Locus coeruleus là gì

Genetic and anatomical isolation of BLA-projection LC-NE neurons

Our previous work demonstrated that increased tonic LC-NE activity induces anxiety-like and aversive behavioral responses [McCall et al., 2015; Siuda et al., 2015a], we next sought to test whether these same behaviors can be generated by stimulating LC-NE fibers at localized projections in the BLA. To examine the potential sources of NE from the LC to the BLA we identified and isolated the projection using two distinct retrograde tracing approaches. First, we injected the tracer Fluorogold into the BLA of wild-type mice [Figure 1A]. Consistent with previous studies, this non-selective retrograde tracing approach revealed known inputs into the BLA from the LC [Robertson et al., 2016; Asan, 1998; Fallon et al., 1978] [Figure 1B]. We next used a dual injection strategy to anatomically isolate BLA-projecting LC-NE+ neurons. To do so, we used mice expressing Cre under the promoter for tyrosine hydroxylase, the rate-limiting enzyme for catecholamine synthesis [ThIRES-Cre mice][Savitt et al., 2005]. Here, we injected a red retrobead tracer into the BLA and the green-labeled adeno-associated virus, AAV5-DIO-Ef1α-ChR2[H134]-eYFP, into the LC [ThIRES-Cre::LC-BLA:ChR2; Figure 1C and D]. In these animals, we identified BLA-projecting Th+ LC neurons with the presence of both fluorophores in the same cells [Figure 1D]. Next, to examine Th+ terminal innervation of the BLA, we injected the cell-filling, Cre-dependent reporter AAV5-DIO-Ef1α-eYFP in the LC of ThIRES-Cre mice [ThIRES-Cre::LC-BLA:eYFP] [Figure 1E]. Here we clearly observed labeled LC neurons [Figure 1F and G] and their projection fibers terminating in the BLA [Figure 1H]. To further corroborate these findings, we examined recent projection experiments performed by the Allen Brain Institute Mouse Connectivity [ABIMC] project that also genetically and anatomically isolate this projection [Oh et al., 2014]. In three different experiments from three different genetic models, we observed LC-BLA projections that are qualitatively similar to our own observations [Figure 1—figure supplement 1A–L]. We present these findings here for clarity and ease of independent comparison, but this work was performed by ABIMC. Together, these anatomical studies identify a discrete projection of Th+ neurons from the LC that potentially release endogenous NE into the BLA.

[A] Cartoon depicting fluorgold tracing strategy. [B] Representative image [selected from three injected mice] shows robust retrograde labeling of the LC from injection in the BLA [green = pseudocolored Fluorgold, tyrosine hydroxylase = red]. Arrowhead indicates example co-localization. Scale bar = 100 µm. 4V = 4th ventricle. The TH- cells dorsal and ventral to the LC are likely part of the medial parabrachial nucleus which has previously identified projections to the BLA [Saper and Loewy, 1980]. [C] Cartoon depicting dual injection tracing strategy for CTB-594 and DIO-ChR2-eYFP. [D] Representative images [selected from three injected mice] shows retrograde labeling in LC of red retrobeads and anterograde labeling of TH+ cells [green] [Nissl=blue]. Arrowhead indicates example co-localization. Scale bar = 100 µm [E] Cartoon depicting anterograde tracing strategy. [F–H] Coronal images depict robust eYFP [yellow] labeling in the LC [F and G] and BLA [H] of the same mouse [scale bars = [F] 50 µm, [G] 10 µm, [H] 20 µm. Inset [F], tyrosine hydroxylase = red, scale bar = 25 µm.

