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Locus coeruleus to basolateral amygdala noradrenergic projections promote anxiety-like behavior

  1. Jordan G McCall Is a corresponding author
  2. Edward R Siuda
  3. Dionnet L Bhatti
  4. Lamley A Lawson
  5. Zoe A McElligott
  6. Garret D Stuber
  7. Michael R Bruchas Is a corresponding author
  1. Washington University School of Medicine, United States
  2. University of North Carolina, United States
  3. Washington University, United States
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Cite as: eLife 2017;6:e18247 doi: 10.7554/eLife.18247

Abstract

Increased tonic activity of locus coeruleus noradrenergic (LC-NE) neurons induces anxiety-like and aversive behavior. While some information is known about the afferent circuitry that endogenously drives this neural activity and behavior, the downstream receptors and anatomical projections that mediate these acute risk aversive behavioral states via the LC-NE system remain unresolved. Here we use a combination of retrograde tracing, fast-scan cyclic voltammetry, electrophysiology, and in vivo optogenetics with localized pharmacology to identify neural substrates downstream of increased tonic LC-NE activity in mice. We demonstrate that photostimulation of LC-NE fibers in the BLA evokes norepinephrine release in the basolateral amygdala (BLA), alters BLA neuronal activity, conditions aversion, and increases anxiety-like behavior. Additionally, we report that β-adrenergic receptors mediate the anxiety-like phenotype of increased NE release in the BLA. These studies begin to illustrate how the complex efferent system of the LC-NE system selectively mediates behavior through distinct receptor and projection-selective mechanisms.

https://doi.org/10.7554/eLife.18247.001

Introduction

The locus coeruleus noradrenergic system (LC-NE) comprises a widespread projection network throughout the central nervous system capable of modulating a diverse range of behaviors including arousal, learning, pain modulation, and stress-induced negative affective states (Berridge and Waterhouse, 2003; Sara, 2009). Understanding the neural circuit basis for how this nearly ubiquitous neuromodulatory network exerts influence on negative affect is a critical step towards therapeutically targeting stress-induced neuropsychiatric disorders (Schwarz and Luo, 2015; Schwarz et al., 2015; Reyes et al., 2015; McCall et al., 2015; Arnsten et al., 2015; Kebschull et al., 2016). One particular efferent projection from the LC is to the basolateral amygdala (BLA). The BLA is an important candidate anatomical substrate for the widely known role of norepinephrine (NE) in affective behaviors (Berridge and Waterhouse, 2003; Schwarz et al., 2015; Davis, 1992; Valentino and Aston-Jones, 2010; Robertson et al., 2016, 2013; Grissom and Bhatnagar, 2011; Siuda et al., 2015a, 2016; Plummer et al., 2015). The BLA is notable for integrating sensory information to encode and drive diverse and opposing affective behaviors including anxiety, fear, aversive, and reward behaviors (Kim et al., 2013; Stuber et al., 2011; Tye et al., 2011; Namburi et al., 2015; Gore et al., 2015; Belova et al., 2007; Beyeler et al., 2016; Wolff et al., 2014; Bermudez and Schultz, 2010; Bruchas et al., 2009; Knoll et al., 2011; Sugase-Miyamoto and Richmond, 2005; Crowley et al., 2016; Sears et al., 2013; Roozendaal et al., 2008, 2006; Miranda et al., 2007). Notable efforts to uncover the role of the BLA and adrenergic signaling in consolidation of fear memories have been reported (Sears et al., 2013; Roozendaal et al., 2008, 2006), as well as recent studies showing that acute stress activates BLA adrenergic receptors (agnostic to the source of NE) to promote anxiety and other stress-related behaviors (Miranda et al., 2007; Buffalari and Grace, 2009a, 2009b; Chang and Grace, 2013). Similarly acute stress paradigms cause selective activation of LC-NE neurons (McCall et al., 2015). Together, there have been significant efforts to examine how source-independent noradrenergic (importantly, there are multiple sources of NE innervating the BLA (Robertson et al., 2016; Plummer et al., 2015) signaling in the BLA can alter synaptic plasticity, fear encoding, and memory consolidation, yet few studies have directly examined how the neuromodulatory LC-NE system utilizes BLA output to alter acute risk averse behaviors, such as anxiety (Grissom and Bhatnagar, 2011; Buffalari and Grace, 2009a, 2009b, 2007).

Downstream and independent of this projection, recent studies have demonstrated that direct activation of both basolateral amygdala (BLA) cell bodies or their projections is both anxiogenic and socially aversive (Siuda et al., 2015a, 2016; Tye et al., 2011; Felix-Ortiz et al., 2013, 2016; Felix-Ortiz and Tye, 2014). Furthermore, it has also been demonstrated that increasing BLA excitatory output through Gαs G-protein activation, and more specifically, β-adrenergic receptor signaling causes acute social anxiety (Siuda et al., 2016). Separately, noradrenergic cell firing in the LC has been shown to increase in the context of stressful stimuli (McCall et al., 2015; Abercrombie and Jacobs, 1987a, 1987b; Aston-Jones et al., 1999; Mana and Grace, 1997). While the anatomical projections from the LC and their cell types have been studied for several years, the precise mechanisms by which fibers from the LC can directly influence BLA function to promote negative affective behaviors are not understood. Specifically, how LC-BLA projections generate affective behavioral responses through specific receptor systems and modulation of cell activity is unknown.

To determine the role of locus coeruleus noradrenergic influence on BLA function and negative affective behavior we optogenetically manipulated LC-NE inputs into the BLA, directly testing whether NE is released from LC terminals into the BLA and whether this terminal stimulation can drive anxiety-like and aversive behavioral responses. We demonstrate that photostimulation of LC projections to the basolateral amygdala releases NE and that this photostimulation evokes downstream modulation of neuronal activity in BLA neurons that project to anxiogenic brain regions. Stimulation of these fibers is sufficient to produce conditioned aversion and the stimulation-induced increased noradrenergic tone is sufficient to produce anxiety-like behavior mediated by local β-adrenergic receptor activity (β-ARs) in the BLA. Taken together, we report a previously undefined role for LC-BLA projections in mediating negative affective behavior through activation of β-ARs.

Results

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.

Figure 1 with 1 supplement see all
Identifying a LC input to 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.

https://doi.org/10.7554/eLife.18247.002

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.

Photostimulation of LC terminals in the BLA releases norepinephrine.

(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]).

https://doi.org/10.7554/eLife.18247.004

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).

Figure 3 with 1 supplement see all
Photostimulation of LC terminals in the BLA alters neuronal activity.

(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).

https://doi.org/10.7554/eLife.18247.005

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.

Figure 4 with 1 supplement see all
Photostimulation of LC terminals in the BLA preferentially activates BLA circuitry associated with anxiety-like behavior.

(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<0.01 95% CI [3.511 to 22.39]; ThIRES-Cre::LC-BLA:ChR2:CTB-CeA vs. ThIRES-Cre::LC-BLA:ChR2:CTB-NAc. Mean difference = 11.25, t(20) = 2.802, *p<0.05 95% CI [0.7605 to 21.74]. Representative images of the BLA expressing cFos after 5 Hz photostimulation in (D) ThIRES-Cre::LC-BLA:ChR2 injected with CTB in NAc, (E) vHPC, or (F) CeA. Scale bar, 100 µm. (G) Confocal images showing colocalization of CTB and cFos after 5 Hz photostimulation in the NAc, vHPC, and CeA. Scale bar, 50 µm.

https://doi.org/10.7554/eLife.18247.007

LC-BLA terminal stimulation is sufficient to elicit a conditioned place aversion

After identifying the LC terminals to the BLA as a projecting source of NE, that photostimulation of these terminals are capable of altering BLA activity, and that this photostimulation appears to bias BLA activation towards circuits involved in negative affect, we next tested whether this same pattern of terminal stimulation can drive an immediate negative valence. Fiber optics were implanted at LC-NE terminal sites in the BLA of ThIRES-Cre mice with AAV5-DIO-Ef1α-ChR2(H134)-eYFP previously injected into the LC (ThIRES-Cre::LC-BLA:ChR2) (Figure 5A, Figure 5—figure supplement 1A). Surprisingly, photostimulation of ThIRES-Cre::LC-BLA:ChR2 terminals using the parameters that induce a real-time place aversion at LC-NE cell bodies (McCall et al., 2015) failed to elicit a real-time aversion in ThIRES-Cre::LC-BLA:ChR2 animals (Figure 5B and C). There was no significant change in place testing behavior (Figure 5C) or locomotor behavior (Figure 5D) within animals or compared to ThIRES-Cre::LC-BLA:eYFP controls at either a physiologically-relevant 5 Hz or an over-driven 60 Hz photostimulation. This finding is particularly interesting in light of previous systemic pharmacology experiments (McCall et al., 2015) that suggested the immediate negative valence induced by LC-NE stimulation is mediated by α1 adrenergic receptor activation. These results indicate that the real-time negative valence of LC-NE stimulation is likely produced via another LC-NE projection and one that is not recruited through antidromic stimulation of BLA-projecting LC neurons.

Figure 5 with 1 supplement see all
Photostimulation of LC terminals in the BLA causes conditioned aversion.

