Engagement of spinal- vs prefrontal-projecting locus coeruleus neurons parses analgesia from aversion & anxiety in rats

The locus coeruleus (LC) projects throughout the brain and spinal cord and is the major source of central noradrenaline. It remains unclear whether the LC acts functionally as a single global effector or as discrete modules. Specifically, while spinal-projections from LC neurons can exert analgesic actions, it is not known whether they can act independently of ascending LC projections. Using viral vectors taken up at axon terminals, we expressed chemogenetic actuators selectively in LC neurons with spinal (LC:SC) or prefrontal cortex (LC:PFC) projections. Activation of the LC:SC module produced robust, lateralised analgesia while activation of LC:PFC produced aversion. In a neuropathic pain model, LC:SC activation reduced hind-limb sensitization and induced conditioned place preference. By contrast, activation of LC:PFC exacerbated spontaneous pain, produced aversion and increased anxiety-like behaviour. This independent, contrasting modulation of pain-related behaviours mediated by distinct noradrenergic modules provides evidence for a discrete functional organisation of the LC.


INTRODUCTION
Neuropathic pain has a population prevalence of 7-8% (Bouhassira et al., 2008, Torrance et al., 2006 and remains difficult to treat (Finnerup et al., 2015) mandating research to improve therapies (von Hehn et al., 2012, Skolnick andVolkow, 2016). Many of the frontline treatments for neuropathic pain modulate spinal noradrenaline, either directly such as noradrenaline re-uptake inhibitors or as part of a common effector pathway such as gabapentinoids and opioids (Nakajima et al., 2012, Hayashida et al., 2007, Hayashida et al., 2008, Jasmin et al., 2003. A functional deficit of noradrenergic control is associated with the development of sensitisation in neuropathic pain models (Hughes et al., 2013, Hughes et al., 2015, De Felice et al., 2011, Xu et al., 1999. However, current systemic pharmacological interventions to correct this imbalance commonly produce CNS side-effects such as anxiety, sleep disturbance, mood shifts and confusion, limiting their therapeutic utility (Finnerup et al., 2015).
The locus coeruleus (LC) is the principal noradrenergic nucleus in the CNS (Dahlstroem and Fuxe, 1965) and is the main source of noradrenergic innervation to the spinal dorsal horn, forming part of a welldescribed analgesic circuit (Millan, 2002, Pertovaara, 2006, Howorth et al., 2009a, Bruinstroop et al., 2012. The LC also projects throughout almost the entire neuraxis and plays a pivotal role in diverse behaviours such as learning and memory (Takeuchi et al., 2016, Martins andFroemke, 2015), strategic behaviour (Tervo et al., 2014), motivation (Varazzani et al., 2015), and arousal (Sara and Bouret, 2012, Carter et al., 2010, Vazey and Aston-Jones, 2014. Furthermore, LC-derived noradrenaline is also causally linked to aversive states like stress, anxiety and depression (McCall et al., 2015, Alba-Delgado et al., 2013. Thus, drug treatments for chronic pain that globally enhance noradrenaline levels or mimic its action are likely to cause adverse effects by acting on LC neuronal circuits that are not directly involved in pain control. The LC has been characterised as a homogeneous cluster of neurons that provide a uniform global signal as part of a central arousal system (Fuxe et al., 2010). There is, however, accumulating evidence for specialization of the LC neurons into subsets on the basis of: 1) anatomical projections (Loughlin et al., 1986, Kebschull et al., 2016, Chandler et al., 2014; 2) electrical properties (Chandler et al., 2014); and 3) co-transmitter content (Simpson and Lin, 2007). In accord with this organisational principle we have demonstrated that distinct populations of LC noradrenergic neurons innervate the spinal cord (LC :SC ) and prefrontal cortex (LC :PFC ) based on anatomy and electrophysiology in vitro and in vivo (Li et al., 2016). Here we selectively target LC neurons from their projections to the spinal cord or prefrontal cortex using the axon terminal uptake and retrograde transport of canine adenovirus type 2 (CAV-2) vectors (Junyent andKremer, 2015, Li et al., 2016). This CAV-2 vector expresses a chemogenetic actuator in noradrenergic neurons specifically, to allow activation of LC :SC or LC :PFC neurons to test whether the beneficial noradrenergic analgesia and stress-like adverse effects seen in pain states are mediated by distinct subpopulations of LC neurons and are therefore dissociable.

Targeting and activation of noradrenergic LC neurons with PSAM
To enable selective activation of LC neurons, the excitatory chemogenetic receptor PSAM (Pharmaco-Selective Actuator Module, PSAM L141F,Y115F:5HT3HC (Magnus et al., 2011)) was expressed in noradrenergic neurons ( Figure 1A-B) using viral vectors with the cell-type specific synthetic promotor PRSx8 (PRS) (Hwang et al., 2001, Lonergan et al., 2005, Howorth et al., 2009a. Efficient and selective targeting of LC neurons was observed after direct injection into the dorsal pons of a lentiviral vector harbouring the PRS-EGFP-2A-PSAM expression cassette. After transduction, EGFP immunofluorescence was detected in 690±38 neurons per LC (N=5) of which 98±0.4% co-expressed dopamine b-hydroxylase (DBH) confirming their identity as noradrenergic ( Figure 1B). To enable post-hoc immuno-localisation, PSAM was tagged with a C-terminal HA. Subsequently, PSAM-HA was detected in the membranes of the LC neurons ( Figure 1C, 82/112 transduced neurons examined from 3 rats). The functionality of the PSAM-HA was verified by Ca 2+ -imaging in cell lines (Figure S 1).
Patch clamp recordings from transduced LC neurons in acute pontine slices showed that the designerligand PSEM308 (pharmaco-selective effector molecule) produced inward currents and a reversible excitation. PSEM308 evoked concentration-dependent inward currents of up to 800pA in voltage clamp (V hold -60 mV, 1-10 μM, Figure 1D). Current-clamp recordings of transduced LC neurons showed an 8-fold increase in firing frequency (0.54±0.21 Hz to 4.22±0.90 Hz, Figure 1E) in response to PSEM308 (3 µM). No response to PSEM308 (doses up to 10 μM) was detected in recordings from nontransduced LC neurons (n=6). We found no difference in intrinsic electrical properties between transduced and non-transduced LC neurons ( Table S 2) suggesting that expression of PSAM is well tolerated. Together, these findings indicate that PSAM is functionally expressed in LC neurons and its activation by its ligand is sufficient to robustly increase action potential discharge.