Optogenetic activation of LC-BLA terminals releases norepinephrine into the BLA

Using the same viral optogenetic strategy as above, we examined whether photostimulation of ThIRES-Cre::LC-BLA:ChR2 projections resulted in NE release at terminals. We validated functional ChR2 expression using whole-cell current clamp recordings of Th+ LC neurons. As, we previously demonstrated [McCall et al., 2015], this targeting method and photostimulation protocol was sufficient to generate action potentials at the LC cell bodies [Figure 2A]. Next, we used a carbon fiber microelectrode [CFME] to perform fast-scan cyclic voltammetry [FSCV] in the BLA during LC-NE terminal stimulation. Using the extended waveform method [see methods] in acute brain slices of ThIRES-Cre::LC-BLA:ChR2 animals, we stimulated slices with 30 5 ms light pulses from a 473 nm LED, at 10 Hz [Figure 2B]. Photostimulation of BLA slices, produced characteristic cyclic voltammograms and uptake consistent with NE [McElligott et al., 2013; Herr et al., 2012] [t1/2 = 2.0 ± 0.2 s, Figure 2C and D]. Following a 20 min baseline, 1 µM reserpine [an inhibitor of vesicular monoamine transporters] was perfused on the slices to deplete catecholamines from the axon terminals [Dahlstroem et al., 1965]. Reserpine treatment significantly attenuated the measured oxidative currents [31.6 ± 1.0% of baseline, Figure 2E and F] further confirming optically-evoked catecholamine release in this isolated ThIRES-Cre::LC-BLA:ChR2 projection. These findings suggest that optogenetic manipulation of LC-BLA terminals causes release of endogenous NE from the LC into the BLA.

[A] LC neuron firing reliably to 10 Hz optical stimulation [CC=whole cell current clamp]. [B] Fast scan cyclic voltammetry [FSCV] schematic. [C–D] Oxidative and reductive currents [scale bar 2 s by 0.4 nA], with representative cyclic voltammograms [inset] and representative color plots [below] in response to photostimulation are attenuated by reserpine [1 μM]. Color plots for baseline and after reserpine [1 µM]: Files were collected over 15 s [X-axis] where the carbon fiber microelectrode was ramped with a triangular waveform from −0.4V to 1.3V and back to −0.4V at 400 V/S [Y-axis] and sampled at 10 Hz. 10 Hz, 473 nm blue LED stimulation onset at 2 s. Oxidative currents [nA] are positive in direction and reductive currents are negative [see color coded scale bar on right]. [E] Attenuation in NE oxidative current in response to reserpine [1 μM] n = 3 pairs; mean ± S.E.M]. [F] Average of first 20 min and last 15 min in [E] [Data represented as mean ± SEM, Paired Student’s t-tests to baseline, Mean difference = 68.56, t[2] = 18.75, **p=0.0028, 95% CI [52.82 to 84.29]].

In vivo photostimulation of LC-BLA terminals modulates BLA activity

BLA neurons have well reported responses to exogenous application of NE [Buffalari and Grace, 2007], but their response to endogenous NE release, explicitly from the LC, has not been previously defined. We next determined whether the optically-evoked endogenous catecholamine release would mimic the responses of BLA neurons to exogenous NE. To do so, we examined BLA single-unit activity in ThIRES-Cre::LC-BLA:ChR2 mice using 16-channel microelectrode arrays coupled to a fiber optic implant [optrode arrays] [McCall et al., 2015; Sparta et al., 2011]. These optrode arrays were used to isolate and record BLA single-unit activity before, during, and after photostimulation of ThIRES-Cre::LC-BLA:ChR2 projections [473 nm, 5 Hz, 10 ms pulse width] [Figure 3A and B]. In these experiments photostimulation of ThIRES-Cre::LC-BLA:ChR2 terminals caused an increase in firing frequency in 21.4% of units recorded in the BLA [Figure 3C–E,H; Figure 3—figure supplement 1A], while some cells [9.5%] displayed inhibitory responses [Figure 3D,F,I; Figure 3—figure supplement 1B]. The remaining neurons [69.0%] appeared to not change in response to photostimulation [Figure 3D,G,J; Figure 3—figure supplement 1C]. Furthermore, in a subset of cells blindly selected [without knowledge of the increase/decrease/static response to photostimulation] following the 5 Hz recordings, we also observed similar increases in firing rates to constant photostimulation, where the overall population of neurons increased firing during stimulation [Figure 3—figure supplement 1D and E]. In cases where the firing rate increased, the mean ± SD latency to fire following each light pulse was 129.3 ± 108.9 ms, suggesting that this change is not due to fast, monosynaptic neurotransmission, but likely a polysynaptic and/or neuromodulatory effect [Figure 3K]. Likewise, in cases where the firing rate decreased the mean latency was slower at 172.6 ± 77.01 ms, and neither case was significantly different to neurons whose firing rate did not change following photostimulation [260.4 ± 165.7] [Figure 3K].