(A) Cartoon of viral and fiber optic delivery strategy and calendar of real-time place testing studies. (B) Representative traces of behavior at different frequencies. (C) Frequency response of RTPT and (D) locomotor activity at 5 and 60 Hz. Data represented as mean ± SEM, n = 4 eYFP, 5 ChR2. (E) Conditioned place aversion (CPA) behavioral calendar. (F) Representative CPA traces. (G) ThIRES-Cre::LC-BLA:ChR2 mice (maroon; n = 9) show a conditioned aversive response to chamber paired with photostimulation compared to ThIRES-Cre::LC-BLA:eYFP controls (grey; n = 8) (Data represented as mean ± SEM, Student’s t-test, Mean difference = 131.3, t(15) = 2.39, *p=0.0303, 95% CI [14.32 to 248.4) following two days of conditioning with (H) no significant differences in locomotor behavior during conditioning day 1 (CD1) and or conditioning day 2 (CD2) in CPA.

https://doi.org/10.7554/eLife.18247.009

It is also possible that the neuromodulatory effects on this circuit require a long-term associative memory component compared to fast-acting, acute activation of similar pathways (Kim et al., 2013; Namburi et al., 2015; Haubensak et al., 2010). To directly test whether long-term learned associations are produced by ThIRES-Cre::LC-BLA:ChR2 stimulation, we employed a Pavlovian conditioned place aversion (CPA) assay similar to our previous work (McCall et al., 2015; Siuda et al., 2015a; Al-Hasani et al., 2013; Siuda et al., 2015b) (Figure 5E, Figure 5—figure supplement 1B). When allowed to freely explore two contextually-differentiated chambers, following two conditioning days of photostimulation of ThIRES-Cre::LC-BLA:ChR2 terminals (5 Hz, 10 ms), animals expressing ChR2-eYFP spent significantly less time in the context that was previously paired with photostimulation compared to the ThIRES-Cre::LC-BLA:eYFP controls (Figure 5F and G). Neither group of mice showed significant changes in locomotor activity before, during, or after conditioning (Figure 5H). Together, these results suggest that there is a learned negative association produced by photostimulation of Th+ fibers in the BLA. This association is capable of producing avoidance from contexts in which the photostimulation previously occurred.

Optogenetic targeting of LC-NE projections to the BLA promotes anxiety-like behavior through beta-adrenergic receptor activity

Following the observation that stimulation of LC-BLA fibers alone is sufficient to cause a conditioned aversion, we next tested whether the same stimulation of these terminals would drive acute anxiety-like behaviors similar to those we recently reported in response to direct LC-NE cell body activation. Photostimulation of ThIRES-Cre::LC-BLA:ChR2 terminals using parameters that induce anxiety-like behavior at LC-NE cell bodies (McCall et al., 2015) resulted in a significant decrease in time spent in the center of the open field (OFT) with no significant change in general locomotor activity (Figure 6A–D, Figure 6—figure supplement 1A). These experiments suggest that stimulation of only the subset of LC-NE fibers that project to the BLA is sufficient to induce anxiety-like behaviors.

Figure 6 with 1 supplement see all
Photostimulation of LC terminals in the BLA promotes anxiety-like behavior through beta-adrenergic receptors.

(A) Calendar of OFT studies. (B) Representative heat maps of activity during OFT. (C) 5 Hz photostimulation causes an anxiety-like phenotype in OFT of ThIRES-Cre::LC-BLA:ChR2 animals compared to ThIRES-Cre::LC-BLA:eYFP controls (Data represented as mean ± SEM, n = 10 eYFP, 11 ChR2; Student’s t-test, Mean difference = 116.9, t(19) = 3.46, **p=0.0026, 95% CI [46.20 to 187.5) with (D) no change in locomotor activity (Data represented as mean ± SEM). (E) Cartoon of viral, cannula, and fiber optic delivery strategy and (F) calendar of EZM behavior. (G) 5 Hz photostimulation causes an anxiety-like phenotype in EZM of ThIRES-Cre::LC-BLA:ChR2 animals compared to ThIRES-Cre::LC-BLA:eYFP controls, which is reversed by intra-BLA propranolol pretreatment (Data represented as mean ± SEM, n = 11 eYFP + Vehicle, n = 9 ChR2 + Vehicle, n = 7 eYFP + Propranolol, n = 8 ChR2 + Propranolol; One-Way ANOVA, Bonferroni’s Multiple Comparison Test, F3,31 = 8.95, p=0.0002; ThIRES-Cre::LC-BLA:eYFP+Vehicle vs. ThIRES-Cre::LC-BLA:ChR2+Vehicle. Mean difference = 14.74, t(2) = 3.94, **p<0.01 95% CI [4.21 to 25.27]; ThIRES-Cre::LC-BLA:ChR2+Vehicle vs. ThIRES-Cre::LC-BLA:ChR2+Propranolol. Mean difference = 16.93, t(2) = 4.19, **p<0.01 95% CI [5.46 to 28.31]; ThIRES-Cre::LC-BLA:eYFP+Propranolol vs. ThIRES-Cre::LC-BLA:ChR2+Vehicle. Mean difference = 18.37, t(2) = 4.39, ***p<0.001 95% CI [6.57 to 30.18). (H) Representative heat maps of activity during EZM.

https://doi.org/10.7554/eLife.18247.011

Our prior work revealed that systemic antagonism of β-ARs blocks the anxiogenic, but not the real-time aversive, components of tonic LC-NE stimulation (McCall et al., 2015). We next tested whether β-AR activity in the BLA is necessary for LC-NE mediated anxiogenesis. To do so we implanted a combined fiberoptic-fluid cannula into the BLA and delivered either an artificial cerebrospinal fluid vehicle or the non-selective β-AR antagonist, Propranolol (1 µg)(Roozendaal et al., 2008, 2006; Chang and Grace, 2013; Shanks et al., 1966), prior to photostimulation (Figure 6E–H, Figure 6—figure supplement 1B). The same photostimulation paradigm that was anxiogenic in the OFT, caused a decrease in time spent in the open area of the elevated zero maze (EZM) of ChR2+ animals that received vehicle, compared to YFP-expressing, vehicle controls (Figure 6G and H). Importantly, when we locally antagonized β-ARs directly in the BLA prior to photostimulation, anxiogenesis was completely blocked in ChR2+ animals with no effect on eYFP-expressing, propranolol controls (Figure 6G and H). These findings suggest that local β-AR activation by NE release from LC-BLA terminals is the substrate responsible for the photostimulation-induced anxiety-like behavior.

Discussion

We aimed to dissect the functional role of LC to BLA inputs, and to determine the specific receptors within the BLA that mediate LC-NE influenced anxiety-like behavior in the region. Taken together, these data suggest that photostimulation of LC-BLA projections releases NE into the BLA and this same photostimulation alters BLA firing. This altered firing appears to preferentially increase activity in BLA neurons that project to area known to modulate negative affect such as the CeA and vHPC. In behaving animals, this stimulation of LC terminals in the BLA facilitates a conditioned aversion and acute anxiety-like behaviors. Importantly, the experiments here cannot determine whether the observed changes in BLA unit activity and the conditioned place aversion are mediated directly by NE release from LC terminals. Instead, these affects could be generated through antidromic stimulation of LC cell bodies (and their diverging efferent projections) or through plastic changes resulting from the repeated and long-term stimulation protocols used during the two 30 min conditioning sessions. However, using local, site-specific pharmacological blockade, we identified that β-ARs within the BLA are necessary for the anxiety-like component of this behavior. These data provide a critical framework for understanding the downstream influence of the LC-NE system in the BLA as it relates to anxiety-like behavioral states.

The BLA is a crucial component in the neural circuitry of negative affective behaviors (Davis, 1992; Davidson, 2002). Activation of BLA cell bodies and their projections can bidirectionally mediate anxiety-like behavior and fear encoding (Davis, 1992; Kim et al., 2013; Tye et al., 2011; Namburi et al., 2015; Beyeler et al., 2016; Wolff et al., 2014; Knoll et al., 2011; Crowley et al., 2016; Sears et al., 2013; Roozendaal et al., 2006; Felix-Ortiz et al., 2013; Debiec and Ledoux, 2004), but how this behavioral control is altered by neuromodulators is not well understood. NE is increased in the BLA following stressful events (Galvez et al., 1996; Hatfield et al., 1999). The BLA receives noradrenergic input from the LC (Asan, 1998; Fallon et al., 1978) as well the Hoxb1+ neuronal populations in the lower brainstem and NTS (Robertson et al., 2016). Despite the LC input being less dense than other NE input (Plummer et al., 2015), here we directly show that molecularly-defined Th+ positive fibers from the LC release catecholamines into the amygdala and alter behavior. Previous studies have demonstrated that noradrenergic tonic cell firing in the LC increases following stressful stimuli (McCall et al., 2015; Abercrombie and Jacobs, 1987a, 1987b; Aston-Jones et al., 1999; Mana and Grace, 1997). We now demonstrate that selective activation of Th+ LC terminals from these neurons modulates BLA neuronal activity in vivo. Despite a diversity of recorded responses, these findings align with prior studies suggesting neurons that increase firing to LC-BLA photostimulation have a higher probability of being BLA projection neurons, while those that decrease have a higher probability of being BLA interneurons (Figure 3L) (Likhtik et al., 2006). Therefore, it seems that it is reasonable that LC-BLA modulation might be selectively increasing activity of BLA projection neurons, though further studies using phototagging or imaging BLA neurons is required. Though it is well established that activation of β-ARs increases BLA activity (Buffalari and Grace, 2007; Huang et al., 1996; Pu et al., 2009), we cannot conclude that this is the only mechanism by which LC-BLA fibers modulate BLA activity. Indeed, it is likely that the diversity of neuronal responses we observed are mediated through different receptors systems (Buffalari and Grace, 2007) and/or local or distal polysynaptic recurrent circuitry (Wolff et al., 2014; Rosenkranz and Grace, 2002, 1999). Furthermore, while we do not know the genetic identity or projection targets of the BLA neurons from which we recorded, it is possible that NE serves to preferentially shift BLA activity in neurons that favor anxiety-like behavior (i.e. those that project to the ventral hippocampus, prefrontal cortex, or central amygdala) rather those that favor positive affect and anxiolysis (i.e. projections to the nucleus accumbens and bed nucleus of the stria terminalis)(Kim et al., 2013; Stuber et al., 2011; Tye et al., 2011; Namburi et al., 2015; Beyeler et al., 2016; Felix-Ortiz et al., 2013, 2016; Felix-Ortiz and Tye, 2014). We attempted to begin to address this question using a retrograde labeling strategy combined with immunohistochemistry for cFos. Our results suggest that there is selective modulation of BLA projections neurons. The combined retrograde labeling and photostimulation studies indicate that, cFos, a secondary marker for neuronal excitation (Madabhushi et al., 2015), is selectively increased in the CeA- and vHPC-projecting BLA neurons (Figure 4). Furthermore, our recent reports demonstrate that increasing the activity of CaMKII+ BLA neurons via chemogenetic or optogenetic activation of Gαs-signaling also produces an anxiogenic-like state, suggesting that excitatory cells possibly mediate the LC-BLA noradrenergic effect (Siuda et al., 2015a, 2016). However, further work will be necessary to understand the cell-type and projection-specific relationship between LC-NE modulation of BLA activity and its ability to drive negative affective behavior through downstream circuits and receptor systems.

