Central Amygdala CRH and Enkephalin Neurons Modulate Anxiety‐like Behavior in Mice
Authors: Paretkar Tanvi 
Published: November 2023

The central nucleus of the amygdala (CeA) is a brain region that regulates a variety of autonomic responses including stress response. The CeA is comprised of GABAergic neurons which also coexpress neuropeptides such as corticotropin‐releasing hormone (CRH) and enkephalin (ENK) as cotransmitters. The ENKergic interneurons within the CeA are inhibitory and modulate the activity of CeA projection neurons that contain the excitatory neuropeptide CRH. Therefore the CeA output and subsequent stress response are affected by the interplay of CRH and ENK subpopulations in the CeA.

We tested the hypothesis that the CRH and ENK neurons in the CeA are functionally distinct neuronal populations in CeA and that their acute activation will have opposing effects on anxiety‐like behavior in mice. We applied chemogenetic approach using cre activated adeno‐associated virus expressing excitatory designer receptor exclusively activated by designer drug (AAV/DREADD/Gs) to acutely excite GABAergic, CRH and ENK neuronal populations in CeA. Acute excitation of the specific neuronal population was achieved by i.p injection of Clozapine N‐Oxide (CNO, 1 mg/kg) in transgenic cre mice (VGAT‐cre, CRH‐cre and ENK‐cre). We investigated the anxiety‐like behavior using the Elevated O‐maze (O‐Maze) and Light‐Dark box (LDB) test. The AAV‐DREADD expression in different cre lines showed differences that matched mRNA expression of CRH and ENK in CeA. Acute excitation of the CRH neurons in CeA produced robust increase in the anxiety‐like behavior. The CRH‐cre mice injected with CNO spent 0 % of their time while the vehicle injected mice spent 25.8 ± 2.8 % of their time in the open arms of the O‐Maze (t8=2.3, p<0.05). In the LDB test, the CNO injected mice spent 4.5 ± 2.2 % of their time and the vehicle injected mice spent 11.4 ± 0.67 % of their time in the light compartment (t7=3.26, p<0.05). Also in the LDB test, time to exit was an average 183.8 ± 29.1 seconds for CNO injected group and 92 ± 21.6 seconds for the vehicle injected group (t8=2.59, p<0.05). Acute excitation of ENK neurons in CeA reduced anxiety‐like behavior. The ENK‐cre mice injected with CNO spent 26.2 ± 3.6 % time in the open arms of O‐Maze whereas the vehicle group spent 14.8 ± 2.8 % of their time in the open arms (t8=2.44, p<0.05). In the LDB test, the CNO injected group spent 31.9 ± 3.5 % of their time in the light compartment whereas the vehicle group spent only 16.9 ± 3.3 % (t9=3.06, p<0.05). Also the time to exit in the LDB test was shorter for CNO injected mice. The CNO injected mice took 25 ± 10.8 seconds to exit whereas the vehicle group exited after 154 ± 47.2 seconds (t9=2.9, p<0.05). However, the acute excitation of the dominant GABAergic population in the CeA did not affect the anxiety‐like behavior in mice.

Our investigation showed that the acute excitation of CRH neurons in CeA increased the anxiety‐like behavior. Very likely this anxiogenic behavior is a consequence of the activation of central amygdalar projections to hypothalamic and brainstem centers. On the other hand, acute excitation of the ENK neurons resulted in reduced anxiety‐like behavior. This can be attributed to the inhibitory role of ENK neurons in CeA which opposes the activation of CRH projection neurons.

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5R01MH105528‐04