Chemogenetic excitation of the LC in vivo
Extracellular recordings with multi-barrelled electrodes were used to determine whether PSEM308 application could excite LC neurons in vivo (after direct LC transduction with LV PRS-EGFP-2A-PSAM ( Figure   1F)). In PSAM expressing rats (N=8), local pressure application of PSEM308, but not saline, caused a time-locked and reproducible excitation in 12 out of the 24 recorded units (Figure 1F), increasing their firing to 234% of baseline (2.63±0.92 Hz to 6.15±1.47 Hz). The remaining cells did not respond (n=6) or showed a small reduction of their firing frequency (-34.6%, 2.83±1.21 to 1.85±0.86 Hz, n=6) in response to local PSEM308 ( Figure 1F). These inhibited units, which were in some cases simultaneously recorded along with LC units whose activity was increased (Figure S 2), likely reflect LC neurons that are not themselves transduced but are inhibited by noradrenaline released from surrounding transduced and hence PSEM308-activated LC neurons (as previously hypothesised by Vazey and Aston-Jones (2014)) These data are in line with pontamine sky blue labelling of the recording sites, which were always surrounded by DBH expressing neurons of which many, but not all, were also EGFP+ ( Figure 1F). Local application of PSEM308 did not alter baseline firing frequency in control LC neurons (2.99±1.55 to 3.15±1.58 Hz, NS, n=7 neurons from 3 non-transduced rats).
The effect of systemic application of PSEM308 was tested on transduced LC units that showed an excitatory response to local PSEM308 (n=5, Figure 1G). Intraperitoneal (i.p) injection of PSEM308 (10 mg/kg, same dose used throughout the rest of the study unless otherwise stated) increased discharge frequency 10-fold from 1.47±0.93 to 14.76±2.8 Hz after 20 min. LC neurons doubled their firing frequency after 5.9 ± 3.9 min and cells reached their maximum discharge frequency after 15.9 ± 6.5 min ( Figure 1G, median 15.8 Hz, range 10.5 to 27.2 Hz). The evoked excitation outlasted the time of experimental recording (30 min) in four out of five neurons.  (black arrowheads) and DBH (white arrowheads). The response to local PSEM308 was categorised as excitation or inhibition if it changed firing rate by more than 3 SD from the baseline rate. Application of PSEM308 produced an excitation in 50% of LC neurons. We found a second group of neurons that showed no response, presumably as they were not transduced. A third group showed an inhibition of spontaneous firing in response to local PSEM308 application. (G) Kinetics of the excitatory response to systemic PSEM308 administration (10 mg/kg i.p). (H) Timeline of conditioned place aversion protocol to assess influence of chemogenetic activation of LC neurons on behaviour. In PSAM expressing rats, PSEM308 (10 mg/kg) caused conditioned place aversion but had no effect on control animals. Representative heat maps of rat position in the pre-test and post-test after PSEM308 with bilateral LC transduction with LV

Chemogenetic activation of the LC alters behaviour
High tonic LC discharge frequencies (5-10Hz) are acutely aversive (McCall et al., 2015). Therefore, we employed a Pavlovian-conditioned place preference (CPP) paradigm to assay whether there was a behavioural consequence to chemogenetic activation of LC neurons. CPP is a voluntary choice task using the innate behaviour of animals to divide their time between environments according to their affective valence. Conditioning with PSEM308 induced avoidance of the drug-paired environment in animals that bilaterally expressed PSAM in LC neurons ( Figure 1H). Such avoidance was not seen with a lower dose of PSEM308 (5 mg/kg), or in animals transduced with a control vector (LV PRS-EGFP ) and administered PSEM308 (10 mg/kg).

Activation of LC :SC but not LC :PFC is analgesic
Based on previous studies we predicted that LC :SC neurons would play a role in anti-nociception (Jones andGebhart, 1986, Howorth et al., 2009b) and hypothesised that the forebrain projecting LC neurons might play a different -potentially pro-nociceptive role (Hickey et al., 2014). To target noradrenergic neurons according to their projection territory, we inserted a PRS-EGFP-2A-PSAM expression cassette into a CAV-2 vector to target LC neurons ( Figure 2A) from their axonal projections (Li et al., 2016, Soudais et al., 2001.  Figure 2A). These LC :SC neurons were located in the ventral aspect of the nucleus. In a second set of animals, CAV2 PRS-EGFP-2A-PSAM bilaterally injected into the PFC transduced similar numbers of neurons (361±72 per LC, N=6). However, these LC :PFC neurons were scattered throughout the LC with a more dorsal location than the LC :SC neurons (representing different groups of neurons as previously noted (Bruinstroop et al., 2012, Howorth et al., 2009a, Li et al., 2016).
In both cases, the transduced LC :SC and LC :PFC neurons were positive for DBH (99.1±0.5% and 99.1±0.7% respectively) indicating efficient and specific retrograde transduction of the noradrenergic neurons (Figure 2A).
We used the Hargreaves' test to measure hind paw thermal withdrawal latency as an assay of nociception. Chemogenetic activation of LC :SC neurons (PSEM308 i.p, N=7 rats) significantly increased thermal withdrawal latency to radiant heat as compared to naïve rats (N=6) (30min post i.p., 11.6±0.9 s LC :SC vs 7.6±0.2 s naïve ( Figure 2B)). This time course of analgesic effect between 15 and 45 min post-dosing is consistent with the kinetics of LC neuron activation after i.p PSEM308 injection ( Figure   1G). Moreover, this LC :SC analgesia was only seen in the hind-limb ipsilateral to the vector injection in the spinal cord ( Figure 2B), indicating an unexpected lateralised organisation of the LC :SC projection.
By contrast, activation of LC :PFC neurons had no effect on the thermal withdrawal latency. This indicates a functional dichotomy in the actions of the ascending and descending LC projections on acute pain.
To investigate the mechanism of LC :SC chemogenetic analgesia, extracellular recordings were made from wide dynamic range (WDR) neurons in the spinal dorsal horn (n=14 from 6 rats, mean depth 415 μm, range 200 to 600 μm) in a preparation that allows the application of drugs directly onto the spinal cord in the irrigating solution ( Figure 2C, (Funai et al., 2014, Furue et al., 1999). WDR neurons encode stimulus intensity, responding to both innocuous and noxious stimuli (Price and Dubner, 1977). The average spontaneous discharge frequency of WDR was 1.86 ± 0.60 Hz, that was increased to 16.52 ± 2.19 Hz by pinch stimulation of the hind paw. In LC :SC transduced animals, perfusion of PSEM308 (10 μM) inhibited the pinch-evoked discharge two-fold to 8.5 ± 1.8 Hz (12/14 WDR neurons, Figure 2C).
However, both spontaneous firing and innocuous, brush-evoked discharge were unaffected by PSEM308 (Figure S 3). These data demonstrate that local activation of terminals of LC :SC neurons in the spinal cord causes a specific inhibition of nociceptive inputs to WDR neurons.
To address the question of whether the LC :SC inhibition of the pinch response was caused by spinal release of noradrenaline, Atipamezole (10 μM, α2-adrenoceptor antagonist) was co-applied with PSEM308 (10 μM). The presence of atipamezole prevented the LC :SC mediated-inhibition of the pinch response ( Figure 2C). Atipamezole alone had no effect on spontaneous discharge, evoked discharge after pinch or brush indicating there is little ongoing spinal release of noradrenaline under anaesthesia (paired t-test p>0.05, n=5 WDR neurons from 2 rats, Figure S 3).