Additionally, we show that photostimulation at the site of LC terminals in the BLA conditions aversive behavior. However, it remains to be seen whether this conditioned aversive behavior is mediated locally in the BLA by NE. This effect may could be due to factors outside of the BLA and distinct from NE release such as antidromic activity at the LC cell bodies or from prolonged changes to LC circuit activity elsewhere that result from long-term, repeated optogenetic stimulation. However, a similar photostimulation paradigm increases anxiety-like behavior that is blocked by local antagonism of β-ARs in the BLA. It is well established that prolonged NE release in the BLA modulates memory storage through β-AR-mediated cAMP production and this effect, in turn, is regulated by α1-AR and α2-AR activity (Galvez et al., 1996; Hatfield et al., 1999; Liang et al., 1990; Ferry and McGaugh, 2008; Ferry et al., 1999a, 1999b). In particular, many groups have shown noradrenergic influence in fear-related memory formation (Sears et al., 2013; Roozendaal et al., 2008, 2006; Debiec and Ledoux, 2004; Garrido Zinn et al., 2016; Skelly et al., 2017). Our findings support these prior works while offering new insight into how this circuitry might acutely modulate negative affect. The observation that the LC-BLA circuitry carries an intrinsically negative valence suggests that this circuit can natively promote negative affect, rather than merely enhancing memory. However, it is possible that without explicit memory cues, our photostimulation paradigm conditions an aversion via recruitment of many other neurotransmitter systems (Brioni et al., 1989; Marsicano et al., 2002). This might not be the case in the presence of a positive cue or reward where the LC-BLA projection may still simply enhance the memory. Further studies will be necessary to fully evaluate the implications of the apparent negative affect promoted by exogenous stimulation of this circuit. While there is some concern that photostimulation of ThIRES-Cre::LC-BLA:ChR2 fibers could cause backpropagating action potentials to LC-NE cell bodies resulting in NE release elsewhere in the brain for the conditioned place aversion, the observation that the anxiety-like phenotype can be blocked with local β-AR antagonism (Figure 6G) suggests there is some degree of circuit isolation in our study. Additionally, the observation that this stimulation does not replicate all of the behavioral phenotypes of cell body activation (McCall et al., 2015) (Figure 5C; i.e. no RTPA) further supports this interpretation. However, we cannot definitively conclude that the observed CPA (Figure 5F and G) is mediated through the same postsynaptic mechanism (i.e. β-AR activation) in the BLA as the anxiety-like phenotype. To this point, we recently demonstrated that Gαs signaling (the same signaling mechanism that β-AR activates) in CaMKIIα+ BLA neurons is sufficient for anxiogenesis, without driving either a real-time or conditioned place aversion (Siuda et al., 2015a, 2016). Additionally, throughout these experiments we have not directly addressed any potential confounds that arise from long-term expression of exogenous opsins (Miyashita et al., 2013; Ferenczi et al., 2016; Warden et al., 2012). Instead, our behavioral controls (eYFP-expression with photostimulation) are aimed at controlling spurious effects of exogenous protein expression, heating, and light delivery to the BLA (Yizhar et al., 2011). Together with our work at LC-NE cell bodies (McCall et al., 2015), these studies and the current findings suggest that LC neurons with fibers in the BLA are capable of conditioning aversion, though possibly through other brain regions and possibly independent of β-AR activation.

The lack of a real-time place aversion is a particularly interesting observation. We previously demonstrated the real-time place aversion elicited by LC-NE cell body activation to be dependent on α1-AR activation (McCall et al., 2015). Together these results suggest that both the anatomical substrate and receptor system responsible for this behavior is distinct from the efferent LC-NE processes responsible for noradrenergic mediated anxiety-like behavior. Furthermore, there are important distinctions between the RTPA and the CPA assays. RTPA is essentially an instrumental conditioning paradigm that relies on operant learning of the subject as it engages the environment, while CPA is a passive, classical conditioning paradigm that possibly recruit distinct brain circuitry during the learning process (Day and Carelli, 2007). Another consideration is the longer time course of NE transmission and receptor activation compared to fast-acting, small molecule neurotransmitters, such as γ-Aminobutyric acid (GABA) and glutamate (Otis and Mody, 1992; Isaacson et al., 1993; Zoli et al., 1998; Szapiro and Barbour, 2007; Agnati et al., 2010; Palij and Stamford, 1994; Courtney and Ford, 2014). These temporal dynamics, combined with the role of NE on memory formation (Sears et al., 2013; Roozendaal et al., 2008, 2006; Debiec and Ledoux, 2004; Sara, 2000), might confound the RTPA assay that relies on distinct pairing of the transition from one chamber to the other with changes in neural activity. With activation of adrenergic receptors, these effects may have slower time dynamics than what is necessary for the place-dependent operant conditioning scheme – despite what we observed through cell body activation (McCall et al., 2015). It is well known, for example, that β-AR signaling through cAMP-gated calcium channels that will likely increase firing acutely, but that they also signal through Arrestin-mediated transduction. The latter can lead to long-term changes in gene and protein expression in addition to changes synaptic plasticity and neuronal activity (Siuda et al., 2015a). All of these molecular considerations may also play a key role in the behavior we observed. While numerous potential circuit-based targets exist, it seems possible that NE projections to the mesolimbic dopamine system have the potential to mediate instrumental learning including real-time aversive behaviors. The LC sends functional projections to the dopaminergic system and numerous groups have demonstrated bidirectional control of real-time place preference and aversion behaviors within this system (Siuda et al., 2015b; Jennings et al., 2013; Lammel et al., 2012; Stamatakis and Stuber, 2012; Tan et al., 2012; van Zessen et al., 2012; Isingrini et al., 2016).

Taken together, our results here and elsewhere (McCall et al., 2015; Siuda et al., 2015a, 2016) demonstrate that LC-NE downstream influence on negative affective behaviors is an elaborate and complex system likely involving many different postsynaptic receptor systems and anatomical projection targets. Importantly, the LC projection to the BLA is a clear functional efferent target for LC-mediated anxiety-like behavior. These findings have broad implications for our understanding of the mechanisms of anxiety and other negative affective disorders and suggest that further study of these noradrenergic circuits and signaling pathways should greatly advance our current understanding of related psychopathologies.

Materials and methods

Animals

Adult (25–35 g) male C57BL/6J (RRID:IMSR_JAX:000664) and ThIRES-Cre (RRID:IMSR_EM:00254) backcrossed to C57BL/6J mice were group-housed, given access to food and water ad libitum and maintained on a 12 hr:12 hr light:dark cycle. All animals were held in a facility in the lab 1 week prior to surgery, post-surgery and throughout the duration of the behavioral assays to minimize stress from transportation and disruption from foot traffic. To determine animal numbers we used G*Power 3 power analysis software (RRID:SCR_013726) (Faul et al., 2007; Charan and Kantharia, 2013) informed by data from our typical animal usage logs from similar experiments to suggest a priori means an standard deviations for each intended statistical methods (i.e. Student’s t-tests, ANOVAs, etc.) or Mead’s resource equation to yield between 10 and 20 degrees of freedom for the error component when no prior data was available (Charan and Kantharia, 2013; Festing and Altman, 2002). Any variation from these approaches was due to behavioral attrition from off-target injections/implants or headcap failures. All procedures were approved by the Animal Care and Use Committee of Washington University and conformed to US National Institutes of Health guidelines.

Viral preparation

Plasmids encoding pAAV-EF1α-DIO-eYFP [final titer 5 × 1012 vg/ml], pAAV-EF1α-double floxed-hChR2(H134R)-eYFP-WPRE-HGHpA [final titer 2 × 1013 vg/ml], were obtained from Addgene (Addgene.org) originally from the Deisseroth Laboratory at Stanford University. The DNA was amplified with a Maxiprep kit (Promega) and packaged into AAV5 serotyped viruses by the WUSTL Hope Center Viral Core.

Stereotaxic surgery

Mice were anaesthetized in an induction chamber (4% isoflurane) and placed in a stereotaxic frame (Kopf Instruments, Model 1900) where they were maintained at 1–2% isoflurane throughout the procedure. A craniotomy was performed and mice were injected as follows. For locus coeruleus terminal studies, 500–1000 nl of AAV5-DIO-ChR2 or AAV5-DIO-eYFP was injected unilaterally (with the exception of two animals for the single-unit electrophysiology experiments that were bilateral) into the locus coeruleus at stereotaxic coordinates from bregma: −5.45 mm anterior-posterior (AP), ±1.25 mm medial-lateral (ML), −3.65 mm dorsal-ventral (DV). Mice were then implanted with chronic fiber optic in the BLA at stereotaxic coordinates from bregma: −1.60 mm AP, ±2.90 mm ML, and −4.75 mm DV. For experiments involving local drug infusion into the BLA, a metal cannula (PlasticsOne) was implanted at −1.60 mm AP, ±2.90 mm ML, and −4.25 mm DV and either an internal infusion cannula or fiber optic was placed to extend another 0.5 mm. The fiber optic or cannula implants were secured using two bone screws (CMA, 743102) and affixed with TitanBond (Horizon Dental Products) and dental cement (Lang Dental)(McCall et al., 2015, 2013). Mice were allowed to recover for at least 6 weeks prior to behavioral testing; this interval permitted optimal AAV expression and Cre recombinase activity. For retrograde studies 400 nl of Flurogold or Retrobeads were injected into the BLA at −1.60 mm AP, ±2.90 mm ML, and −4.75 mm DV. Mice were allowed to recover for one week for fluorogold and two weeks for retrobeads, prior to perfusion for histological examination. Cholera Toxin subunit b AlexaFluor 555 (Invitrogen; CTB-555) was injected using a Neuros Hamilton Synringe (32 gauge, beveled) to the NAc (+1.25 mm AP, ±0.75 mm ML, −4.30 mm DV; 200 nl), CeM (−0.80 mm AP, ±2.35 mm ML, −5.20 mm DV; 150 nL), vHPC (−3.55 mm AP, +3.50 ML, −4.00 mm DV; 250 nL). The beveled syringe was inserted facing caudally for both NAc and vHPC and medially for CeA injections. CTB was prepared fresh for each series of experiments (Conte et al., 2009).

cFos induction

ThIRES-Cre::LC-BLA:ChR2 mice were injected with CTB-555 (Invitrogen, Carlsbad, CA) into the NAc, CeM, or vHPC at least 6 weeks following AAV5-DIO-ChR2-eYFP injection into the LC. Mice were then implanted with a chronic fiber optic in the BLA as described above. Mice were allowed to recover for 5 days and habituated to a chamber and fiber optic tether over 2 days. Seven days following CTB injection, 5 Hz photostimulation (10 ms pulses, 10 mW) was delivered to the BLA for 20 min. Mice were then sacrificed for immunohistochemistry 90 min following the onset of photostimulation. Following immunohistochemistry and imaging, images were background subtracted using ImageJ Fiji (RRID:SCR_002285). Cells were manually counted by an experimenter blind to the experimental conditions.

