Why do we do what we do? What agent drives our decision-making? What motivates one person to work hard and another to stay in bed all day? These questions have no simple answers (if any at all), but neuroscience researchers believe they can access the underpinnings of motivated and reward-seeking behavior by studying the neural circuits that drive such behaviors. The lab of Dr. Garret D. Stuber at University of North Carolina at Chapel Hill does just that.
Using techniques in-vitro (after an animal has been sacrificed for study) and in-vivo (while the animal is alive and behaving), scientists in the Stuber lab try to identify and isolate neural circuits that guide behavior in health and disease. In this post, we will look at a recent publication examining a particular circuit implicated in integrating a range of opposing emotional states and guiding behavior. Specifically, the authors focus on neuronal projections from the bed nucleus of the stria terminalis (BNST) to the ventral tegmental area (VTA), by first examining the electophysiological properties of this circuit, and then manipulating the circuit in-vivo to confirm that their predictions about its function can be observed in an awake, behaving mouse.
The BNST is considered to be a part of the extended amygdala. Many people readily recognize the amygdala as an important brain center for emotional processing (in fear and anxiety) and, naturally, decision making related to those emotions. The VTA is a group of neurons that include dopaminergic projections to various brain regions, and is functionally implicated in both reward and aversion. Thus, studying the connections between the BNST and the VTA could shed light on the neural substrates for integrating differing emotional states and driving anxiety-related behavior. Elucidating these mechanisms could have marked implications for diseases such as addiction, anxiety, and depression.
Since neurons in the brain can project to many different areas, the authors first had to ensure that they could identify neurons that reside in the BNST (particularly the ventral BNST: vBNST) and project to the VTA. To do this, the authors used a technique called optogenetics, wherein special ion channels that can be activated by light are expressed in neurons of interest. To ensure and categorize functional connections between the two areas, the authors used a clever trick: they stimulated, sequentially, with light, the cell bodies in the vBNST and their axon terminals in the VTA while recording from the vBNST. Because action potentials have no directional preference, but cannot travel through sections of axon that are in a refractory period (having just propagated an action potential), functional connections stimulated under precise conditions would induce “action potential collision.” The recording schematic is illustrated in Fig 1b, with an example recording in Fig 1c, and Fig 1d shows the attenuation of the antidromic spike (propagating backwards along the axon), indicating “spike collision”.
Because the behavior of neurons projecting from vBNST to VTA is not homogenous, the authors next separated the population into a subset of excitatory projecting neurons (glutamatergic) and inhibitory projecting neurons (GABA-ergic). They did this by using promoters for Vglut2 or Vgat, which express differentially in the two populations, and recorded EPSCs and IPSCs in VTA neurons, respectively. To examine the significance of these two populations, the authors recorded from each type of neuron during aversive foot-shock stimuli and found that excitatory glutamatergic neurons increased their firing during foot shocks and subsequent relevant cues, while inhibitory neurons decreased their firing to aversive foot shock (Jennings et al, 2013).
To test whether this observed pathway would be sufficient to drive anxiety-related behaviors, the scientists next activated these subsets of neurons in-vivo. By expressing ChR2 in excitatory vBNST neurons, they could stimulate the vBNST-VTA pathway with light pulses when the mouse was in a specific context, in this case one of two chambers. They observed that mice significantly avoided the chamber paired with the photostimulation, indicating an anxiety-like response. To corroborate this observation, an injection of DNQX, which blocks glutamate activity, abolished this avoidance. Furthermore, mice that were photostimulated in an open-field test spent significantly more time in corners of the observation table versus control mice (Fig 4, Jennings et al 2013).
As activating the excitatory pathway to VTA resulted in avoidance and anxiety-like behavior, the scientists also tested whether there would be behavioral consequence to activating the analogous inhibitory pathway. Indeed, stimulating the inhibitory pathway resulted in significant place preference to the chamber that was paired with the stimulation; again, this result was abolished by GABAzine, a GABA inhibitor. Mice even nose poked to receive the photostimulation, indicating a reward-related behavior. In fact, this pathway was successful in alleviating anxiety-related freezing produced by aversive foot shocks, providing strong evidence that these vBNST-VTA neurons are strongly related to anxiety or anxiety buffering (Jennings et al 2013).
This study by Dr. Stuber’s lab provides strong evidence for a neural pathway that regulates anxiety and reward-seeking behavior. Identifying these pathways could be a promising lead towards identifying physiological disruptions that cause anxiety, depression, and addiction. If you’d like to find out more about these experiments (and perhaps other, even more recent studies), come listen to Dr. Garret D. Stuber on Tuesday, February 3 in the Center for Neural Circuits and Behavior Marylin C. Farquhar Conference Room!
Uri Magaram is a first year graduate student in the UCSD Neuroscience program.
Jennings, J. H., Sparta, D. R., Stamatakis, A. M., Ung, R. L., Pleil, K. E., Kash, T. L., & Stuber, G. D. (2013). Distinct extended amygdala circuits for divergent motivational states. Nature, 496(7444), 224-228. doi:10.1038/nature12041