Activation of LC :PFC module is aversive and anxiogenic
Because activation of LC :PFC neurons did not change nociceptive thresholds we investigated whether activation of these neurons contributes to aversion or to the anxiety-like behaviour ascribed to the LC by McCall and colleagues (2015). We found that activation of LC :PFC caused avoidance of the drugpaired chamber in the CPP paradigm indicating a negative affective valence ( Figure 3A).

Figure 3: Chemogenetic activation of LC :PFC neurons is aversive and anxiogenic unlike LC :SC (A) Representative heat maps showing rat location in the place preference arena before and after conditioning for LC :SC (left) and LC :PFC (right) transduced rats (outer compartments 30x30cm). Activation of LC :PFC with PSEM308 (10 mg/kg) caused conditioned aversion to the paired chamber (2-way repeated measures ANOVA with Bonferroni's multiple comparison between the conditioned and unconditioned chamber, *** P<0.001) and had no effect on LC :SC transduced rats. Pre-test data pooled for graphical presentation (B) Cross-over design for open field test (OFT) and tracks showing activity in the open field test (OFT)
after PSEM308 (10 mg/kg) or saline (inner square 30x30 cm). PSEM308 reduced the time LC :PFC rats spent in the centre of the arena and increased the time these rats were immobile. In contrast, PSEM308 had no effect on LC :SC transduced rats (2-way RM-ANOVA with Bonferroni's multiple comparison test, PSEM308 vs saline, * P<0.05, ** P<0.01). (See also Figure S 5) The open field test uses the innate behaviour of rodents to avoid exposed areas as a measure of anxiety. Anxiogenic stressors typically reduce the time rodents spend in the centre of the arena and increase the time animals are immobile. Chemogenetic activation of LC :PFC reduced centre time by 62.6% and doubled immobility time in the open field test after PSEM308 i.p ( Figure 3B). These findings indicate that chemogenetic activation of the LC :PFC neurons changes behaviour to produce anxiety/aversion and promoted immobility (as previously reported for high frequency stimulation of the whole LC (Carter et al., 2010)).
In contrast, when the LC :SC neurons were transduced, conditioning with PSEM308 was not aversive in the CPP paradigm ( Figure 3A) and did not produce anxiety-like behaviour in the open field test ( Figure   3B). There was no difference in the total distance animals travelled between drug or saline injection in the open field test for LC :SC animals (1894±90 PSEM308 vs 1885±137 cm saline, paired t-test p=0.94) suggesting there was no gross locomotor deficit. This indicates that chemogenetic activation of the LC :SC module does not produce the same negative affective behaviour that is seen with the LC :PFC module.

Engagement of LC :SC module attenuates neuropathic sensitisation
To test for possible analgesic benefit of chemogenetic activation of the LC :SC module in neuropathic pain, we employed the tibial nerve transection model ( Figure 4A and Figure S 4). This procedure causes the progressive development of punctate mechanical (von Frey threshold lowered from 15.72±1.3 to 3.2±1.4 g, N=17) and cold hypersensitivity (increased frequency of paw withdrawals from 14.1±3.7 to 76.5±6.2 %, N=17) over 3 weeks (Figure S 4). Chemogenetic activation of the LC :SC module increased the mechanical withdrawal threshold after nerve injury (from 1.2±0.3 baseline to 11.7±3.3 g, t=30 min after PSEM308 i.p, N=6 ( Figure 4B)). Similarly, we found activation of LC :SC produced a significant improvement in weight-bearing on the nerve-injured limb; linking the reduced evoked mechanical hypersensitivity to a spontaneous behaviour (Figure S 4). The chemogenetic analgesic effect on mechanical and cold allodynia was dose-dependent (1-10 mg/kg) and completely blocked by prior intrathecal (i.t) injection of the a2-adrenoceptor antagonist yohimbine (60 ng, 10.0±2.6 g PSEM308+vehicle to 1.2±0.4 g PSEM308+yohimbine, N=5 (Figure 4D)). These findings indicate, that activation of LC :SC neurons is analgesic in this model of neuropathic pain and that the effect is mediated by noradrenaline acting on a2-adrenoceptors in the spinal cord.