Immunohistochemistry

Immunohistochemistry was performed as described (McCall et al., 2015; Siuda et al., 2015a, 2016). Briefly, mice were anesthetized with pentobarbital and transcardially perfused with ice-cold 4% paraformaldehyde in phosphate buffer (PB). Brains were dissected, post-fixed for 24 hr at 4°C and cryoprotected with solution of 30% sucrose in 0.1M PB at 4°C for at least 24 hr, cut into 30 µm sections and processed for immunostaining. 30 µm brain sections were washed three times in PBS and blocked in PBS containing 0.5% Triton X-100% and 5% normal goat serum. Sections were then incubated for ~16 hr at room temperature in chicken anti-TH (1:2000, Aves Labs, Tigard, OR) or rabbit anti-phospho-cFos (1:500, Cell Signaling Technology, Danvers, MA). Following incubation, sections were washed three times in PBS and then incubated for 2 hr at room temperature goat anti-chicken or anti-rabbit Alexa Fluor 594 or 633 (1:500, Invitrogen, Carlsbad, CA) were then washed three times in PBS and followed by three 10 min rinses in PB and mounted on glass slides with Hardset Vectashield (Vector Labs, Burlingame, CA) (RRID:AB_2336787) for microscopy. All sections were imaged on both epifluorescent and confocal microscopes. Gain and exposure time were constant throughout each experiment, and all image groups were processed in parallel using Adobe Photoshop CS5 (Adobe Systems, San Jose, CA) (RRID:SCR_014199).

AntibodySpeciesDilutionSourceRRID
THChicken1:2000Aves LabsRRID:AB_10013440
Alexa Fluor 594 anti-chicken IgGGoat1:500InvitrogenRRID:AB_142803
Alexa Fluor 633 anti-chicken IgGGoat1:500InvitrogenRRID:AB_1500591
Alexa Fluor 633 anti-rabbit IgGGoat1:500InvitrogenRRID:AB_2535731
Phospho-cFosRabbit1:500Cell SignalingRRID:AB_10557109

Slice preparation

Slice electrophysiology and voltammetry experiments were performed as previously described (Stamatakis et al., 2013). Briefly, mice were anesthetized (Euthasol) and perfused with ice-cold sucrose aCSF (in mM: 225 sucrose, 119 NaCl, 1.0 NaH2PO4, 4.9 MgCl2, 0.1 CaCl2, 26.2 NaHCo3, 1.25 glucose). Following rapid decapitation, coronal slices containing LC and BLA were prepared on a vibratome (VT-1200, Leica Microsystems, Wetzlar, Germany) and allowed to rest for at least 30 min in oxygenated aCSF (in mM: 119 NaCl, 2.5 KCl, 1.0 NaH2PO4, 1.3 MgCl2, 2.5 CaCl2, 26.2 NaHCO3, and 11 glucose) at 35°C prior to placement in the recording chamber where slices were perfused with oxygenated aCSF at 37°C at a rate of 2 ml/min.

Slice electrophysiology

Whole-cell current clamp recordings were made in LC neurons expressing ChR2-eYFP with a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) (RRID:SCR_011323). Since LC neurons are spontaneously active, current was injected such that the cell was resting at −65 mV. To elicit action potentials, slices were stimulated with 5 pulses of blue LED at a rate of 10 Hz (Thorlabs, 473 nm, 5 ms pulse width, 1 mW) via the 40X objective.

Fast scan cyclic voltammetry

Carbon fiber microelectrodes (CFME, 75 μm in length) were lowered into 300 μM coronal slices and placed where the densest ChR2-eYFP expression was observed (CeA/BLA border) ThIRES-Cre::LC-BLA:ChR2 mice. To detect NE, the CFME was ramped from −0.4 V to 1.3 V versus a Ag/AgCl reference electrode (in the bath) at a rate of 400 V/s at 10 Hz. Slices were stimulated with 30 pulses of a blue LED (Thorlabs, 473 nm, 5 ms pulse width, 1 mW) via a 40X objective at 10 Hz every 5 min to release NE. Electrochemical data was collected and analyzed using a combination of Tar Heel CV (ESA, Chelmsford, MA) (Robinson and Wightman, 2007), HDCV (http://www.chem.unc.edu/facilities/electronics_software.html) (Bucher et al., 2013), and Labview (RRID:SCR_014325). Following collection, background subtracted cyclic voltammograms (CVs) were smoothed one time with a Fast Fourier Transformation (Bucher et al., 2013). CVs had characteristic oxidation and reduction peaks coinciding with catecholamine detection (ox: 600–700 mV red: −200–300 mV). Oxidative currents were analyzed at the peak of the oxidative potential for individual experiments. Clearance half-life (t1/2) was measured in Clampfit 10.2 (Molecular devices) as previously described (McElligott et al., 2013).

In vivo electrophysiology

Spontaneous single unit activity was recorded as previously described (McCall et al., 2015; Siuda et al., 2015a). Briefly, mice were lightly anesthetized (1% isoflurane), placed in a stereotactic frame and two skull screws were placed on either side of the midline to ground the electrode array. The recording apparatus consisted of a 16-channel (35 μm tungsten wires, 150 μm spacing between wires, 150–33 μm spacing between rows, Innovative Physiology, Durham, NC) electrode array. This array was epoxied to a fiber optic and lowered into the BLA (stereotaxic coordinates from bregma: −1.3 mm (AP),±2.9 mm (ML) and −4.9 mm (DV). Extracellular recordings were taken from ThIRES-Cre::LC-BLA:ChR2 mice. Spontaneous and photostimulated neuronal activity was recorded from each electrode, bandpass-filtered with activity between 250 and 8000 Hz, and analyzed as spikes. Voltage signals were amplified and digitally converted using OmniPlex and PlexControl (Plexon, Dallas, TX) (RRID:SCR_014803). For ThIRES-Cre::LC-BLA:ChR2 recordings, 3 min of baseline recordings were made followed by 3 min of 5 Hz photostimulation (10 ms pulses, 10 mW) and then another 3 min post- stimulation (off, on, off). Principle component analysis and/or evaluation of t-distribution with expectation maximization was used to sort spikes using Offline Sorter (Plexon)(RRID:SCR_000012) and only cells with distinct clusters away from the noise that remained firing throughout the duration of the recording were included. To assess firing rate changes for each cell, all spikes were binned into 1 s bins. Using z-scores (z= (x-μ)/σ; where x is the sampled firing rate of the neuron per bin), firing rates were normalized to the mean baseline firing rate during the 60 s before the start of the photostimulation. Neurons were classified as increasing firing during photostimulation if their average z-score during photostimulation was greater than 1. Similarly, neurons were classified as decreasing firing if their average z-score during photostimulation was less than −1. All units with average z-scores from −1 to 1 during photostimulation were classified as no change. To determine the latency to fire, we calculated the average time from the onset of first photostimulation to the next spike from each cell, independent of whether the cell classified as increasing, decreasing, or static. To determine baseline firing rate, all spikes were binned into 1 s bins and the mean of the first 180 s of the recording (prior to any photostimulation) was used. Average waveforms were computed from each isolated unit using the first 540 s of each recording session. Subsequently, we performed linear correlations between each neuron of a designated class (i.e. increasing, decreasing, or static) where r = 1 would be an identical waveform. Bursts were defined as at least two spikes with an interspike interval (ISI) of <170 ms to start a burst, and two spikes with an ISI > 300 ms to terminate a burst. Percent spikes in bursts was quantified by recording the number of spikes in bursts out of the total number of spikes from a unit.

General behavior notes

Behavioral assays were performed in a sound attenuated room maintained at 23°C. Lighting was measured and stabilized at ~4 lux for anxiety tests and ~200 lux for place testing. All behavioral apparatuses were cleaned with 70% ethanol in between animals. In each assay, animals received constant photostimulation throughout the entire trial. AAV5-DIO-ChR2 and AAV5-DIO-eYFP animals received 5 ms pulses of 10 Hz photostimulation (473 nm). Movements were video recorded and analyzed using Ethovision Software (RRID:SCR_000441).

Real time place testing (RTPT)

Mice were placed into a custom-made unbiased, balanced two-compartment conditioning apparatus (52.5 × 25.5 × 25.5 cm) as previously described (McCall et al., 2015; Siuda et al., 2015b; Al-Hasani et al., 2015) and allowed to freely roam the entire apparatus for 20 min. Entry into one compartment triggered constant photostimulation (5 Hz for LC-BLA; ~10 mW light power) while the animal remained in the light-paired chamber. Entry into the other chamber ended the photostimulation. The side paired with photostimulation was counterbalanced across mice. Time spent in each chamber and total distance traveled for the entire 20 min trial was measured using Ethovision 8.5 (Noldus, Wageningen, Netherlands). Data are expressed as mean ± S.E.M percent time spent in photostimulation-paired chamber.

