The Tong lab studies how neurocircuitry in the brain controls feeding, energy expenditure and glucose homeostasis. To tackle the complexity in the hypothalamic neural network in the regulation of energy balance, we have generated mouse models with site-specific expression of Cre and loxP-dependent gene-deletion restricted to defined groups of brain neurons. Using these animal models, as well as an array of techniques including immunohistochemistry, in situ hybridization, neuronal tracing and stereotaxic delivery of viruses, optogenetics, chemogenetics, in vivo fiber photometry, and a wide variety of behavioral techniques, we delineate neural pathways in the brain underlying food intake, body weight homeostasis and glucose homeostasis, and provide a framework for an effective drug design against the current epidemics of obesity and its associated syndromes.
To study this, we use a wide variety of genetic, molecular, endocrinological, and behavioral methods:
OPTOGENETICS AND BEHAVIORAL TECHNIQUES
Using transgenic mice expressing tissue specific Cre (such as Vgat-ires-cre, where Cre is only expressed in GABAergic neurons) and stereotaxic neurosurgery for targeted transfection with adeno-associated viral vectors bearing plasmids with Cre-dependent expression of protein (e.g. DIO-ChR2-EYFP), we achieve neuroanatomical and neuronal type-specific expression of ChR2. Then, an optic fiber is implanted over projecting terminals of these neurons, and 473nm laser light is pulsed to induce a wide variety of behaviors. We employ many behavioral assays and add new ones as needed to assess mouse hunger, affective state, and preference for optogenetic stimulus.
Lateral hypothalamic projections to the paraventricular hypothalamic nucleus
(A) Schematic demonstrating virus delivery and optic fiber placement (B) LH expression of DIO-ChR2-EYFP and projections to the PVH (C) LH expression of DIO-ChR2-EYFP
(Left) Optogenetic stimulation of LH GABAergic terminals in the PVH causes feeding (Right) Activation of LH glutamatergic terminals in the PVH causes grooming
IN VIVO FIBER PHOTOMETRY OF CALCIUM-DEPENDENT FLUORESCENCE
Recent technical advancement in genetically-encoded calcium indicators (GECIs), especially of the GFP-Calmodulin-M13-peptide 6 slow and fast version (GCaMP6s and GCaMP6f) has allowed second-to-second precision in recording alterations in calcium levels in neuron cell bodies of freely behaving mice. This allows ethologically relevant coupling of neural activity changes with natural behavior.
Increase in neuronal population activity due to anxiety
Graph demonstrates increase in calcium-dependent fluorescence of a set of GABAergic neurons in response to looming and approach of experimenter (red arrow indicates onset, looming lasts 5 seconds), indicating that these neurons increase in activity when the animal is vigilant and anxious, and remain activated for a period of time afterwards. Also note that on repeated testing, the baseline activity of these neurons also increases, indicating increased overall anxious state.
EX VIVO BRAIN SLICE ELECTROPHYSIOLOGY
Given the deeply interwoven structure of subcortical brain tissues, it is necessary to evaluate neural activity patterns at the single cell basis using the suite of techniques termed channelrhodopsin-assisted circuit mapping (CRACM). Using a combination of Cre-dependent expression of ChR2 in presynaptic neurons and Cre-On, Cre-Off differential labeling of postsynaptic neurons, it is possible to both prove that a monosynaptic connection between two regions exists, and the molecular characteristics of the downstream target.(A) Schematic demonstrating the green, ChR2-EYFP-expressing presynaptic neuronal terminals being stimulated over the orange, mCherry-expressing postsynaptic cell body which has a patch-clamp electrode in voltage-clamp configuration (B) EYFP+ positive fibers and synaptic terminals amidst mCherry+ postsynaptic cell bodies (C) 20x objective microscopy of mCherry+ cell body which was then patched (D) An inhibitory post-synaptic current (oIPSC) is seen when blue light is shined on the brain slice, and this was not eliminated with blockers of endogenous synaptic release (tetrodotoxin (TTX) and 4-aminopyridine (4-AP)) indicating it is monosynaptic. (E) Application of a GABA receptor antagonist, GABAzine, blocked the light-induced current; washout with artificial cerebospinal fluid (ACSF) caused some current recovery.