutinin/cholera toxin subunit b (PHAL/ parts of the anterior paratenial and paraventricular nuclei (PTa/ CTb)—were placed stereotaxically in the medial ACB (Figs. 1 A–C PVTa) (Fig. 4C), rostral mediodorsal nucleus (MDr), and lateral and Fig. S1). The sources of extrinsic axonal inputs specifically to habenula (LH) (Fig. 3D). In addition, a descending medial fore- ACBdmt in these double-COIN topography experiments (Fig. 1A; brain bundle pathway innervates several regions forming critical the filled purple area is representative) were simpler that those components of the fight-or-flight defensive behavior system (16)— described for the whole ACB (4, 5). Only three major cerebral hemisphere regions were retrogradely labeled with CTb (Fig. 2): (i) the cortical infralimbic area (ILA; Brodmann area 25) (Fig. Author contributions: R.H.T. and L.W.S. designed research; R.H.T. performed research; 1D), where deep electrical stimulation relieves symptoms in R.H.T. and L.W.S. analyzed data; and R.H.T. and L.W.S. wrote the paper. chronically depressed patients (6); (ii) the hippocampal ventral The authors declare no conflict of interest. subiculum (SUBv), which modulates hypothalamic stress respon- 1To whom correspondence should be addressed. E-mail: [email protected]. ses (7) and also projects directly to ILA (8); and (iii) the amygdalar This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. basomedial nucleus (BMA), which receives olfactory information 1073/pnas.1009112107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1009112107 PNAS Early Edition | 1of5 Downloaded by guest on September 24, 2021 the dorsal premammillary nucleus (bilaterally) (Fig. 4E) and several periaqueductal gray (PAG) components (Fig. 2)—and multiple components of the behavior state control system, including the VTA, IF, and DR. To confirm and extend these foundational ACBdmt-related connections, we used a second double-COIN strategy with injections placed in opposite corners of a hypothesized four-node circuit (Fig. 2, colored circles). In the first configuration, PHAL/ CTb was iontophoresed into the ILA, and BDA/FG was iontophoresed into the LHAa (Fig. 5G and Fig. S2 A and B). These experiments confirmed an ILA > ACBdmt corticostriatal projection with PHAL anterograde tracing (Fig. 1D) and an overlapping ACBdmt > LHAa striatohypothalamic projection with FG retrograde tracing (Figs. 4B and 5E). The experiments also extended earlier results by providing direct structural support* for a closed forebrain chain involving the ACBdmt, LHAa, PTa/ PVTa, and ILA (Fig. 5H). The ILA and LHAa COINs are in opposite or alternate nodes of a potential four-link circuit, and when the two COINs are accurately targeted in a single animal, overlapping anterograde-retrograde labeling was observed in the uninjected nodes, the ACBdmt (Fig. 4F) and PTa/PVTa (Fig. S2C). Therefore, the evidence thus far suggests (see Fig. 2) that the ACBdmt is part of a closed cortico > striato > hypothalamo > thalamo > cortical loop; that this loop receives direct input from subdivisions of hippocampus, amygdala, and brainstem aminer- gic groups; and that this circuitry provides output to hypothalamic stress and ingestive behavior systems (from the ventral subiculum and BSTamg), the fight-or-flight system (from the LHAa), and widespread parts of the hypothalamus, PAG, and visceral sensorimotor system [from the ILA (17), confirmed here with PHAL]. As a control, the injection sites in these double-COIN circuit- tracing experiments were “rotated” 90 degrees, so that double COINs were now aimed for ACBdmt and PTa/PVTa (Fig. 5 A, B, and H). As hypothesized, mixed anterograde-retrograde labeling was observed in the uninjected LHAa and ILA (Fig. 5 C and D), independently confirming results of the alternate COIN injection scheme for a closed forebrain chain at the level of confocal microscopy. But more interestingly, the rotated double- COIN experiment revealed that the closed chain also incorpo-

*Structural evidence here for functional interactions consists of confocal microscopy images of direct appositions. Proof of functional synaptic interactions requires a combina- tion of electron microscopy observation of synaptic specializations (presynaptic and postsynaptic densities) and electrophysiological evidence of functional interaction (changes in membrane potential).