Conditioned place aversion (CPA)

Mice were trained in an unbiased, balanced three compartment conditioning apparatus as previously described (McCall et al., 2015). Briefly, mice were pre-tested by placing individual animals in the small central compartment and allowing them to explore the entire apparatus for 30 min. Time spent in each compartment was recorded with a video camera (ZR90; Canon, Tokyo, Japan) and analyzed using Ethovision 8.5 (Noldus). Mice were randomly assigned to light and no-light compartments and received no light in the morning and light (5 Hz for LC-BLA) in the afternoon at least 4 hr after the morning training on two consecutive days. CPA was assessed on day 4 by allowing the mice to roam freely in all three compartments and recording the time spent in each. Scores were calculated by subtracting the time spent in the light stimulus-paired compartment post-test minus the pre-test.

Open field test (OFT)

OFT testing was performed as described (McCall et al., 2015) in a square enclosure (50 × 50 cm). We connected ThIRES-Cre::LC-BLA:ChR2 mice to fiber optics and allowed them to roam freely for 21 min. Photostimulation alternated between off and on (5 Hz (10 ms width)) photostimulation (~10 mW light power)) states in 3 min time segments, beginning with 3 min of no stimulation. The open field was cleaned with 70% ethanol between each trial. The center was defined as a square comprised of 25% the total area of the OFT (i.e. each length was 50% that of the total OFT).

Elevated zero maze (EZM)

EZM testing was performed as described (McCall et al., 2015), the EZM (Harvard Apparatus, Holliston, MA) was made of grey plastic, 200 cm in circumference, comprised of four 50 cm sections (two opened and two closed). The maze was elevated 50 cm above the floor and had a path width of 4 cm with a 0.5 cm lip on each open section. ThIRES-Cre::LC-BLA:ChR2 animals were connected to cables coupled to a function generator, positioned head first into a closed arm, and allowed to roam freely for 7 min. Animals received 5 Hz (10 ms width) photostimulation (~10 mW light power). For the β-AR antagonism experiment, mice were injected into the BLA with Propranolol (1 µg, intra-BLA, 30 min prior to behavior, Tocris, Bristol, United Kingdom). Mean open arm time was the primary measure of anxiety-like behavior.

Genotyping of mouse lines

DNA was isolated from tail tissue obtained from weanling mice (21–28 days of age), and PCR screening was performed using the following primers: Cre recombinase (forward: 5’- GCA TTA CCG GTC GAT GCA ACG AGT GAT GAG-3’ and reverse: 5’- GAG TGA ACG AAC CTG GTC GAA ATC AGT GCG-3’) yielding a 400 bp PCR product in Cre positive animals. Fatty acid-binding protein intestinal primers (forward: 5’- TGG ACA GGA CTG GAC CTC TGC TTT CCT AGA-3’ and reverse: 5’- TAG AGC TTT GCC ACA TCA CAG GTC ATT CAG-3’) were used as positive controls and yield a 200 bp PCR product.

Statistics/data analysis

All summary data are expressed as mean ± SEM. Statistical significance was taken as *p<0.05, **p<0.01, ***p<0.001, as determined by the Student’s t-test (paired and unpaired): One-Way Analysis of Variance (ANOVA) or One-Way Repeated Measures ANOVA, followed by Dunnett’s or Bonferroni post hoc tests as appropriate. In cases where data failed the D’Agostino and Pearson omnibus normality test, non-parametric analyses were used. Statistical analyses were performed in GraphPad Prism 5.0 (RRID:SCR_002798).

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Decision letter

  1. Liqun Luo
    Reviewing Editor; Howard Hughes Medical Institute, Stanford University, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Activation of locus coeruleus to basolateral amygdala projections drives anxiety and aversion" for consideration by eLife. Your article has been favorably evaluated by a Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal her identity: Kay M Tye (Reviewer #2).

As you see from the reviews below, all reviewers are enthusiastic about the topic and findings, and each has raised a number of recommendations for revision. After discussion among all three reviewers, we would like to invite you to submit a revised manuscript that can satisfactorily address the following:

1) We feel that the in vivo recording data in Figure 3 need to be significantly strengthened. Specifically, "n" should be increased to have more examples of up- or down-modulated BLA neurons. It will be ideal if in these experiments you can learn more about the recorded cells' other properties, such as their response to natural stimuli and modulations by propranolol, but we do not hold these as essential for the acceptance for a revised manuscript.

2) We would also request that you perform the propranolol experiment in the aversion assay (Figure 4), which will strengthen your claim that the effect on aversion is mediated by LC-NE neuron projection to BLA rather than via antidromic stimulation elsewhere. If this additional data cannot be obtained, you should significantly tone down the claim that aversion is related to LC-NE modulation of BLA, including in the title.

3) We believe that most of the other critiques can be addressed by further analyses of existing data, and by textual revisions. You should try to address these as thoroughly as possible.

Reviewer #1:

McCall et al. characterized the action of locus coeruleus norepinephrine (LC-NE) neurons at one of their numerous target sites: the basolateral amygdala (BLA), in the context of anxiety and aversion. They confirmed previous findings that LC-NE neurons project and arborize axons at BLA, showed that photoactivation of ChR2-expressing LC-NE neurons resulted in NE release at BLA as measured by fast scan cyclic voltammetry, characterized firing pattern changes of BLA neurons in response to photostimulation of LC-NE neurons using in vivo recording, found that terminal stimulation of LC-NE fibers at BLA caused conditioned (but not real time) place aversion and enhanced anxiety-like behaviors, and showed that the anxiety-like behaviors can be suppressed by application of propranolol, an antagonist of β-adrenergic receptor.

Overall this is a nice and timely set of experiments addressing an important question: how do LC-NE neurons affect the physiology and function of a specific target area, in this case BLA that is well known for its function in regulating emotion and anxiety. Some of the experiments and analyses could go further.

1) The in vivo recording (Figure 3) of BLA neurons in response to photostimulation of LC-NE neurons is perhaps the least satisfactory experiment. I will start with a few questions about the description. There were 20 cells in Figure 3C, but only 18 were further analyzed in Figure 3D (and onwards?). What were the selection criteria? How was "latency" measured (Figure 3K) for cells that had no change in firing rate? Do Figure 3L and 3M contain new cells (legend for 3M says n=10), or a subset of cells in Figure 3C or Figure 3D? Again, how are these cells selected?

Given the clear heterogeneity of response, it will be more satisfying if the authors have some more knowledge about these cells-for example are there any signatures that distinguish three types of neurons in the PCA analysis? Do they respond differently to natural stimuli? Do they respond differently to propranolol, which will provide a nice link to Figure 5? Increasing N is desirable as the examples are only 4 and 2 for neurons whose firing rate is up- or down-modulated by LC-NE neuron photostimulation.

2) Given the broad projection patterns of LC-NE neurons, the behavioral effect of LC-NE photostimulation in Figure 4 and Figure 5 could in principle be due to antidromic effects in other target areas. The propranolol effect caused by local application in Figure 5 supports a BLA local action. The propranolol experiment should be extended to Figure 4 as well.

Reviewer #2:

Overall, I was delighted to read this interesting and important manuscript. I have relatively minor comments.

I found it intriguing that the authors observed such heterogeneous responses to LC-BLA photostimulation and wonder if the differences in responses of BLA neurons to NE is related to either a) their projection target, b) cell type (projection or interneuron) or c) their role during anxiety-related behaviors. This is beyond the scope of the current manuscript and I do not expect the authors to respond to this query with further experiments, but if the authors have any other data regarding basal spike rate, waveform, peak firing rates, burst patterns or activity during anxiety behaviors it would be nice to see. The authors must have the firing profile data already, and just need to analyze and display this.

The Authors state that "Downstream and independent of this projection, recent studies have demonstrated that direct activation of both basolateral amygdala (BLA) cell bodies or their projections is both anxiogenic and socially aversive" and should consider adding the following papers to the references already listed: Felix-Ortiz, Neuron, 2013; Felix-Ortiz, J Neurosci, 2014; Felix-Ortiz, Neuroscience, 2015. These citations should also be added to the references following the sentence "Furthermore, while we do not know the genetic identity or projection targets of the BLA neurons from which we recorded, it is possible that NE serves to preferentially shift BLA activity in neurons that favor anxiety-like behavior (i.e. those that project to the ventral hippocampus, prefrontal cortex, or central amygdala) rather those that favor positive affect and anxiolysis (i.e. projections to the nucleus accumbens and bed nucleus of the stria terminalis)".

The finding that LC-BLA inputs drive conditioned place aversion potentially place previous studies concluding that NE in the BLA contributes to fear memory formation/storage. The authors should discuss this body of research and clearly address how their data may challenge this existing notion. Specifically, the finding that this circuit is intrinsically of negative valence may actually provide an alternative explanation for a number of results previously thought to support a role in memory formation (associated with fear). The authors discuss these results as being consistent, though, it's not clear to me that this is the case.

It was curious that they were not able to produce real-time place aversion but were able to observe conditioned place aversion. The authors interpret this as an issue of the involvement of long-term memory. An alternative interpretation would be that this effect is specific to Pavlovian and not instrumental conditioning. Real-time place aversion is explicitly different in that the animal must enter the paired side of the chamber to receive photostimulation. Thus, a number of other issues may also be involved. First, this is essentially an instrumental conditioning task, as the photostimulation is contingent on animal behavior. Second, this may be related to the lingering effects of NE (as the authors hint at, but don't directly discuss).

The appropriate control for the propranolol experiment would be a saline/vehicle injection (both in combination with ChR2 stimulation), as well as a light-off condition wherein ChR2 is still expressed. ChR2 expression can (with long-term expression) change basal behavior even in the absence of light (see Warden et al., Nature, 2012). It seems as if they may have done this based on the supplementary figure, but the main text was confusing. Please clarify.

In Figure 1B, what is above the ventricle? There is some retrograde labeling from the BLA that is TH-. Also, it is hard to see if this exists in D, but it seems to. Finally, some quantification of the TH overlap with cre-dependent fluorophore expression should be included. Fibers in BLA are scarcely visible in 1L, I am surprised this level of expression was sufficient to evoke behavioral responses – was this image representative?

In Figure 3F, the firing rate appears to be dropping prior to photostimulation – which makes it unclear whether the effect we are observing is actually related to the optogenetic manipulation.

