Joel D. Hahn

Cyto- and chemoarchitecture of the hypothalamic paraventricular nucleus in the C57BL/6J male mouse: A study of immunostaining and multiple fluorescent tract tracing

The paraventricular nucleus of the hypothalamus (PVH) plays a critical role in the regulation of autonomic, neuroendocrine, and behavioral activities. This understanding has come from extensive characterization of the PVH in rats, and for this mammalian species we now have a robust model of basic PVH neuroanatomy and function. However, in mice, whose use as a model research animal has burgeoned with the increasing sophistication of tools for genetic manipulation, a comparable level of PVH characterization has not been achieved. To address this, we employed a variety of fluorescent tract tracing and immunostaining techniques in several different combinations to determine the neuronal connections and cyto‐ and chemoarchitecture of the PVH in the commonly used C57BL/6J male mouse. Our findings reveal a distinct organization in the mouse PVH that is substantially different from the PVH of male rats. The differences are particularly evident with respect to the spatial relations of two principal neuroendocrine divisions (magnocellular and parvicellular) and three descending preautonomic populations in the PVH. We discuss these data in relation to what is known about PVH function and provide the work as a resource for further studies of the neuronal architecture and function of the mouse PVH. J. Comp. Neurol., 2012.

Distinct patterns of neuronal inputs and outputs of the juxtaparaventricular and suprafornical regions of the lateral hypothalamic area in the male rat.

We have analyzed at high resolution the neuroanatomical connections of the juxtaparaventricular region of the lateral hypothalamic area (LHAjp); as a control and in comparison to this, we also performed a preliminary analysis of a nearby LHA region that is dorsal to the fornix, namely the LHA suprafornical region (LHAs). The connections of these LHA regions were revealed with a coinjection tract-tracing technique involving a retrograde (cholera toxin B subunit) and anterograde (Phaseolus vulgaris leucoagglutinin) tracer. The LHAjp and LHAs together connect with almost every major division of the cerebrum and cerebrospinal trunk, but their connection profiles are markedly different and distinct. In simple terms, the connections of the LHAjp indicate a possible primary role in the modulation of defensive behavior; for the LHAs, a role in the modulation of ingestive behavior is suggested. However, the relation of the LHAjp and LHAs to potential modulation of these behaviors, as indicated by their neuroanatomical connections, appears to be highly integrative as it includes each of the major functional divisions of the nervous system that together determine behavior, i.e., cognitive, state, sensory, and motor. Furthermore, although a primary role is indicated for each region with respect to a particular mode of behavior, intermode modulation of behavior is also indicated. In summary, the extrinsic connections of the LHAjp and LHAs (so far as we have described them) suggest that these regions have a profoundly integrative role in which they may participate in the orchestrated modulation of elaborate behavioral repertoires.

Hippocampal GLP-1 Receptors Influence Food Intake, Meal Size, and Effort-Based Responding for Food through Volume Transmission

Glucagon-like peptide-1 (GLP-1) is produced in the small intestines and in nucleus tractus solitarius (NTS) neurons. Activation of central GLP-1 receptors (GLP-1Rs) reduces feeding and body weight. The neural circuits mediating these effects are only partially understood. Here we investigate the inhibition of food intake and motivated responding for food in rats following GLP-1R activation in the ventral hippocampal formation (HPFv), a region only recently highlighted in food intake control. Increased HPFv GLP-1R activity following exendin-4 administration potently reduced food intake (both chow and Western diet) and body weight, whereas HPFv GLP-1R blockade increased food intake. These hypophagic effects were based on reduced meal size, and likely do not involve nausea as HPFv exendin-4 did not induce a conditioned flavor avoidance. HPFv GLP-1R activation also reduced effort-based responding for food under an operant progressive ratio reinforcement schedule, but did not affect food conditioned place preference expression. To investigate possible routes of HPFv GLP-1 signaling, immunohistochemical analysis revealed the absence of GLP-1 axon terminals in the HPFv, suggesting volume transmission as a mechanism of action. Consistent with this, the presence of active GLP-1 was detected in both the cerebrospinal fluid (CSF) and the HPFv. The source of CSF GLP-1 may be NTS GLP-1-producing neurons, as, (1) ∼30% of NTS GLP-1 neurons colocalized with the retrograde tracer fluorogold (FG) following lateral ventricle FG injection, and (2) GLP-1-immunoreactive axon terminals were observed adjacent to the ventricular ependymal layer. Collectively these findings illuminate novel neuronal and behavioral mechanisms mediating food intake reduction by GLP-1.