[A] Schematic illustrating single-unit extracellular recording paradigm of BLA neurons modulated by ChR2-expressing LC-BLA terminals. [B] Representative principal component analysis plot showing the first two principal components with clear clustering of a single unit [maroon] from the noise [grey]. Inset shows the waveform and spikes making up the isolated unit. Y-scale is 150 microvolts and x-scale is 500 ms. [C] Recordings from eight hemispheres of six Th-CreLC-BLA:ChR2 mice show the distribution of firing rates present in BLA neurons prior to and following LC-BLA terminal photostimulation [473 nm, 5 Hz, 3 min]. [D] Average normalized firing rate of neurons that increase [maroon], decrease [grey], or do not change [black] firing rate in response to photostimulation. Inset, shows number of neurons in each group. Representative histograms [1 s bins] of isolated single-units showing increase [E] or decrease [F], or no change [G] in neuronal firing in response to photostimulation [473 nm, 5 Hz, 3 min]. Z-scored population responses of neurons showing increase [H] or decrease [I], or no change [J] in neuronal firing in response to photostimulation. [K] Response latency following onset of photostimulation for cells that did not alter firing [=] [n = 29], increased firing [+] [n = 9], or decreased firing [-] [n = 4]. [Data represented as mean ± SD]. [L] The same cells sorted by baseline firing rate. [Data represented as mean ± SEM. Kruskal-Wallis test one-way ANOVA for non-parametric data with Dunn’s multiple comparisons test, Kruskal-Wallis statistic = 6.536, p=0.0381; + vs. – Mean rank difference = −18.75, adjusted *p=0.0329; + vs. + Mean rank difference = −6.828, adjusted p=0.4341; - vs. = Mean rank difference = 11.92, adjusted p=0.2053.] [M] Waveform similarity, within group distribution of linear correlations. Inset, every average waveform for each recorded unit separated by response profile [= black, + maroon, - grey]. [N–P] Bursting profiles for each recorded neuron. [N] Number of bursts per second. [Data represented as mean ± SEM]. [O] Mean firing rate within bursts for each neuron. [Data represented as mean ± SEM]. [P] Proportion of recorded spikes that occurred during bursts. [Data represented as mean ± SEM].

We next sought to determine whether these differences in neuronal response arise from distinct subsets of BLA neurons. To do so, we examined the baseline firing rate, waveforms, and bursting properties of recorded units. Neurons that increase firing in response to LC-BA terminal stimulation have a significantly lower mean basal firing rate [2.194 ± 2.22 SD] than those that decrease firing [13.06 ± 11.82 SD], however neither group is distinguishable from non-responsive neurons [Figure 3L]. To quantifiably assess the waveform shapes of the recorded neurons, we calculated the average waveform for each neuron and performed a linear correlation on these values within each group of neurons. These analyses demonstrate that all neurons that increase firing to LC-BLA photostimulation have an r value greater than 0.835 and all neurons that decrease have an r value above 0.820, while the population of neurons that did not significantly respond to photostimulation have an r above 0.515. These results indicate that neurons that do not respond to photostimulation are less internally similar, while the excited and inhibited cells are more similar within groups, suggesting that these modulated neurons are more likely to each be part of single class of neurons [Figure 3M]. Finally, we examined the bursting properties of the recorded neurons. While most neurons exhibited some bursting properties, no differences were found between groups in terms of frequency of bursting [Figure 3N], mean firing rate during bursts [Figure 3O], or the percentage of spikes from the recording sessions that occurred within a burst [Figure 3P]. Together, these heterogeneous firing properties are consistent with previous studies using iontophoresis of NE into the BLA, and further highlight the complex pharmacological and circuit activity that NE modulates within the BLA [Buffalari and Grace, 2007; Ferry et al., 1997; Huang et al., 1996]. These results suggest that photostimulation of LC terminals in the BLA causes varied neuronal firing rate responses, with the majority of photostimulation-responsive neurons increasing firing in response to the LC-NE terminal photostimulation.