Reviewer #3:

Major conclusions of paper:

McCall et al. combine in vivo optogenetics, physiology, pharmacology, and behavior, as well as FSCV, to characterize a specific sub-circuit of the extensive LC-NE system. Their experiments identify novel examples of heterogeneity within the LC→ BLA circuit, substantially build on existing work in the field, and have important implications for understanding how NE modulates anxiety/aversion. While interesting, I felt that some of their conclusions regarding specificity within this circuit were not experimentally demonstrated as strong as they could be, but hopefully, this can be addressed during revision. I also had some technical questions regarding experiments for Figure 3.

Main concerns:

Figure 3:

1) Sample size seems low for statistical analysis (4 cells in increased firing group, 2 cells in decreased group).

2) I was confused by 3C. Is each dot a cell? How do they correspond to the pie chart in 3D?

3) It says in the Materials and methods that injections were unilateral, but according to Figure 3 legend, data collection was from "4 hemispheres from 2 animals". Almost all LC projections to forebrain are ipsilateral (though I'm not sure about projections to BLA), so it doesn't seem possible that both hemispheres could be used for these experiments if only one LC was injected.

4) I'm unsure if the statistical comparisons are appropriate; i.e. – pre-sorting of the "increased" and "decreased" cells based on average firing.

5) It would be nice to expand on the identity of the NE-responding BLA neurons. What if adrenergic inhibitors are applied to the BLA during their optrode recordings? How does this affect the heterogeneous NE-mediated activity they observed? Alternatively, could CRACM-style experiments be performed in slice, where recordings are performed in BLA neurons when ChR2-expressing LC fibers are stimulated? This could provide higher resolution for the relative size of these different NE-responding populations, as well as their identity (via post-recording cell fill and immunolabeling?)

Regarding optogenetic stimulation of fibers/related to Figure 4: The authors find that stim. of BLA→ LC fibers produces negative association over time, which isn't present in real-time place aversion. But is it known if avoidance association is specific to the BLA→LC projection? It also seems possible that chronic activation of LC terminals could cause a change in LC cell body activity, and thus broadly affect the LC-NE circuit. This is mentioned in the Discussion, but maybe it's possible to experimentally address this concern. For instance, does local infusion of an adrenergic receptor antagonist into the BLA during or after conditioning block the avoidance (similar to the experiment done in Figure 5G)?

https://doi.org/10.7554/eLife.18247.013

Author response

As you see from the reviews below, all reviewers are enthusiastic about the topic and findings, and each has raised a number of recommendations for revision. After discussion among all three reviewers, we would like to invite you to submit a revised manuscript that can satisfactorily address the following:

1) We feel that the in vivo recording data in Figure 3 need to be significantly strengthened. Specifically, "n" should be increased to have more examples of up- or down-modulated BLA neurons. It will be ideal if in these experiments you can learn more about the recorded cells' other properties, such as their response to natural stimuli and modulations by propranolol, but we do not hold these as essential for the acceptance for a revised manuscript.

We appreciate the concerns regarding Figure 3 and agree that these experiments needed bolstering. Due to the above cited consideration on mouse number we were unable to perform all the suggested experiments, but we were able to more than double the n from the original submission and perform the important request for expanded analyses on this data. While were unable to test the responses of these cells to natural stimuli, we were able to follow-up with a functional anatomy experiment that provides some clarity to the projection targets of the modulated cells. In particular, we used retrograde labeling combined with cFos staining (a secondary marker for neuronal activation) to identify that LC-BLA stimulation appears to bias increased activity to BLA circuits known to promote aniety-like behavior (i.e. the central amygdala and the ventral hippocampus). Together, we hope these new data are more comprehensive and well powered than what was presented in the initial submission.

2) We would also request that you perform the propranolol experiment in the aversion assay (Figure 4), which will strengthen your claim that the effect on aversion is mediated by LC-NE neuron projection to BLA rather than via antidromic stimulation elsewhere. If this additional data cannot be obtained, you should significantly tone down the claim that aversion is related to LC-NE modulation of BLA, including in the title.

In this particular case of the conditioned place aversion experiments, we were unable to perform the necessary experiments to fully control for effects outside of the BLA from antidromic stimulation due to limited mice expand the in vivo recordings, which were indicated by reviewers and review summary as the top priority for the revision. We do agree that optogenetic manipulations are terminal sites come with many caveats of interpretation and we have taken every effort to tone down our conclusions regarding these experiments and have therefore removed this “aversion” concept from the title of the article. More details are included below.

3) We believe that most of the other critiques can be addressed by further analyses of existing data, and by textual revisions. You should try to address these as thoroughly as possible.

We appreciate these other concerns raised and have tried to address these directly and completely. More details are included below.

Reviewer #1:

[…] 1) The in vivo recording (Figure 3) of BLA neurons in response to photostimulation of LC-NE neurons is perhaps the least satisfactory experiment. I will start with a few questions about the description. There were 20 cells in Figure 3C, but only 18 were further analyzed in Figure 3D (and onwards?).

We appreciate this attention to detail by the reviewer and apologize for the mistake. Figure 3C includes two cells firing at a low rate that reached a firing rate zero for the remainder of the recording session such that we judged them to be lost, from the electrode rather than simply not firing. They should have not been included in 3C in the original submission and have been removed from this submission.

What were the selection criteria?

We have adjusted the wording in the Materials and methods section to be more explicitly clear as to how single cells were selected. It now reads, “Principle component analysis and/or evaluation of t-distribution with expectation maximization was used to sort spikes using Offline Sorter (Plexon) and only cells with distinct clusters away from the noise that remained firing throughout the duration of the recording were included.”

How was "latency" measured (Figure 3K) for cells that had no change in firing rate?

We regret that this was not clearer in the initial submission. The latency presented here is simply the time to the first spike following the first photostimulation. Therefore a ‘latency’ exists for all included cells. We have added this explanation to the Materials and methods section which reads: “To determine the latency to fire, we calculated the average time from the onset of first photostimulation to the next spike from each cell, independent of whether the cell classified as increasing, decreasing, or static.”

Do Figure 3L and 3M contain new cells (legend for 3M says n=10), or a subset of cells in Figure 3C or Figure 3D? Again, how are these cells selected?

These cells represent a subset of 3C/3D where the recording was judged sufficiently stable to continue recording for an additional manipulation. These recordings were done prior to the analysis to determine whether each cell increased or decreased (or stayed the same) to the 5 Hz stimulation. To be clearer, we have altered the text to read: “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).”

Given the clear heterogeneity of response, it will be more satisfying if the authors have some more knowledge about these cells-for example are there any signatures that distinguish three types of neurons in the PCA analysis? Do they respond differently to natural stimuli? Do they respond differently to propranolol, which will provide a nice link to Figure 5? Increasing N is desirable as the examples are only 4 and 2 for neurons whose firing rate is up- or down-modulated by LC-NE neuron photostimulation.

We appreciate the concerns regarding Figure 3 and agree that these experiments needed bolstering. Due to the above cited consideration on mouse number we were unable to perform all of the suggested experiments, however we were able to more than double the n of the original submission and perform the requested expanded analyses on this larger data set. Notably, this doubling preserved the ratio of up- and down-modulated cells almost exactly as the smaller n. We also examined more properties of the existing and newly acquired cells, including sorting them by baseline firing rate, determining the similarity of the waveforms within each group, and quantifying their bursting properties. In the case of sorting the neurons by baseline firing, we can now observe that up-modulated cells have a lower baseline firing rate than down-modulated cells. This finding suggests that it may be more likely that these up-modulated cells are BLA projection neurons, rather than interneurons (Likhtik et al., Journal of Neurophysiology, 2006). However, this is only a likelihood and further experiments would be necessary to fully draw this conclusion. We have adjusted the figures and text accordingly to reflect these observations.

While were unable to test the responses of these cells to natural stimuli, we did expand the study with a follow-up functional anatomy experiment that provides some clarity to the projection targets of the modulated cells. In particular, we used retrograde labeling combined with cFos staining to identify that LC-BLA stimulation appears to bias increased activity to BLA circuits known to promote aniety-like behavior (i.e. the central amygdala and the ventral hippocampus). Together, we think these new data are more satisfying than what was presented in the initial submission.

2) Given the broad projection patterns of LC-NE neurons, the behavioral effect of LC-NE photostimulation in Figure 4 and Figure 5 could in principle be due to antidromic effects in other target areas. The propranolol effect caused by local application in Figure 5 supports a BLA local action. The propranolol experiment should be extended to Figure 4 as well.

We agree with the reviewer that this is a critical experiment for the interpretation of the aversion experiments. Unfortunately, we were not able to secure sufficient TH-IRES-Cre mouse cohorts on a reasonable timeline to complete these experiments properly with all controls and implants (~40+ Cre+ animals). Instead, we have taken every effort to tone down our conclusions throughout the paper regarding the aversion experiments in (the original) Figure 4 and have also appropriately removed aversion from the title of the paper, focusing instead on the observed anxiety-like behaviors. Future experiments (including those to eliminate the confound of backpropagating action potentials) will be necessary to determine the precise role of LC-NE fibers in the BLA for aversive behaviors.

Reviewer #2:

Overall, I was delighted to read this interesting and important manuscript. I have relatively minor comments.

I found it intriguing that the authors observed such heterogeneous responses to LC-BLA photostimulation and wonder if the differences in responses of BLA neurons to NE is related to either a) their projection target, b) cell type (projection or interneuron) or c) their role during anxiety-related behaviors. This is beyond the scope of the current manuscript and I do not expect the authors to respond to this query with further experiments, but if the authors have any other data regarding basal spike rate, waveform, peak firing rates, burst patterns or activity during anxiety behaviors it would be nice to see. The authors must have the firing profile data already, and just need to analyze and display this.