Connections of the lateral hypothalamic area juxtadorsomedial region in the male rat

The connections of the lateral hypothalamic area juxtadorsomedial region (LHAjd) were investigated in a series of pathway‐tracing experiments involving iontophoretic co‐injection of the tracers Phaseolus vulgaris‐leucoagglutinin (PHA‐L; for outputs) and cholera toxin B subunit (CTB; for inputs). Results revealed that the LHAjd has connections with some 318 distinct gray matter regions encompassing all four subsystems—motor, sensory, cognitive, and behavioral state—included in a basic structure–function network model of the nervous system. Integration of these subsystems is necessary for the coordination and control of emotion and behavior, and in that regard the connections of the LHAjd indicate that it may have a prominent role. Furthermore, the LHAjd connections, together with the connections of other LHA differentiations studied similarly to date, indicate a distinct topographic organization that suggests each LHA differentiation has specifically differing degrees of involvement in the control of multiple behaviors. For the LHAjd, its involvement to a high degree in the control of defensive behavior, and to a lesser degree in the control of other behaviors, including ingestive and reproductive, is suggested. Moreover, the connections of the LHAjd suggest that its possible role in the control of these behaviors may be very broad in scope because they involve the somatic, neuroendocrine, and autonomic divisions of the nervous system. In addition, we suggest that connections between LHA differentiations may provide, at the level of the hypothalamus, a neuronal substrate for the coordinated control of multiple themes in the behavioral repertoire. J. Comp. Neurol. 520:1831–1890, 2012.

Hippocampus ghrelin signaling mediates appetite through lateral hypothalamic orexin pathways

Feeding behavior rarely occurs in direct response to metabolic deficit, yet the overwhelming majority of research on the biology of food intake control has focused on basic metabolic and homeostatic neurobiological substrates. Most animals, including humans, have habitual feeding patterns in which meals are consumed based on learned and/or environmental factors. Here we illuminate a novel neural system regulating higher-order aspects of feeding through which the gut-derived hormone ghrelin communicates with ventral hippocampus (vHP) neurons to stimulate meal-entrained conditioned appetite. Additional results show that the lateral hypothalamus (LHA) is a critical downstream substrate for vHP ghrelin-mediated hyperphagia and that vHP ghrelin activated neurons communicate directly with neurons in the LHA that express the neuropeptide, orexin. Furthermore, activation of downstream orexin-1 receptors is required for vHP ghrelin-mediated hyperphagia. These findings reveal novel neurobiological circuitry regulating appetite through which ghrelin signaling in hippocampal neurons engages LHA orexin signaling. DOI: http://dx.doi.org/10.7554/eLife.11190.001

Connections of the juxtaventromedial region of the lateral hypothalamic area in the male rat.