Figure 4. Chemogenetic activation of LC :SC is analgesic via a spinal a2-mechanism (A) Experimental timeline of vector injection and the tibial nerve transection model of neuropathic pain (s = sural, cp = common peroneal and ti = tibial nerves). (B-C) Quantification of mechanical withdrawal threshold (von Frey, after Chaplan (1994)). (B) Nerve injured animals showed a mechanical sensitisation of the ipsilateral limb. Activation of LC
Therefore, we predicted that the noradrenaline reuptake inhibitor reboxetine (1mg/kg i.p), a dose too low to cause an analgesic effect on its own, would enhance chemogenetically-evoked LC :SC analgesia. Co-administration of reboxetine amplified and prolonged the analgesic effect of PSEM308 and completely reversed mechanical hypersensitivity (17.8±3.2 g) as compared to sham animals (17.9± 1.1g, (Figure 4C)).
In marked contrast to LC :SC , activation of the LC :PFC module by PSEM308 had either no effect or tended to increase evoked mechanical or cold responses in the nerve injury model and did not

Bidirectional modulation of spontaneous pain and pain-related behaviours by LC modules
We assayed the frequency of spontaneous foot-lifts as an index of ongoing pain caused by nerve injury.
As anticipated, we found that nerve injury significantly increased the number of spontaneous footlifts over 5 min (6.2±0.7 sham, N=6 vs 30.7±3.9 TNT, N=17 (Figure 5B + Video S 1 )). Activation of LC :SC more than halved the number of spontaneous foot-lifts in nerve injured rats (13.1±2.9), consistent with a reduction of ongoing pain. By contrast, activation of LC :PFC neurons almost doubled the number of foot-lifts (52.6±9.3 (Figure 5B)) suggesting a diametric modulation of spontaneous pain by these distinct subsets of LC neurons.
If this dichotomous action of the LC on spontaneous pain is also reflected in the affective experience of pain, then we anticipated that chemogenetic stimulation of the LC :SC module would reduce the negative pain-associated affect whilst LC :PFC activation should increase pain affect. In rats with neuropathic sensitisation, activation of LC :SC increased the time spent in the PSAM-paired chamber whilst LC :PFC activation induced aversion ( Figure 5A). These data indicate that the neuropathic animals obtain benefit from LC :SC activation but that their situation is worsened by LC :PFC activation.
A link between the signs of ongoing pain and the affective component of pain is provided by the correlation between the PSEM308-induced modulation of chamber preference and the incidence of spontaneous foot-lifts across all groups of animals ( Figure 5C). No correlation was found between chamber preference and PSEM308-induced changes in evoked responses (data not shown) suggesting that modulation of ongoing pain is a better predictor of the perceived benefit of this intervention. (5.4±1.6% saline to 2.0±0.7% PSEM308, N=7) and increased immobility by 139% compared to saline (47.3±6.0% saline to 65.9±7.7% PSEM308, N=7 (Figure S 5)). Furthermore, the effect on immobility caused by activation LC :PFC neurons was further enhanced by 135% by co-administration reboxetine (1 mg/kg) (61.3±8.0% PSEM308 to 82.8±3.9% PSEM308+reboxetine, N=6) indicating that the effect was caused by release of noradrenaline. In contrast, PSEM308 application had no effect on open field performance in LC :SC transduced or sham animals (Figure S 5).