We agree that these suggestions are key components to better understanding the system. We have taken two approaches to addressing these concerns. First, we added a new experiment and figure to the manuscript attempting to address, as a first pass, whether BLA neurons that increase in activity following LC-BLA photostimulation are segregated by projection target. To do so, we retrogradely labeled BLA neurons with a fluorophore-tagged Cholera Toxin B in either the ventral hippocampus, central amygdala, or nucleus accumbens, stimulated LC-BLA fibers, and observed co-localization between the CTB label and the presence of cfos, a secondary marker for neuronal activation. These studies indicate a bias towards activation of BLA neurons that project to ventral hippocampus and central amygdala, rather than the nucleus accumbens. While these experiments suggest that LC-BLA biases activation towards these projection targets that are known to promote anxiety-like behavior, much more work will be necessary in subsequent studies to clearly elucidate the intricacies of these tri-synaptic circuits. In addition to these new experiments, we have also added more recordings (as discussed elsewhere) and replotted our findings based on the baseline firing profile of these neurons, their average waveform, and we have quantified their bursting properties by group as well. As stated previously, the sorting the neurons by baseline firing demonstrates that cells that increase in response to the photostimulation have a lower baseline firing rate than down-modulated cells. This finding suggests that it may be more likely that these up-modulated cells are BLA projection neurons, rather than interneurons (Likhtik et al., Journal of Neurophysiology, 2006). However, this is only a likelihood and further experiments would be necessary to fully draw this conclusion. We have adjusted the figures and text accordingly to reflect these observations.

The Authors state that "Downstream and independent of this projection, recent studies have demonstrated that direct activation of both basolateral amygdala (BLA) cell bodies or their projections is both anxiogenic and socially aversive" and should consider adding the following papers to the references already listed: Felix-Ortiz, Neuron, 2013; Felix-Ortiz, J Neurosci, 2014; Felix-Ortiz, Neuroscience, 2015. These citations should also be added to the references following the sentence "Furthermore, while we do not know the genetic identity or projection targets of the BLA neurons from which we recorded, it is possible that NE serves to preferentially shift BLA activity in neurons that favor anxiety-like behavior (i.e. those that project to the ventral hippocampus, prefrontal cortex, or central amygdala) rather those that favor positive affect and anxiolysis (i.e. projections to the nucleus accumbens and bed nucleus of the stria terminalis)".

We appreciate this attention to detail by the reviewer. While we had already cited Felix-Ortiz, Neuron, 2013 and Felix-Ortiz, Neuroscience, 2015, we had previously failed to cite Felix-Ortiz, J Neurosci, 2014. That citation is now included in both locations of the manuscript.

The finding that LC-BLA inputs drive conditioned place aversion potentially place previous studies concluding that NE in the BLA contributes to fear memory formation/storage. The authors should discuss this body of research and clearly address how their data may challenge this existing notion. Specifically, the finding that this circuit is intrinsically of negative valence may actually provide an alternative explanation for a number of results previously thought to support a role in memory formation (associated with fear). The authors discuss these results as being consistent, though, it's not clear to me that this is the case.

This interpretation is an important consideration we did not highlight it clearly enough in our original submission. We have altered the text to provide more detail on the existing body of research as well as elaborating on our intended interpretation. The new text reads: “It is well established that prolonged NE release in the BLA modulates memory storage through β-AR-mediated cAMP production and this effect, in turn, is regulated by α1-AR and α2-AR activity (Debiec and Ledoux, 2004; Galves, Mesches and McGaugh, 1996; Madabhushi et al., 2015; Liang, McGaugh and Yao, 1990; Ferry and McGaugh, 2008; Ferry, Roozendaal and McGaugh, 1999). […] Further studies will be necessary to fully evaluate the implications of the apparent negative affect promoted by exogenous stimulation of this circuit.”

It was curious that they were not able to produce real-time place aversion but were able to observe conditioned place aversion. The authors interpret this as an issue of the involvement of long-term memory. An alternative interpretation would be that this effect is specific to Pavlovian and not instrumental conditioning. Real-time place aversion is explicitly different in that the animal must enter the paired side of the chamber to receive photostimulation. Thus, a number of other issues may also be involved. First, this is essentially an instrumental conditioning task, as the photostimulation is contingent on animal behavior. Second, this may be related to the lingering effects of NE (as the authors hint at, but don't directly discuss).

These are importantalternative considerations and we have integrated them into the Discussion as such: “Furthermore, there are important distinctions between the RTPA and the CPA assays. […] All of these molecular considerations may also play a key role in the behavior we observed.”

The appropriate control for the propranolol experiment would be a saline/vehicle injection (both in combination with ChR2 stimulation), as well as a light-off condition wherein ChR2 is still expressed. ChR2 expression can (with long-term expression) change basal behavior even in the absence of light (see Warden et al., Nature, 2012). It seems as if they may have done this based on the supplementary figure, but the main text was confusing. Please clarify.

We apologize that this was not clearer in the initial submission. The EZM experiment had four groups eYFP+Vehicle, ChR2+Vehicle, eYFP+Propranolol, and ChR2+ Propranolol. This approach allows comparisons to detect a change evoked from photostimulation (eYFP+Vehicle vs. ChR2+Vehicle) as well as an effect from the pharmacological treatment (ChR2+Vehicle vs. ChR2+ Propranolol) while controlling for any effects of the antagonist independent of photostimulation (eYFP+Propranolol vs. ChR2+ Propranolol). We have revised the Results section to more clearly state these details, it now reads: “To do so we implanted a combined fiber-optic fluid cannula into the BLA and delivered either an artificial cerebrospinal fluid vehicle or the non-selective β-AR antagonist, Propranolol (1 µg)(Sears et al., 2013; Roozendaal et al., 2006; Buffalari and Grace, 2009; Buffalari and Grace, 2007), prior to photostimulation (Figure 6E and F, Figure 6—figure supplement 1B). […] Importantly, when we locally antagonized β-ARs directly in the BLA prior to photostimulation, anxiogenesis was completely blocked in ChR2+ animals with no effect on eYFP-expressing, propranolol controls (Figure 6G).”

Furthermore, we do agree that controls for the long-term expression of ChR2 are important, however we were unfortunately unable to incorporate these experiments into this submission due to limited animal numbers. We have addressed this limitation now in the Discussion, stating: “Additionally, throughout these experiments we have not directly addressed any potential confounds that arise from long-term expression of exogenous opsins (Galves, Mesches and McGaugh, 1996; Hatfield, Spanis and McGaugh, 1999; Likhtik et al., 2006). Instead, our behavioral controls (eYFP-expression with photostimulation) are aimed at controlling spurious effects of exogenous protein expression, heating, and light delivery to the BLA (68).”

In Figure 1B, what is above the ventricle? There is some retrograde labeling from the BLA that is TH-. Also, it is hard to see if this exists in D, but it seems to.

It seems as though the initial image/labeling on the image caused confusion. There is nothing above the ventricle (labeled 4V on image). However, there is retrograde labeling to TH- cells that are dorsal and ventral to the LC itself (top right of the image in 1B). These cells are likely part of the medial parabrachial nucleus which have previously identified projections to the BLA (Saper & Loewy, Brain Research, 1980). We have made a note regarding this labeling in the Figure 1 legend.

Finally, some quantification of the TH overlap with cre-dependent fluorophore expression should be included.

We agree that using any Cre-driver line for cell-type selective modulation should be well documented for its selectivity. For this concern, we point the reviewer to our previous manuscript where we indeed included a detailed description of this overlap for noradrenergic, DBH+ cells. In particular, the data presented in Figures S2C, 5E & S5A of McCall et al., Neuron, 2015 directly addresses this question by comparing viral expression to dopamine β hydroxylase (DBH) staining with essentially 100% of Cre-dependent viral expression overlapping with DBH+ cells. We therefore felt it was redundant to do this again.

Fibers in BLA are scarcely visible in 1L, I am surprised this level of expression was sufficient to evoke behavioral responses – was this image representative?

We appreciate this attention to detail by the reviewer. The images used throughout the manuscript were selected to be representative of what is typically seen. We have included the data from the Allen Institute (now Figure 1—figure supplement 1) to support these observations as well. We see similar expression in the BLA as has been observed elsewhere (e.g. Plummer et al., Development, 2015). We agree that it is remarkable that such diffuse innervation has meaningful behavioral effects, but believe this to be a characteristic feature of the noradrenergic system through its known volume transmission properties (e.g. Courtney & Ford, Journal of Neuroscience, 2014). A further important consideration for neuromodulation, acting via metabotropic receptors is that density of inputs, fluorescence, or fiber labeling do not necessarily predict how well coupling amplification can happen after a GPCR is activated. For example, in many cases a receptor pool of say 100 receptors, only needs to be occupied by 10% by norepinephrine to have a maximal possible effect on a neuronal response. This concept, termed ‘spare receptors’ is because of large amplification that happens after the G-protein coupling event, cAMP generation, and channel modulations. So, while we appreciate this is a visually weak input, there are numerous other examples in both the central and peripheral nervous system where modulatory inputs (including by NE) by visualization alone look weak, yet these fibers carry and amplify robust messages that dramatically effect behavior and circuits. This is also as an aside, why interpreting viral tracing density visually as a measure of function can be a confound in some cases.

In Figure 3F, the firing rate appears to be dropping prior to photostimulation – which makes it unclear whether the effect we are observing is actually related to the optogenetic manipulation.

We appreciate this observation by the reviewer, with additional recordings we no longer see this ‘anticipatory’ decrease in firing. We do note, however, that while the photostimulation-induced increase in firing returns to baseline quickly following the end of the photostimulation, the suppression of firing appears to be maintained long after stimulation ends.

Reviewer #3:

Major conclusions of paper:

McCall et al. combine in vivo optogenetics, physiology, pharmacology, and behavior, as well as FSCV, to characterize a specific sub-circuit of the extensive LC-NE system. Their experiments identify novel examples of heterogeneity within the LC→ BLA circuit, substantially build on existing work in the field, and have important implications for understanding how NE modulates anxiety/aversion. While interesting, I felt that some of their conclusions regarding specificity within this circuit were not experimentally demonstrated as strong as they could be, but hopefully, this can be addressed during revision. I also had some technical questions regarding experiments for Figure 3.

We appreciate this positive assessment of our work and hope that you will find this revised manuscript a stronger contribution to the field.

Main concerns:

Figure 3:

1) Sample size seems low for statistical analysis (4 cells in increased firing group, 2 cells in decreased group).

We appreciate concerns that the initial data set was too limited. We were able to now more than double the n of the original submission and perform the requested expanded analyses on this data. Notably, this doubling preserved the ratio of increasing and decreasing cells almost exactly as the smaller n.

2) I was confused by 3C. Is each dot a cell? How do they correspond to the pie chart in 3D?