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

2of5 | www.pnas.org/cgi/doi/10.1073/pnas.1009112107 Thompson and Swanson Downloaded by guest on September 24, 2021 glu BMA Olfactory cortex glu glu SUBv CA1v Polysensory cortex (cognitive) glu glu ∼ SCH Retina glu ne NTS Pain, viscerosensory glu ne glu ILA PTa/ (Brodmann area 25) PVTa MDr gaba glu LH

glu 1 glu 4 ADP 2 BSTamg 3 glu PAG DR glu glu glu glu IF SIdmtr 5-HT ACBdmt LHAa VTA gaba glu da gaba glu gaba PMd glu glu glu

Visceromotor glu glu Medial hypothalamus

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 neuro-

transmitters not documented in the text (5-HT, serotonin; da, dopamine; gaba; glu, glutamate; ne, norepinephrine) is provided in SI Materials and Methods. NEUROSCIENCE

rates a “classical” (18) cortico > striato > pallido > thalamo > overlapping anterograde-retrograde labeling was observed in the cortical closed chain involving the SIdmtr (Fig. 2). That is, SIdmtr (Fig. 5D), labeled by the same COINs that generated the

B C D

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 sche- matically 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.)

Thompson and Swanson PNAS Early Edition | 3of5 Downloaded by guest on September 24, 2021 sm A B C V3t ABC fx CP VL sm

PVT PT

ACB opt so aco DEF D sm E

MH PMd

sm GH V3m

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, Fig. 5. All major ACBdmt axonal projections contribute to a single thalamo > medial habenula; opt, optic tract; PMd, dorsal premammillary nucleus; PT, cortico > striatal closed chain (circuit). Shown are analyses of circuit con- paratenial nucleus; PVT, thalamic paraventricular nucleus; sm, stria medul- nections based on placement of matched pairs of COINs (A and B)(Fig. S2 A laris; SO, supraoptic nucleus; V3m, third ventricle mammillary recess; V3t, and B) and the resulting interactions between labeled tracers (C–F)(Fig. S2C). third ventricle thalamic part; VL, lateral ventricle. (Scale bars: 100 μm.) 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 “ ” fi two possible sets of indirectly connected, or opposite, nodes (X/Y or A/B) to speci c interactions in both the LHAa and ILA (Fig. 5 C and D). detect the presence or absence of interactions (blue circles), indicated by The resulting configuration, a bifurcated circuit or closed chain overlapping anterograde and retrograde labeling in both “adjacent” nodes. with one parallel node (Fig. 5H, Right), requires further struc- If opposite nodes are connected, then both X/Y COINs will contribute to tural characterization (e.g., with double COINS in the SIdmtr interactions in each of the A/B nodes, whereas the complementary COIN pair and LHAa), as well as careful functional characterization within in A/B will label corresponding interactions in nodes X/Y. Because an ILA > the context of the network outlined in Fig. 2. ACBdm > LHAa > PTa/PVTa > ILA circuit was hypothesized (H), the X/Y double- Two more features of this circuitry are informative. First, the COIN experiment targeted PTa/PVTa (A, Inset, AL23) and ACBdm (B; AL13), > predicting contacts in the LHAa (C; AL25) and ILA (D; AL9). The complemen- massive ACBdm LH projection is critical, because the LH then tary A/B pair corresponds to COINs in ILA and LHAa (Fig. S2 A and B) and shows projects (19) to three behavioral state system components (20, robust interactions not only in the PTa (Fig. S2C) and ACBdmt (E; AL13), but 21) with ascending forebrain projections (VTA, IF, and DR) also in SI/ventral pallidum (F; AL16). In A–F, the yellow letters in the upper (Fig. 2). Thus, the LHAa > LH projection may be part of right refer to corresponding positions in the generic circuit (G). Panels a feedback loop to the behavioral state system representing in showing tracer interactions also include the relationship to adjacent nodes part a negative reward/motivational value (22), whereas the and, by extension, the direction of tracer transport, indicated by arrows. In all LHAa > PTa/PVTa projection is part of a feedback loop to the 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 ven- cerebral cortex. Second, the viscerosensory nucleus of the soli- tricle thalamic part. (Scale bars: A and B,100μm; C,5μm; D–F,25μm.) tary tract (NTS) projects directly to the behavioral state system (23, 24), the PTA/PVTa, and the ILA (Fig. 2), the latter two of which are noradrenergic (25, 26). Aside from the pharmacolog- minergic input arises not from the VTA, but apparently from the ical evidence of a central noradrenergic influence on stress, de- medially adjacent IF instead. Furthermore, although inputs to the pression, and appetite (27), this is interesting because the ILA ACBdmt from the ILA, BMA, and SUBv have been demonstrated projects directly to the NTS, providing the substrate for a possi- previously, other reported accumbens inputs specifically avoid the ble very long cortico > NTS feedback loop (Fig. 2). ACBdmt (4, 5). Finally, in the context of the ACBdm as a hedonic hot spot, other relevant SUBv outputs (Fig. 2) are to medial hy- Discussion pothalamic regions involved in stress and ingestive behavioral Our findings establish the overall structural organization of responses, both directly and through the ventral lateral septal nu- a unique neural network that specifically involves the ACBdmt, is cleus and BSTamg (10, 14, 28), as well as to the BMA (10). relatively simple by limbic forebrain standards, and thus is relatively Our double-COIN strategy directly suggests that the ACBdmt amenable to experimental functional analysis, in part because is part of a large, previously unappreciated bifurcated feedback network nodes typically are quite localized and restricted to sub- circuit in the forebrain (Fig. 5H). One core feature is the inclusion divisions of well-known brain regions. Unexpectedly, the ACBdmt of all four components of a classical cortical > basal ganglia > projects heavily to the LHAa but not to the VTA, and its dopa- thalamo > cortical circuit, confirming that this motif applies