Discussion
By modifying LC cells using an intersectional vector strategy, based on transcriptional profile and projection territory, we rendered defined subgroups of noradrenergic neurons chemogeneticallyactivatable both in vitro and in vivo. Selective activation of the LC :SC or LC :PFC modules produced distinct changes in behaviour that were functionally antithetical in the context of pain states. The activation of the LC :SC neurons evokes an a2-adrenoceptor-mediated analgesic action that is sustained in a model of chronic neuropathic pain. This amelioration of the pain phenotype by LC :SC activation is seen for both evoked and spontaneous behaviour and was accompanied by reduced negative pain affect. In contrast activation of the LC :PFC produces an anxiety-like pattern of behaviour with negative affective valency and does not alter nociceptive sensitivity but instead increases expression of the signs of spontaneous pain after nerve injury. These data indicate that there is a modular functional organisation for these specific output targets of the LC and raises the question of whether this organisational principle is generalizable to other targets of the LC. Additionally, this approach in modulating a specific LC module establishes a proof of concept template for chronic pain treatment approaches with the potential for reduced side effects.
Within the pain field there has been a long interest in descending noradrenergic control which has been well characterised pharmacologically in vitro and in vivo (Millan, 2002, Yoshimura and Furue, 2006, Pertovaara, 2006, Jones, 1991. However, it has been difficult to selectively engage this system either clinically or in animal models because of its anatomical location, nuclear organisation, and lack of pharmacological targets to enable differentiated control with drugs. Here we use chemogenetic engineering approaches (Magnus et al., 2011) to manipulate and investigate the system with systemic drug administration. In targeting the LC :SC neurons we see an anatomical organisation that is similar to that previously described (Bruinstroop et al., 2012, Howorth et al., 2009a, Li et al., 2016, Westlund et al., 1983 with labelling in a cluster of around 15% of the LC neurons located in the ventral pole of the nucleus. LC :SC activation in vivo was anti-nociceptive in a heat withdrawal assay that was lateralised to the side of CAV2 PRS-EGFP-2A-PSAM vector injection in the spinal cord. Given that LC neurons were transduced bilaterally in the pons after this injection (as expected (Howorth et al., 2009a)), this indicates that there is a novel lateralised functional organisation for the control of nociception.
In a neuropathic pain model, activation of the LC :SC attenuated the degree of allodynia and hyperalgesia. This was seen for all sensory modalities tested and also applied to spontaneous measures of sensitisation i.e. foot-lifts. This benefit from engaging the LC :SC extends the findings from previous chronic intrathecal dosing studies with noradrenaline re-uptake inhibitors that also suppressed the neuropathic phenotype (Hughes et al., 2015). Given that we had documented a depletion of LC fibres in the spinal cord at the level of the nerve afferent input (Hughes et al., 2013) then it was not known whether there would be the potential to restore noradrenergic inhibition with the retrograde chemogenetic strategy. The demonstration of preserved actions indicates that both release of noradrenaline and the a2-adrenoceptor mechanism required are still functional. This is an important consideration for this kind of intervention as a therapeutic strategy; although we note that this strategy would have be successfully implemented after nerve injury (rather than transduced before nerve injury as here) if it were to be deployable clinically.
Pain is an emotive experience and relief of pain is a strong learning signal with positive affective valence i.e. animals display a preference for conditions where their pain is alleviated (King et al., 2009). Therefore, the preference of nerve-injured animals for with LC :SC activation indicates that this was analgesic rather than simply suppressing a reflex motor withdrawal -this parallels previous studies with intrathecal clonidine/noradrenaline re-uptake inhibitors that have shown a conditioned place preference (Hughes et al., 2015, Wei et al., 2013 or drive for self-administration (Martin et al., 2006).
Additionally, no preference for LC :SC activation was seen in naïve rats, indicating that it is the analgesic action in the neuropathic model that has a positive valence rather than intrinsic rewarding effect of LC :SC stimulation. This is consistent with the sparse forebrain innervation (particularly of rewardrelated sites) for these LC :SC neurons (Howorth et al., 2009a, Li et al., 2016. Engagement of the forebrain projecting LC :PFC produced a contrasting profile without any action on evoked-nociception and evidence of a facilitation of spontaneous pain behaviours in the neuropathic pain model. Additionally, activation of the LC :PFC was aversive and anxiogenic in both naïve and neuropathic animals. This aversive action agrees with previous studies of the LC (McCall et al., 2015) and may reflect high tonic levels of discharge produced by the chemogenetic activation approach.
Such aversion and anxiety is a common component of chronic pain syndromes which likely contributes to the hypervigilance and catastrophizing. An increase in noradrenaline in the PFC has been shown in neuropathic pain models (Suto et al., 2014) that has been associated with an impairment of attention.
Similarly, lesion studies of the LC :PFC have shown a reduction in pain associated anxiety and negative affect without any change in nociception (Bravo et al., 2016) which supports our finding that activation of LC :PFC produces negative affect and identifies this projection from the LC as a key player in the negative cognitive consequences of chronic pain.
These two opposing actions of the LC :SC and LC :PFC neurons to either suppress nociceptive input or to produce negative affective responses reflects the fact that they are distinct functional modules. This mirrors their anatomical segregation (Li et al., 2016) and implies that the modules could be capable of independent action, with recruitment in different behavioural contexts. We speculate that it may be the balance between recruitment of the 'restorative' LC :SC limb versus the 'alarmist' LC :PFC projection that determines whether pain following an injury resolves or persists into chronic pain (see also (Arora et al., 2016, Brightwell and Taylor, 2009, Taylor and Westlund, 2017). Further the recruitment of either module may actually reciprocally inhibit the other given the a2-adrenoceptor-mediated feedback inhibition within the locus coeruleus (Aghajanian et al., 1977). This would also require each module to have different afferent drives that were independently regulated. At present, there is little evidence for such an organisation but this is an emerging territory for which the necessary neurobiological toolboxes are only now being developed (Schwarz et al., 2015). Their TRIO approach using CAV in combination with rabies vectors suggested that there was a distinction in the afferent innervation of the 'medullary projecting' LC neurons that we think likely represents collaterals from the LC :SC . This provides preliminary evidence that such an input -output specialisation may be present within the LC.
Previous studies have employed subtractive methods to study the role of the LC in pain regulation in animal models. These methods using toxins, knock outs or lesions to inhibit the noradrenergic system typically show subtle and sometimes contradictory effects on nociception (Martin et al., 1999, Jasmin et al., 2003, Jasmin et al., 2002, Tsuruoka and Willis, 1996, Howorth et al., 2009b. This may be in part ascribed to the long-term adaptation to the loss of a population of neurons. However, our findings of antithetical LC actions reported herein (and Hickey (2014)) provide an alternative explanation; namely that the LC is not a single functional entity with respect to pain regulation. Rather there are counterbalancing actions of individual modules and consequently the net effect of subtractive interventions will be difficult to predict/interpret. This consideration also applies to attempts to obtain therapeutic benefit from systemic dosing of noradrenaline re-uptake inhibitors in patients -while some benefit, a majority either obtain no benefit or suffer side effects that are often related to anxiety, changes in affect and sleep disturbances that are likely due to actions on ascending LC projections (Finnerup et al., 2015). Our findings indicate that targeting just the spinal projection could separate analgesia from many of the side effects.
In demonstrating this functional and anatomical dichotomy within the LC modules we have provided behavioural evidence that indicates that the LC has levels of sub-specialisation according to modalities. This builds upon prior work indicating anatomical and cellular specialisation within the LC (Loughlin et al., 1986, Kebschull et al., 2016, Chandler et al., 2014, Howorth et al., 2009a, Li et al., 2016. Given that the LC has been implicated in diverse neuronal behaviours such as learning and memory (Takeuchi et al., 2016, Martins andFroemke, 2015), strategy (Tervo et al., 2014), arousal (Carter et al., 2010) and anxiety (McCall et al., 2015). In each example the role of the LC in that behaviour has been considered as acting on a specific CNS substrate (i.e. hippocampus, auditory cortex, prefrontal cortex and amygdala) by inference somewhat in isolation. We speculate that the organisation of the LC outputs into modules allows these influences in different behavioural contexts to be independently selected and regulated. Given this organisational structure it may be that that the Locus Coeruleus would be more appropriately named Loci Coeruleus reflecting the fact that it appears to be a collection of noradrenergic modules each with distinctive paths and roles in regulating brain function.