Evolutionary conservation of the hypothalamus attests to its critical role in the control of fundamental behaviors. However, our knowledge of hypothalamic connections is incomplete, particularly for the lateral hypothalamic area (LHA). Here we present the results of neuronal pathway-tracing experiments to investigate connections of the LHA juxtaventromedial region, which is parceled into dorsal (LHAjvd) and ventral (LHAjvv) zones. Phaseolus vulgaris leucoagglutinin (PHAL, for outputs) and cholera toxin B subunit (CTB, for inputs) coinjections were targeted stereotaxically to the LHAjvd/v. Results: LHAjvd/v connections overlapped highly but not uniformly. Major joint outputs included: Bed nuc. stria terminalis (BST), interfascicular nuc. (BSTif) and BST anteromedial area, rostral lateral septal (LSr)- and ventromedial hypothalamic (VMH) nuc., and periaqueductal gray. Prominent joint LHAjvd/v input sources included: BSTif, BST principal nuc., LSr, VMH, anterior hypothalamic-, ventral premammillary-, and medial amygdalar nuc., and hippocampal formation (HPF) field CA1. However, LHAjvd HPF retrograde labeling was markedly more abundant than from the LHAjvv; in the LSr this was reversed. Furthermore, robust LHAjvv (but not LHAjvd) targets included posterior- and basomedial amygdalar nuc., whereas the midbrain reticular nuc. received a dense input from the LHAjvd alone. Our analyses indicate the existence of about 500 LHAjvd and LHAjvv connections with about 200 distinct regions of the cerebral cortex, cerebral nuclei, and cerebrospinal trunk. Several highly LHAjvd/v-connected regions have a prominent role in reproductive behavior. These findings contrast with those from our previous pathway-tracing studies of other LHA medial and perifornical tier regions, with different connectional behavioral relations. The emerging picture is of a highly differentiated LHA with extensive and far-reaching connections that point to a role as a central coordinator of behavioral control.

A hippocampus to prefrontal cortex neural pathway inhibits food motivation through glucagon-like peptide-1 signaling

The hippocampus and the medial prefrontal cortex (mPFC) are traditionally associated with regulating memory and executive function, respectively. The contribution of these brain regions to food intake control, however, is poorly understood. The present study identifies a novel neural pathway through which monosynaptic glutamatergic ventral hippocampal field CA1 (vCA1) to mPFC connectivity inhibits food-motivated behaviors through vCA1 glucagon-like peptide-1 receptor (GLP-1R). Results demonstrate that vCA1-targeted RNA interference-mediated GLP-1R knockdown increases motivated operant responding for palatable food. Chemogenetic disconnection of monosynaptic glutamatergic vCA1 to mPFC projections using designer receptors exclusively activated by designer drugs (DREADDs)-mediated synaptic silencing ablates the food intake and body weight reduction following vCA1 GLP-1R activation. Neuropharmacological experiments further reveal that vCA1 GLP-1R activation reduces food intake and inhibits impulsive operant responding for palatable food via downstream communication to mPFC NMDA receptors. Overall these findings identify a novel neural pathway regulating higher-order cognitive aspects of feeding behavior.

Amylin Acts in the Lateral Dorsal Tegmental Nucleus to Regulate Energy Balance Through Gamma-Aminobutyric Acid Signaling

BACKGROUND The pancreatic- and brain-derived hormone amylin promotes negative energy balance and is receiving increasing attention as a promising obesity therapeutic. However, the neurobiological substrates mediating amylins effects are not fully characterized. We postulated that amylin acts in the lateral dorsal tegmental nucleus (LDTg), an understudied neural processing hub for reward and homeostatic feeding signals. METHODS We used immunohistochemical and quantitative polymerase chain reaction analyses to examine expression of the amylin receptor complex in rat LDTg tissue. Behavioral experiments were performed to examine the mechanisms underlying the hypophagic effects of amylin receptor activation in the LDTg. RESULTS Immunohistochemical and quantitative polymerase chain reaction analyses show expression of the amylin receptor complex in the LDTg. Activation of LDTg amylin receptors by the agonist salmon calcitonin dose-dependently reduces body weight, food intake, and motivated feeding behaviors. Acute pharmacological studies and longer-term adeno-associated viral knockdown experiments indicate that LDTg amylin receptor signaling is physiologically and potentially preclinically relevant for energy balance control. Finally, immunohistochemical data indicate that LDTg amylin receptors are expressed on gamma-aminobutyric acidergic neurons, and behavioral results suggest that local gamma-aminobutyric acid receptor signaling mediates the hypophagia after LDTg amylin receptor activation. CONCLUSIONS These findings identify the LDTg as a novel nucleus with therapeutic potential in mediating amylins effects on energy balance through gamma-aminobutyric acid receptor signaling.