4of5 | www.pnas.org/cgi/doi/10.1073/pnas.1009112107 Thompson and Swanson Downloaded by guest on September 24, 2021 generally to cerebral functions as diverse as eye movement control with all of their synaptic relationships. For example, connection (18) and affective tone (29). Another core feature is the circuit analysis at the level of neuron type is required to determine bifurcation, which allows alternate, parallel routes between the whether the ACB connections to GABAergic neurons in the SI ACBdmt and PTa/PVTa, one through the SIdmtr (ventral pal- and glutamatergic neurons in the LHA arise from a single neuron lidum) and the other through the LHAa. The thalamic PTa/PVTa population with branched axons or from two ACB populations, is critically placed because it receives GABAergic input from the one to the SI and the other to the LHA. The same strategy is then SIdmtr and presumably glutamatergic input from the LHAa (30) applied systematically to adjacent nodes in the circuitry. (SI Materials and Methods), as well as noradrenergic inputs from the viscerosensory NTS and a major input from the circadian Materials and Methods rhythm generator suprachiasmatic nucleus (25, 31). All coinjection experiments involved two central iontophoretic injections, The double-COIN strategy also labels all other known axonal each containing one anterograde and one retrograde tract tracer (four dif- inputs and outputs of injected gray matter regions, indicating that ferent tracers in all) combined in solution and delivered via the same pipette. circuitry involving the ACBdm extends through the nervous sys- All tracers, either individually or in various combinations, were immunolabeled tem as a longitudinal series of circuits (Fig. 2). Because “higher” and visualized with either colorimetric or fluorescence methods. Fluorescent and “lower” are difficult to define objectively in this organization, signals were imaged with confocal scanning laser microscopy configured in a network model of organization (e.g., the Internet) is clearly a multitrack format and acquired as separate channels in a single file. In- favored over a hierarchical model (e.g., the US Army) (20). dividual channels were consistently false-colored by a tracer. Fig. 1B is Further evidence to clarify this distinction can be obtained by a composite image of six panels, each a projection of nine slices for each of the simply expanding systematically the double-COIN analysis started four tracers. Adobe Photoshop CS3 was used to assemble the final image and here to eventually include all gray matter regions of the nervous to adjust the brightness and contrast of all images. The background image in system. Double-COIN analysis is in fact a hypothesis-driven, in- Fig. 1B was created by duplicating the individual channels of the BDA tracer ternally controlled, experimental strategy for mapping systemat- labeling and applying a second, extreme adjustment of brightness and con- ically the nervous system’s global wiring diagram, which can be trast. Summary and schematic diagrams were created using Adobe Illustrator abstracted to a formal connection matrix known as the con- CS3. Additional details are provided in SI Materials and Methods. nectome. In principle, this can be done at successively greater ACKNOWLEDGMENTS. We thank Hong-Wei Dong, Samantha Butler, and levels of resolution, ranging from the macroconnection level of especially Eveline Bijlard. We also thank Olympus America, Inc., for the use of an gray matter regions (as done here) to the mesoconnection level of Olympus FV1000 confocal microscope. This work was supported by the National

neuron type and the microconnection level of individual neurons Institutes of Health, National Institute of Neurological Disorders and Stroke. NEUROSCIENCE

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