EXPERIMENTAL MODEL AND SUBJECT DETAILS
All procedures conformed to the UK Animals (Scientific Procedures) Act 1986 and were approved by

Generation of CAV2 PRS-EGFP-2A-PSAM
The EGFP-2A-PSAM HA cassette was excised from the custom synthesised plasmid (pCMV-EGFP-2A-PSAM HA ) with ApaI and HpaI. The CAV2 shuttle vector pTCAV2-PRS-ChR2-mCherry (previously described (Li et al., 2016)) was cut with ApaI/HpaI to remove ChR2-mCherry and subsequently ligated to generate pTCAV2-PRS-EGFP-2A-PSAM HA . The resulting plasmids were then purified and digested with BamHI/NotI for homologous recombination into the SwaI linearized CAV2 genome (pCAVΔE3Sce) in BJ5183 cells (Agilent Technologies) following the manufacturer's protocol. CAV2 vectors were produced using previously described methods (Ibanes and Kremer, 2013). Briefly, DK-SceI dog kidney cells constitutively expressing the fusion protein of the endonuclease SceI and the estrogen receptor (SceI-ER) were grown in complete DMEM (with 10% fetal bovine serum, nonessential amino acids, L-glutamine, penicillin and streptamycin and G418) on 10cm dishes to 80% confluency. Subsequently, cells were treated with 1mM OH-tamoxifen for 4h to induce translocation of SceI-ER into the nucleus. The tamoxifen pre-treated DK-SceI cells (8x10 6 ) were seeded in 10cm dishes containing 1mM OH-tamoxifen and immediately transfected with 15 µg of the supercoiled pCAV genome using 30µl turbofect. The transfection medium was replaced by complete DMEM the following morning. Cells were harvested and virus released by freeze-thaw cycles to prepare initial virus containing stocks for vector amplification in DK-SceI cells (Ibanes and Kremer, 2013). Vector stocks were titrated in functional transducing units (TU) per ml as previously described (Li et al., 2016, Ibanes and Kremer, 2013, Kremer et al., 2000 and diluted to 0.6x10 12 TU before injection.

Stereotaxic injections
The procedures for viral vector injections into the LC, the PFC or the SC have been described in detail previously (Hickey et al., 2014, Howorth et al., 2009a, Li et al., 2016. In brief, rats were anesthetized by intraperitoneal injection of ketamine (5 mg/100 g, Vetalar; Pharmacia) and medetomidine (30 μg/100 g, Domitor; Pfizer) until loss of paw withdrawal reflex. The rat was placed in a stereotaxic frame. Burr holes and microinjections were made using a Drill and Injection Robot with Wireless Capillary Nanoinjector (NEUROSTAR, Germany).
Locus coeruleus targeting in adult rats (120-200g): A dorso-ventral injection track was started at 1.2mm lateral and -2.1mm posterior to lambda with 10˚ rostral angulation. Three injections of 0.3µl were made at -5.3mm, -5.5mm and 5.8mm depth. In rats intended for in vivo LC recordings a screw was inserted into the burr hole to simplify later CNS access.
Prefrontal cortex: Four burr holes were drilled bilaterally, 0.7mm lateral to the midline, at +2mm, +1.2 mm, +0.8 mm and -0.2mm from bregma. At each site 0.2µl of vector was injected at 1.0mm and 1.5mm depth to the brain surface. At +2mm further injections were made at -5 and -5.5 mm depth from the brain surface Spinal cord: The vertebral column was exposed and clamped at vertebral segment L3. A laminectomy was performed at T12-L1 and 0.4µl CAV2 vector injected bilaterally into the L3-L4 segment of the spinal cord (SC) at six injection sites in total. These injections were located 0.5mm lateral from the midline and 0.5mm deep and spread in pairs over the segmental extent. For unilateral injections (pain studies) six injections of (0.4µl) were made unilaterally into the right lumbar dorsal horn.

Electrophysiology
Patch clamp recordings from acute pontine slices: Pontine slices were prepared from juvenile (age p28-p32) Wistar rats 7-14 days after lentiviral transduction as previously described (Hickey et al., 2014). Rats were deeply anesthetized with isoflurane 5%, decapitated and the brain quickly removed and immediately chilled in ice-cold cutting solution (similar to the recording solution but NaCl was reduced from 126mM to 85 mM and substituted with sucrose 58.4mM) and the brainstem blocked. Horizontal slices (300-350μm thick) of the pons were cut from dorsal to ventral using a vibratome (Linearslicer Pro 7; DSK) in cold (4°C) cutting solution. After cutting, slices were kept at room temperature in carbogenated recording solution (NaCl 126 mM, KCl 2.5 mM, NaHCO 3 26 mM, NaH 2 PO 4 1.25 mM, MgCl 2 2 mM, CaCl 2 2 mM, and D-glucose 10 mM saturated with 95%O 2 /5%CO 2 , pH7.3, osmolality 290mOsm/L) for at least 1 h to recover before recording. Pontine slices were transferred into the recording chamber of an upright fluorescence microscope (DMLFSA; Leica Microsystems), superfused with artificial CSF at a rate of 4-8 ml/min heated to 35°C. Borosilicate glass patch pipettes (Harvard Apparatus GC120F-10, resistances of 4-6MΩ) were filled with internal solution (K-gluconate 130 mM, KCl 10 mM, Na-HEPES 10 mM, MgATP 4 mM, EGTA 0.2 mM, and Na 2 GTP 0.3 mM). Cells were identified under gradient contrast illumination and examined for transduction by epifluorescence illumination. Whole cell voltage and current clamp recordings were obtained with a Multiclamp 700A amplifier (Axon instruments). All membrane potentials were corrected for a junction potential of 13 mV. Drugs were infused in the bath solution. Spike2 software (CED, Cambridge Electronic Design) with customized scripts was used to acquire and store data.
In vivo extracellular recordings from the locus coeruleus and local application of PSEM308 Anaesthesia was induced with 1.2-2 g/kg urethane (Sigma) i.p to achieve loss of paw withdrawal reflex. Once induced, urethane anaesthesia typically remained stable for 5-7 hours. The animal was placed in a stereotaxic frame, the skull exposed and the screw above the viral injection site removed to gain access to the CNS. In vivo single units were recorded with preloaded quadruple-barrelled microelectrodes (Kation Scientific, Carbostar-4 recording/iontophoresis microelectrode, E1041 standard) connected to a drug injector (Parker, Picospritzer II). Recordings were referenced against a silver chloride electrode pellet that was inserted between muscles and skin near the recording site.
Neuronal activity was recorded via a Multiclamp 700A amplifier (with #CV-7b headstage, Axon instruments) filtered at 3 kHz and digitized at 10 kHz using a 1401micro and Spike2 software (CED, Cambridge Electronic Design) with custom scripts was used to store data and to control the drug delivery. Locus coeruleus cells were identified by their wide (~1 ms), large amplitude action potentials, the slow spontaneous firing frequency (0.5-5Hz) and a characteristic burst of action potentials in response to contralateral hind-paw pinch (Hickey et al., 2014, Cedarbaum andAghajanian, 1976). Action potential discharge frequency was measured in 5 s bins and compared before, during, and after drug application. The average depth of electrode placement for LC recordings was 5.7±0.14 mm from the cerebellar surface.