Network architecture of the cerebral nuclei (basal ganglia) association and commissural connectome

Significance The cerebral nuclei together with the cerebral cortex form the cerebral hemispheres that are critically important for the control of voluntary behavior and motivation. Network analysis of microscopic connectional data collected since the 1970s in a small, intensely studied mammal provides a new way to understand overall design features of circuitry coordinating activity in the various parts of the cerebral nuclei on both sides of the brain. Basically, intracerebral nuclei circuitry is organized into four modules on each side of the brain, with connections within and between modules on one side being quite dense and connections between the cerebral nuclei on either side being quite sparse. The results provide insight into cerebral nuclei structure and function. The cerebral nuclei form the ventral division of the cerebral hemisphere and are thought to play an important role in neural systems controlling somatic movement and motivation. Network analysis was used to define global architectural features of intrinsic cerebral nuclei circuitry in one hemisphere (association connections) and between hemispheres (commissural connections). The analysis was based on more than 4,000 reports of histologically defined axonal connections involving all 45 gray matter regions of the rat cerebral nuclei and revealed the existence of four asymmetrically interconnected modules. The modules form four topographically distinct longitudinal columns that only partly correspond to previous interpretations of cerebral nuclei structure–function organization. The network of connections within and between modules in one hemisphere or the other is quite dense (about 40% of all possible connections), whereas the network of connections between hemispheres is weak and sparse (only about 5% of all possible connections). Particularly highly interconnected regions (rich club and hubs within it) form a topologically continuous band extending through two of the modules. Connection path lengths among numerous pairs of regions, and among some of the network’s modules, are relatively long, thus accounting for low global efficiency in network communication. These results provide a starting point for reexamining the connectional organization of the cerebral hemispheres as a whole (right and left cerebral cortex and cerebral nuclei together) and their relation to the rest of the nervous system.

Organizing principles for the cerebral cortex network of commissural and association connections

Significance The cerebral cortex supports cognition and is a structure common to all mammals. The major cortical subdivisions (its gray matter regions) are connected by a complex network of axonal connections that includes connections between regions in the same hemisphere (association connections on the right or left side) and those between hemispheres (commissural connections between opposite sides). A database of over 5,000 connections in the cortical network was extracted from the literature, and network analysis revealed three identical cortical modules (neural subsystems) on each side. One appears to deal especially with the external world, one with the viscera, and one with planning, prioritization, and self-awareness. A set of general organizing principles for association and commissural connections also emerged from the analysis. Cognition is supported by a network of axonal connections between gray matter regions within and between right and left cerebral cortex. Global organizing principles of this circuitry were examined with network analysis tools applied to monosynaptic association (within one side) and commissural (between sides) connections between all 77 cortical gray matter regions in each hemisphere of the rat brain. The analysis used 32,350 connection reports expertly collated from published pathway tracing experiments, and 5,394 connections of a possible 23,562 were identified, for a connection density of 23%—of which 20% (1,084) were commissural. Network community detection yielded a stable bihemispheric six-module solution, with an identical set in each hemisphere of three modules topographically forming a lateral core and medial shell arrangement of cortical regions. Functional correlations suggest the lateral module deals preferentially with environmental sensory-motor interactions and the ventromedial module deals preferentially with visceral control, affect, and short-term memory, whereas the dorsomedial module resembles the default mode network. Analysis of commissural connections revealed a set of unexpected rules to help generate hypotheses. Most notably, there is an order of magnitude more heterotopic than homotopic projections; all cortical regions send more association than commissural connections, and for each region, the latter are always a subset of the former; the number of association connections from each cortical region strongly correlates with the number of its commissural connections; and the module (dorsomedial) lying closest to the corpus callosum has the most complete set of commissural connections—and apparently the most complex function.