Shedding Light on Serotonergic Neurotransmission in Amygdala Circuits
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Material below summarizes the article Control of Amygdala Circuits by 5-HT Neurons via 5-HT and Glutamate Cotransmission, published on February 15, 2017, in JNeurosci and authored by Ayesha Sengupta, Marco Bocchio, David M. Bannerman, Trevor Sharp, and Marco Capogna.
Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter implicated in a vast array of sensory, motor, and cognitive functions. Its role in emotional regulation is well-known because selective serotonin reuptake inhibitors (SSRIs), drugs that increase extracellular 5-HT levels, are a widely prescribed treatment for depression and anxiety.
5-HT release in the amygdala is of particular interest. Evidence from human studies suggests that the amygdala is involved in pathological states of depression and anxiety. Moreover, SSRIs and genetic variations in the 5-HT transporter influence amygdala activation to aversive stimuli, as well as anxiety-related personality traits and susceptibility to affective disorders. Finally, in animal models, manipulations of 5-HT signaling in the amygdala have been shown to alter fear and anxiety behavior.
5-HT neurons have cell bodies located in the dorsal raphe nucleus (DRN), and some of these send axonal projections to the amygdala. Within the amygdala, the basal nucleus (BA) receives particularly dense 5-HT inputs. BA circuits comprise a majority of excitatory principal output neurons that release glutamate and a minority of various local inhibitory interneurons that release GABA.
Pharmacological approaches provide functional evidence for the presence of 5-HT receptors in the BA. In these experiments, we applied exogenous 5-HT onto brain slices while we recorded electrophysiological responses of BA neurons. This showed that 5-HT can directly excite interneurons via 5-HT2A receptors, which in turn increases inhibitory inputs onto principal neurons.
Understanding the role of 5-HT in the BA is challenging, however, because histological studies have revealed that several 5-HT receptor types of excitatory and inhibitory nature are expressed in different neuron populations.
These earlier studies took the first steps towards characterizing the mechanisms of 5-HT neurotransmission in the BA. However, because they rely on pharmacological application of 5-HT, the observed effects may have been influenced by prolonged exposure, non-physiological concentrations, activation of extrasynaptic receptors, and the absence of potential co-released transmitters.
To progress toward a more physiological approach, we expressed the light-gated cation channel Channelrhodopsin-2 (ChR2) in DRN 5-HT neurons. This allowed us to selectively excite 5-HT projections in the BA by delivering light stimulation, a technique called optogenetics. Thus, we could stimulate the release of endogenous 5-HT and detect postsynaptic responses in BA neurons using whole-cell patch-clamp electrophysiology.
First, we ensured that our optogenetic approach was working correctly. This involved four steps: verifying ChR2 expression in the DRN, quantifying the specificity of ChR2 expression to 5-HT neurons, validating the functionality of ChR2 in 5-HT cell bodies, and checking ChR2 expression in 5-HT axons in the BA.
We found strong ChR2 expression in the DRN, which we could readily visualize because ChR2 was tagged with a fluorescent protein reporter. We carried out an immunohistochemical stain for 5-HT to confirm all cells expressing ChR2 contained 5-HT. By recording the electrophysiological activity of 5-HT cells in acute brain slices, we determined that light stimulation effectively caused excitation in these cells. Finally, we ascertained that ChR2 expression was also detected in 5-HT axons in the BA.
After validating our optogenetic approach, we proceeded to monitor the responses of BA neurons to photostimulation of 5-HT axons. Since previous studies had reported effects of 5-HT on BA interneurons, we started by analyzing the responses of this cell population.
Delivering single, short light pulses produced a fast excitatory response in 55 percent of interneurons. Delivering light pulses in a train caused the response to reduce in amplitude over time, and this amplitude depression was enhanced at higher stimulation frequencies. The fast excitatory response was abolished by the application of glutamate receptor antagonists. This indicates that these axons can release glutamate as well as 5-HT, and that with short light pulses, glutamate release is wholly responsible for the fast excitatory response.
An immunohistochemical stain for a vesicular glutamate transporter confirmed this finding. We detected the glutamate transporter in 5-HT axons in the BA, suggesting the molecular machinery for glutamate transmission was present in these axons.
The photostimulation experiment also indicated that trains of stimuli were necessary to trigger 5-HT responses. Light pulse trains evoked slow excitatory and slow inhibitory responses in 53 percent and 37 percent of BA interneurons, respectively. The excitatory response was abolished by a 5-HT2A receptor antagonist, whereas the inhibitory response was blocked by a 5-HT1A receptor antagonist. Hence, the slower effects were mediated by different types of 5-HT receptors, which produced either excitatory or inhibitory modulation of BA interneurons.
Remarkably, the amplitude of both 5-HT responses increased at higher stimulation frequencies, opposite to what was observed for the glutamatergic response.
We also found evidence for cell-type-specific modulation of BA interneurons, because the amplitudes of the fast glutamatergic and the slow 5-HT responses were significantly greater in fast-spiking versus non-fast-spiking interneurons. The signaling mechanisms are therefore preferentially targeted to distinct BA interneuron types.
Finally, we recorded from principal neurons to understand how 5-HT neurons shape the overall BA excitatory output. These experiments corroborated the finding that 5-HT neurons act in a cell-type-specific manner. Unlike in interneurons, we did not detect any fast glutamatergic or slow 5-HT2A-mediated excitation, but found only a slow, 5-HT1A-mediated inhibition in 93 percent of principal neurons.
Additionally, we observed a light-evoked increase in inhibitory input events in principal neurons — these were mediated by GABAergic signaling. This was likely due to the recruitment of local inhibitory interneurons by 5-HT projections, imposing an indirect modulation reported previously in the literature.
Overall, direct 5-HT1A and indirect GABAergic inhibition by 5-HT axons dampened the firing rate of principal neurons. This indicates 5-HT neurons are able to inhibit the output of the BA to other brain regions.
In conclusion, we discovered novel mechanisms through which DRN 5-HT neurons modulate the BA. Our findings demonstrate 5-HT projections co-release 5-HT and glutamate in a cell-type-specific manner. Our observations also suggest lower firing rates of DRN 5-HT neurons could favor glutamate transmission, whereas higher rates could enhance 5-HT. This result is behaviorally relevant because DRN 5-HT neurons increase their firing rate in vivo during salient environmental events.
Previously, elucidating the role of 5-HT transmission has understandably focused on isolating the actions of 5-HT. Our data highlight the necessity to incorporate concepts of co-transmission into future investigations of the 5-HT system in health and disease.
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Control of Amygdala Circuits by 5-HT Neurons via 5-HT and Glutamate Cotransmission. Ayesha Sengupta, Marco Bocchio, David M. Bannerman, Trevor Sharp, Marco Capogna. JNeurosci Feb 2017 DOI: https://doi.org/10.1523/JNEUROSCI.2238-16.2016