In vivo single unit recordings from the dorsal horn
Animals were terminally anaesthetised with urethane (1.2-2 g/kg i.p, Sigma) The spinal cord was exposed by laminectomy over the L1-L4 spinal segment for recordings (Funai et al., 2014, Furue et al., 1999. The animal was placed in a stereotaxic frame and the spinal cord stabilised with two spinal clamps at T1 and L5 and a bath was formed by skin elevation. Warmed (36˚C), Krebs solution was used to continually superfuse the spinal cord (NaCl 117 mM, KCl 3.6 mM, NaHCO 3 25 mM, NaH 2 PO 4 1.2 mM, MgCl 2 1.2 mM, CaCl 2 2.5 mM and D-glucose 11 mM saturated with 95%O 2 /5%CO 2 , pH7.3, osmolality 290mOsm/L). The dura mater was removed under binocular vision (Leica MZ6). A reference electrode (World Precision Instruments, Ag/AgCl Electrode pellets, EP2) was inserted into the muscle layer near the recording area and spinal units were recorded with a stainless-steel microelectrode (FHC, UESSEGSEFNNM, 8-8.8MΩ) that was positioned ~500 µm from the midline.
Neuronal activity was amplified and low pass filtered at 3 kHz using a Multiclamp 700A amplifier (Axon instruments with #CV-7b headstage). The data were digitized at 10 kHz (CED Power1401) and stored using Spike2 software (CED, Cambridge Electronic Design) with customized scripts to monitor pinch force applied from custom calibrated forceps with tip pads of area 3.5mm 2 . These forceps were used to ensure that the pinch stimulus was standardised between applications and across animals. The forceps were equipped with strain gauges and a pinch pressure of 70 g mm -2 (Drake et al., 2014) was used throughout all experiments to compare pinch-evoked neuronal activity (NB this pressure was above the threshold for pain when applied to the experimenter's index finger).
Wide dynamic range (WDR) neurons were identified in the deep dorsal horn by graded responses to both non-noxious touch and noxious pinch. Their discharge frequency was measured in response to light touch with a brush or to sustained pinch pressure over five second time bins immediately after stimulus application.

Tibial nerve transection model
Rats were anesthetized by intraperitoneal injection of ketamine (5 mg/100 g, Vetalar; Pharmacia) and medetomidine (30 μg/100 g, Domitor; Pfizer) until loss of paw withdrawal reflex. A 1.5-2cm long skin incision was made in the right hind leg in line with the femur (Pertin et al., 2012). The fascial plane between the gluteus superficialis and the biceps femoris was located and blunt dissected to expose the three branches of the sciatic nerve (i.e. common peroneal, sural and tibial nerves). The tibial nerve was carefully exposed. Subsequently, two tight ligations (5-0 silk) were made approximately 2mm apart and the neve segment in between transected and removed. The nerve and muscle were placed back into the correct position and the skin sutured. For sham surgery, the same procedure without ligation and transection of the tibial nerve was performed.

Intrathecal injection after nerve injury
The injection procedure was similar to a previous report (De la Calle and Paino, 2002) Rats, 6 weeks post TNT (400-500g), were briefly induced with isoflurane in medical oxygen before transferring into a face mask with 1.5-2% isoflurane. The rat was placed in prone position on a 5cm tall plastic board so that the hind legs hang over the board to arch the lower back and to open the intervertebral space between L4-L5 spinal segments. Subsequently, a 25G needle was inserted transcutaneously to the vertebral canal (typically producing a tail twitch) and 10-20ul of vehicle or yohimbine (3µg/µl in sterile saline with 20% DMSO) injected. PSEM308 was injected i.p immediately after recovery from anaesthesia and sensory testing began 30min thereafter.
As previously described (Hughes et al., 2013), we observed that the mechanical withdrawal thresholds fell in the contralateral paw after intrathecal injection of yohimbine in nerve injured animals. This phenomenon was used as an internal control for successful intrathecal targeting because it was more reliable than a tail flick response (Figure S 4).

Behaviour
Rats underwent two 30 minute habituation sessions (unless otherwise stated) on consecutive days before testing. Animals were placed into Plexiglas chambers 20 minutes before sensory assessment began on the test day. All experiments, apart from conditioned place preference (CPP), were conducted in a cross-over design comparing saline and PSEM308 (10 mg/kg, unless otherwise specified). The experimenter was always blind to the animal group and to the administered solution.

Hargreaves' Test
Thermal withdrawal latencies were measured for the hind paw using the method described by Hargreaves et al. (1988). Animals were placed into a Plexiglas chamber on top of a glass plate. The radiant heat source (Ugo Basile Plantar test) was focused onto the plantar surface of the hind paw and the time to withdrawal recorded from 40 min before and up to 60 minutes after intraperitoneal injection of the selective agonist PSEM308 or saline. Each withdrawal value was the mean of 2 tests and tests were repeated every 15 minutes. A 24 second cut-off value was used to avoid tissue damage. The heat source intensity was adjusted so that control rats responded with a latency of 7-8 seconds.

Spontaneous foot lifts
Animals were placed into a Plexiglas chamber on a metal grid and a webcam (Logitech HD Pro Webcam C920) positioned underneath. The number of spontaneous footlifts and flinches was recorded over a 5 minute period 20 minutes after i.p injection of PSEM308 / saline.

von Frey test
To assess punctate mechanical sensitivity animals were placed into a Plexiglas chamber on a metal grid and von Frey filaments (0.4g-26g) were applied to the lateral aspect of the plantar surface of the hind paw. The 50% paw withdrawal threshold was determined using the Massey-Dixon up-down method (Chaplan et al., 1994). This procedure was repeated every 30 minutes starting 1h prior and up to 4h after i.p injection of PSEM308 (1, 5 and 10 mg/kg) or saline.