LC-BLA terminal stimulation biases activation towards anxiety-promoting BLA neurons

The BLA is thought to be a heterogeneous hub for emotional processing containing separable populations for regulating either positive or negative affect. Recent studies have suggested that these opposing populations are distinct in either their projection target or their cell-type [Stuber et al., 2011; Namburi et al., 2015; Beyeler et al., 2016; Felix-Ortiz et al., 2016; Burgos-Robles et al., 2017; Kim et al., 2016, 2017; Correia et al., 2016]. Given the role of BLA adrenergic receptors in modulating anxiety-like and aversion behaviors [Siuda et al., 2015a, 2016], we hypothesized that LC-NE innervation of BLA neurons may preferentially bias activation of neurons that promote anxiety-like behavior such as the ventral hippocampus [vHPC]- and central amygdala [CeA]- projecting BLA neurons as opposed to projections that promote positive affect and anxiolysis such as nucleus accumbens [NAc]-projecting BLA neurons. Using a combination of retrograde viral tracing, immunohistochemistry, and optogenetic stimulation in Th-CreLC-BLA:ChR2 mice, we assessed these potential tri-synaptic circuits that may underlie anxiety-like behaviors [Figure 4A]. Photostimulation of LC-NE terminals in the BLA [5 Hz, 10 ms] significantly increases the number of cFos-expressing BLA neurons in ThIRES-Cre::LC-BLA:ChR2 animals compared to ChR2-lacking [Figure 4B; Figure 4—figure supplement 1A–D] and contralateral BLA controls following photostimulation [Figure 4—figure supplement 1E]. To assess whether the cFos+ BLA neurons resulting from LC-NE terminal activation were biased toward a particular class of BLA projection neurons we next repeated the experiment with the retrograde tracer Cholera toxin subunit B [CTB] injected into BLA projection regions [vHPC, CeA, and NAc]. These immunohistochemistry studies reveal that the cFos present following LC-NE terminal activation in the BLA, overlaps significantly more with vHPC- and CeA-targeted compared to NAc-targeted CTB in the BLA [Figure 4C–G; Figure 4—figure supplement 1F–H]. These results suggest that LC-NE terminals in the BLA preferentially activate vHPC- and CeA- projecting BLA neurons thought to be involved in modulating negative valence and affective behaviors.

[A] Diagram of viral and optogenetic strategy. [B] 5 Hz photostimulation increases cFos expression within the BLA in ThIRES-Cre::LC-BLA:ChR2+ animals compared to ThIRES-Cre::LC-BLA:ChR2- controls [Data represented as mean ± SEM, n = 9 ChR2, n = 4 Ctrl; average of 3 sections/mouse; Student’s t-test, Mean difference = 19.17, t[10] = 4.005, **p=0.0040, 95% CI [−35.47 to −10.11]. [C] 5 Hz photostimulation increases cFos expression significantly more in BLA neurons projecting to the vHPC and CeA compared to NAc in ThIRES-Cre::LC-BLA:ChR2 animals [Data represented as mean ± SEM, n = 9 vHPCCTB, n = 6 CeACTB, n = 9 CeACTB, 3 sections per mouse; One-Way ANOVA, Bonferroni’s Multiple Comparison Test, F2,20 = 7.199, **p=0.0044; ThIRES-Cre::LC-BLA:ChR2:CTB-vHPC vs. ThIRES-Cre::LC-BLA:ChR2:CTB-NAc Mean difference = 12.95, t[20] = 3.585, **p