We apologize for any confusion as originally presented. We have altered the figure to connect the dots from pre- and during stimulation in 3C. For clarity, we have also segregated the cells into increasing, decreasing, and static and have presented these in separate graphs in the new Figure 3—figure supplement 1. We hope this expanded view is clearer.

3) It says in the Materials and methods that injections were unilateral, but according to Figure 3 legend, data collection was from "4 hemispheres from 2 animals". Almost all LC projections to forebrain are ipsilateral (though I'm not sure about projections to BLA), so it doesn't seem possible that both hemispheres could be used for these experiments if only one LC was injected.

We appreciate this attention to detail and apologize for the minor error in reporting. All animals were injected unilaterally except for the two used for single unit electrophysiology the initial submission. The new animals recorded and reported in this submission were all injected unilaterally and recorded from the ipsilateral BLA. We have noted these two exceptions in the Materials and methods, stating: “For locus coeruleus terminal studies, 500-1000 nl of AAV5-DIO-ChR2 or AAV5-DIO-eYFP was injected unilaterally (with the exception of two animals for the single-unit electrophysiology experiments that were bilateral) into the locus coeruleus at stereotaxic coordinates from bregma: -5.45 mm anterior-posterior (AP), ± 1.25 mm medial-lateral (ML), -3.65 mm dorsal-ventral (DV).”

4) I'm unsure if the statistical comparisons are appropriate; i.e. – pre-sorting of the "increased" and "decreased" cells based on average firing.

We agree that statistical comparisons based on pre-sorted groups are inappropriate. Based on editorial comments from a presubmission inquiry, we removed any direct statistical comparison based on the firing rate of neurons (the feature used to classify the cells). Instead we employed the z-score method that we (Jennings et al., Nature, 2013) and others (e.g. Wolff et al., Nature, 2014) have used previously to identify gross changes in neuronal activity. The inclusion of statistics for the basal frequency measure is still appropriate as this feature was not part the classification scheme and the descriptive statistics for latencies remain for informative purposes. All other statistics in this figure and its supplement are done on the whole sample without any pre-classification.

5) It would be nice to expand on the identity of the NE-responding BLA neurons. What if adrenergic inhibitors are applied to the BLA during their optrode recordings? How does this affect the heterogeneous NE-mediated activity they observed? Alternatively, could CRACM-style experiments be performed in slice, where recordings are performed in BLA neurons when ChR2-expressing LC fibers are stimulated? This could provide higher resolution for the relative size of these different NE-responding populations, as well as their identity (via post-recording cell fill and immunolabeling?)

We appreciated these elegant suggestions to improve our work. Unfortunately, most of these suggestions were outside of the scope of what was possible for this study given the struggles we had with animal breeding. While the adrenergic inhibitors would be an important addition to this story, the practical limitations of recording in anesthetized mice meant that we would need many fold more animals than we had available after exhausting all possible sources/supplies. With the pressing need to increase our n for the recording experiments, we focused efforts on acquiring that new data with the hopes of following up with adrenergic compounds, however, we were unable to add sufficient numbers of recordings with the pharmacological treatments, so no conclusion could be made and these are not included in the manuscript. We do, however, believe that the new figure detailing LC-BLA photostimulation-induced increases in cfos to be a first step in answering some of the concerns raised here. While it lacks the temporal precision that would be provided by the suggested CRACM-style slice recordings, the new experiment suggests that there is projection selectivity in the modulation of BLA activity by the LC. Future work will endeavor to answer these questions more completely.

Regarding optogenetic stimulation of fibers/related to Figure 4: The authors find that stim. of BLA→ LC fibers produces negative association over time, which isn't present in real-time place aversion. But is it known if avoidance association is specific to the BLA→LC projection? It also seems possible that chronic activation of LC terminals could cause a change in LC cell body activity, and thus broadly affect the LC-NE circuit. This is mentioned in the Discussion, but maybe it's possible to experimentally address this concern. For instance, does local infusion of an adrenergic receptor antagonist into the BLA during or after conditioning block the avoidance (similar to the experiment done in Figure 5G)?

As previously discussed, we agree with the reviewer that this is a critical experiment for the interpretation of the aversion experiments. Unfortunately, we were not able to secure sufficient TH-IRES-Cre mice on a reasonable timeline to complete these experiments. Instead, we have taken every effort to tone down our conclusions throughout the paper regarding the experiments in (the original) Figure 4 and have removed aversion from the title of the paper, focusing instead on the observed anxiety-like behaviors. Future experiments (including those to eliminate the confounding effect of potential backpropagating action potentials) will be necessary to determine the precise role of LC-NE fibers in the BLA for aversive behaviors.

https://doi.org/10.7554/eLife.18247.014

Article and author information

Author details

  1. Jordan G McCall

    1. Department of Anesthesiology, Division of Basic Research, Washington University School of Medicine, St. Louis, United States
    2. Washington University Pain Center, Washington University School of Medicine, St. Louis, United States
    3. Department of Neuroscience, Washington University School of Medicine, St. Louis, United States
    4. Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, United States
    Present address
    1. Department of Anesthesiology, Washington University, St. Louis, United States
    2. Department of Pharmaceutical and Administrative Sciences, St. Louis College of Pharmacy, St. Louis, United States
    3. Center for Clinical Pharmacology, Washington University School of Medicine and St. Louis College of Pharmacy, St. Louis, United States
    Contribution
    JGM, Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Edward R Siuda
    For correspondence
    jordangmccall@wustl.edu
    Competing interests
    No competing interests declared.
    ORCID icon 0000-0001-8295-0664
  2. Edward R Siuda

    1. Department of Anesthesiology, Division of Basic Research, Washington University School of Medicine, St. Louis, United States
    2. Washington University Pain Center, Washington University School of Medicine, St. Louis, United States
    3. Department of Neuroscience, Washington University School of Medicine, St. Louis, United States
    4. Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, United States
    Present address
    Trevena Inc., King of Prussia, United States
    Contribution
    ERS, Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Contributed equally with
    Jordan G McCall
    Competing interests
    No competing interests declared.
  3. Dionnet L Bhatti

    1. Department of Anesthesiology, Division of Basic Research, Washington University School of Medicine, St. Louis, United States
    2. Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, United States
    Contribution
    DLB, Data curation, Investigation, Visualization, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared.
    ORCID icon 0000-0003-3031-2067
  4. Lamley A Lawson

    Department of Anesthesiology, Division of Basic Research, Washington University School of Medicine, St. Louis, United States
    Contribution
    LAL, Investigation
    Competing interests
    No competing interests declared.
  5. Zoe A McElligott

    1. Department of Psychiatry, University of North Carolina, Chapel Hill, United States
    2. Bowles Center for Alcohol Studies, University of North Carolina, Chapel Hill, United States
    Contribution
    ZAM, Investigation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared.
    ORCID icon 0000-0002-4717-5698
  6. Garret D Stuber

    1. Department of Psychiatry, University of North Carolina, Chapel Hill, United States
    2. Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, United States
    3. Neuroscience Center, University of North Carolina, Chapel Hill, United States
    Contribution
    GDS, Supervision, Methodology
    Competing interests
    No competing interests declared.
    ORCID icon 0000-0003-1730-4855
  7. Michael R Bruchas

    1. Department of Anesthesiology, Division of Basic Research, Washington University School of Medicine, St. Louis, United States
    2. Washington University Pain Center, Washington University School of Medicine, St. Louis, United States
    3. Department of Neuroscience, Washington University School of Medicine, St. Louis, United States
    4. Division of Biology and Biomedical Sciences, Washington University School of Medicine, St. Louis, United States
    5. Department of Biomedical Engineering, Washington University, St. Louis, United States
    Contribution
    MRB, Conceptualization, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    bruchasm@wustl.edu
    Competing interests
    MRB: Michael R. Bruchas, PhD is a co-founder of Neurolux, Inc, a company that is making wireless optogenetic and various neuroscience-related probes. None of the work in this manuscript used these devices or is related to any of the company's activities, but we list this information here in full disclosure.
    ORCID icon 0000-0003-4713-7816

Funding

National Institute of Mental Health (MH101956)

  • Jordan G McCall

Washington University in St. Louis

  • Jordan G McCall
  • Edward R Siuda

National Institute on Alcohol Abuse and Alcoholism (AA023555)

  • Zoe A McElligott

Alcohol Beverage Medical Research Foundation

  • Zoe A McElligott

National Institute on Drug Abuse (DA032750)

  • Garret D Stuber

National Institute on Drug Abuse (DA035144)

  • Michael R Bruchas

McDonnell Center for Systems Neuroscience

  • Michael R Bruchas

National Institute of Mental Health (MH112355)

  • Michael R Bruchas

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work is supported by NIMH R01 MH112355 (MRB), and early work in this study by NIDA R21 DA035144 (MRB), the McDonnell Center for Systems Neuroscience (MRB), NIMH F31 MH101956 (JGM), WUSTL DBBS (JGM, ERS), start-up funds from Washington University in St. Louis (JGM), NIAAA K01 AA023555 (ZAM), and the Alcohol Beverage Medical Research Foundation (ZAM). We thank all of the Bruchas Laboratory for their helpful insight and discussion throughout the preparation of the manuscript. We also thank Thomas J Baranski, N Gautam, Robert W Gereau, IV, Erik D Herzog, Timothy E Holy, Joseph D Dougherty, and Joe Henry Steinbach for helpful discussions. We thank Karl Deisseroth (Stanford University) for the channelrhodopsin-2 (H134) construct. We also thank The WUSTL HOPE Center viral vector core for related viral preparations (NINDS, P30NS057105).

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols at Washington University in St. Louis. The protocol was approved by the Animal Studies Committee at Washington University in St. Louis (Protocol Number: 20130219; expiration date: 15/10/2016). All surgery was performed under isoflurane anesthesia, and every effort was made to minimize suffering.

Reviewing Editor

  1. Liqun Luo, Reviewing Editor, Howard Hughes Medical Institute, Stanford University, United States

Publication history

  1. Received: May 27, 2016
  2. Accepted: July 13, 2017
  3. Accepted Manuscript published: July 14, 2017 (version 1)
  4. Version of Record published: August 9, 2017 (version 2)

Copyright

© 2017, McCall et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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