Acetone Test
To assess cold sensitivity animals were placed into a Plexiglas chamber on a metal grid and an acetone drop on top of a 1ml syringe was presented to the hind paw from underneath 5 times, 2 minutes apart. Data are expressed as the percentage of withdrawals. Animals were tested prior to and 45 minutes after i.p injection of PSEM308 or saline.

Incapacitance Test
The distribution of weight between the hind-limbs was measured with an incapacitance tester (Linton Instrumentation). Animals were habituated to the Plexiglas chamber with their hind paws on two force transducers in 4 sessions (each lasting 3 minutes) on two consecutive days. Measures were taken 45 minutes after injection of PSEM308 or saline. The percentage of weight borne on the injured leg was averaged over these 4 measures.

Conditioned place preference/aversion
All rats were habituated to an unbiased three-compartment box, which contained a central neutral chamber with different association chambers either side. The outside compartments have visual and tactile cues e.g. "bars" or perforated "holes" as flooring and differently patterned (but equal luminosity) wallpapers to maximise differentiation between the compartments. On the first day of the experiment animals were allowed to roam freely through all three compartments and a Basler camera (acA1300-60gm) with a varifocal lens (Computar H3Z4512CS-IR) connected to EthovisionXT (Noldus) was used to record the time rats spent in either of the outer compartments as a baseline value. Subsequently, rats underwent four conditioning sessions in which the outside compartments were paired with PSEM308 injection or saline injection. Two pairing sessions per day at least 4h apart on two consecutive days were conducted in a counterbalanced design. For each conditioning session the animals were placed into the locked outer compartment 5 minutes after the i.p injection and left for 30min. On the test day, the doors between the compartments were opened and the time spent in the outer compartments measured once more. It is expected that the choice of the animal's preferred environment in the post-test is influenced according to the valence of the chemogenetic stimulation. Noldus EthoVision® XT was used to analyse recorded videos and to quantify the time animals spent in the paired chambers.

Open field test
On the first day of testing animals were randomly assigned into two groups and received PSEM308 or saline 20-25 minutes before placing the subjects into the open field arena (56.5x56.5cm with an inner zone of 30x30cm) for 6 minutes. Exploration behaviour (distance travelled, location in central / perimeter areas and immobility (velocity < 1.75cm/s)) was tracked for 5 minutes starting 60 seconds after entering the arena using EthoVisionXT. One week later the open field experiment was repeated using the other solution.

Histology Tissue Collection and Processing
Rats were killed with an overdose of pentobarbital (Euthatal, 20 mg/100 g, i.p; Merial Animal Health) and perfused transcardially with 0.9% NaCl (1 ml/g) followed by 4% formaldehyde (Sigma) in phosphate buffer (PB; pH 7.4, 1 ml/g). The brain and spinal cord were removed and post-fixed overnight before cryoprotection in 30% sucrose in phosphate buffer. Coronal tissue sections were cut at 40μm intervals using a freezing microtome and left free floating for fluorescence immunohistochemistry.

Immunofluorescence
Tissue sections were blocked for 45minutes in phosphate buffer containing 0.3% Triton X-100 (Sigma) and 5% normal goat serum (Sigma). Incubated on a shaking platform with primary antibodies to detect EGFP (1:3000), DBH (1:2000), or HA tag (1:500-1:1000) for 14-18 h at room temperature. After washing, sections were then incubated for 3 h with appropriate Alexa Fluor® secondary antibodies. A Leica DMI6000 inverted epifluorescence microscope equipped with Leica DFC365FX monochrome digital camera and Leica LAS-X acquisition software was used for widefield microscopy. For confocal and tile scan confocal imaging, a Leica SP5-II confocal laser-scanning microscope with multi-position scanning stage (Märzhäuser, Germany) was utilized. Images were exported as TIF files before being processed, analysed and prepared for presentation using Fiji-ImageJ software.
Stereological cell counts (Miller et al., 2014) were made by stepping through a confocal z-stack with 1μm spacing and EGFP positive cells were only counted if the top of the soma was unambiguously located within the imaged dissector volume of the section (12μm). The average section thickness after histological processing was 18.0±0.7μm (30 sections from 3 animals) and dual guard zones of 2μM were applied. Therefore, the height sampling factor was 0.66 (hsf = dissector height/average section thickness). The section sampling factor was 0.25 (ssf = 1 / number of serial sections). The correction was made using the following formula.

= * 1
Where N is the estimated number of cells and C the sum of all counted cells. The volume fraction is the product of ssf, hsf and asf (area sampling fraction). Because all EGFP positive neurons in the LC area were counted the area sampling fraction was omitted from the volume fraction.

QUANTIFICATION AND STATISTICAL ANALYSIS
All statistical analysis was conducted using GraphPad Prism 7. All data are presented as mean ± SEM (unless otherwise specified). Sample size estimates were based either on previous data from similar experiments (Hughes et al., 2015, King et al., 2009, McCall et al., 2015 or for novel experiments based on an initial pilot experiment to obtain an estimate of effect size and variance for a power calculation with alpha <0.05 and beta 0.8 (using G*Power) to inform subsequent definitive experiments. T-tests, one-way or two-way ANOVA were used to compare groups as appropriate.
Bonferroni's post-test was used for all comparisons between multiple groups. Differences were Exclusions: One animal was excluded from conditioned place preference experiments because it learnt to escape from the arena. Data from four spinal cord WDR neurons were excluded because the recording was lost before the end of the protocol. One animal (in the LC :PFC group) was excluded from analysis because of a failure of vector transduction discovered on post hoc histology.   (C) Co-application of reboxetine (1 mg/kg) and PSEM308 (10 mg/kg) in LC :PFC rats increased immobility time, decreased the distance travelled and decreased the time spent in the centre of the arena as compared to reboxetine alone. Co-application of reboxetine and PSEM308 (10 mg/kg) also increased immobility as compared to PSEM308 alone (paired t-test PSEM308 vs PSEM308 + Reboxetine, + P<0.05).