A novel role for the cholinergic basal forebrain nucleus of Meynert in chronic pain via modulation of the prelimbic cortex

The basal nucleus of Meynert (NBM) subserves critically important functions in attention, arousal and cognition via its profound modulation of neocortical activity and is emerging as a key target in Alzheimer’s and Parkinson’s dementias. Despite the crucial role of neocortical domains in pain perception, however, the NBM has not been studied in chronic pain. Here, using in vivo tetrode recordings in behaving mice, we report that beta and gamma oscillatory activity is evoked in the NBM by noxious stimuli and is facilitated at peak inflammatory pain. Optogenetic and chemogenetic cell-specific, reversible manipulations of NBM cholinergic-GABAergic neurons reveal their role in endogenous control of nociceptive hypersensitivity, which are manifest via projections to the prelimbic cortex, resulting in layer 5-mediated antinociception. Our data unravel the importance of the NBM in top-down control of neocortical processing of pain and suggest a potential for its therapeutic modulation via neurostimulation strategies in chronic pain disorders. in and to eight-month heterozygous Chat-IRES-Cre mice ; 57 with a C57BL/6 and ChAT-Cre mice. Cre-ve littermates used for control experiments. four-month-old male and female Rbp4-Cre animals (B6.FVB/CD1-Tg(Rbp4cre) KL100Gsat/Uhg for targeting C57BL/6J

GABAergic neurons reveal their role in endogenous control of nociceptive hypersensitivity, which are manifest via projections to the prelimbic cortex, resulting in layer 5-mediated antinociception. Our data unravel the importance of the NBM in top-down control of neocortical processing of pain and suggest a potential for its therapeutic modulation via neurostimulation strategies in chronic pain disorders.

Introduction:
A major hindrance to adequate therapy of chronic pain disorders is given by incomplete knowledge on brain circuits underlying the perception of pain and their modulation over transition from acute to chronic pain. Their elucidation is therefore important for yielding mechanistic insights as well as for therapeutic advance. Recent studies on functional interrogation of brain circuits have led to breakthroughs on structure-function properties of some brain networks involved in pain, and particularly revealed key roles for neocortical domains 1 . Pain perception is subject to profound modulation by contextual, environmental and psychosocial factors. As contributing neural mechanisms, insights are now emerging on modulation of neocortical processing by afferent input from GABAergic, dopaminergic and serotonergic pathways 2 .
In comparison, very little is known about the scope and functions of cholinergic pathways in the brain in modulating pain perception. This is in contrast to extensive pharmacological studies from the past two decades, which report effects of cholinergic signaling via both ionotropic nicotinic receptors as well as metabotropic muscarinic receptors on pain and analgesia 3 . Systemic, peripheral, as well as spinal administration of cholinergic ligands modulates nociception and studies with central administration have implicated cholinergic signaling in opioidergic analgesia and descending modulatory systems. However, there has been very little progress in exploiting cholinergic modulation towards pain relief, owing primarily to major gaps in understanding the underlying circuitry, particularly with respect to the delineation of the origin of cholinergic inputs. This is particularly important, because both facilitatory and inhibitory effects are associated with pharmacological modulation of cholinergic receptors, which can be attributed not only to diversity of receptor-mediated signaling but also to the locus of cholinergic modulation in the nervous system.
In the brain, cholinergic neurons are abundant either in form of local interneurons in specific areas, such as the caudate putamen, or organized in the cholinergic nuclei Ch1-Ch6 of the basal forebrain and brainstem to function as projection neurons with distant targets 4 . Amongst these, the basal forebrain system comprises discrete groups of cholinergic cells (Ch1-Ch4), with neurons in the medial septum (MS) and the vertical limb of the diagonal band of Broca (vDB) primarily targeting the hippocampus, while the neurons in Ch4 largely account for the cholinergic input into the neocortical mantle and also project to the amygdala 4 . In the rodent brain, the structure most analogous to the Ch4 is given by the nucleus basalis magnocellularis (NBM; basal nucleus of Meynert), also extending into a band ventral to the anterior commissure called the substantia innominata, which are collectively referred to under the term 4 NBM in this study, consistent with several other published studies (e.g., 5 ; schematic view in Fig. 1a). This sector contains the largest component of corticopetal projections from the basal forebrain and is overwhelmingly cholinergic in nature. The NBM has been ascribed a modulatory role in specific key functions, such as arousal, attention, fear and social interactions, including social recognition memory 5 . Furthermore, the NBM has been implicated in sharpening the acuity of sensory processing by enhancing the 'signal-to-noise' ratio in cortical circuits via nicotinic and muscarinic mechanisms involving both pyramidal neurons and GABAergic interneurons 6,7 . These properties potentially place the NBM in a critical position to modulate pain perception and its plasticity, given the importance of neocortical processing in pain 1 . Surprisingly, however, the NBM has hardly been studied in the context of pain, barring a few studies with excitotoxic lesions and broad toxin-mediated ablation of cholinergic groups. Importantly, there have been no studies functionally delineating the underlying native circuitry. Moreover, it remains unknown whether and how activity patterns in the NBM change in association with pain and the NBM undergoes plasticity during the transition to chronic pain in vivo.
Here, we performed in vivo recordings using tetrodes to dynamically capture changes in activity of single neurons as well as oscillatory field rhythms in the NBM in freely-moving, behaving mice during nociception and the transition to inflammatory hypersensitivity. We report specific responses of the NBM to pain-inducing (noxious) stimuli, which demonstrate a switch in responsivity to low intensity stimuli in inflammatory pain, thus mirroring behavioral hypersensitivity. Simultaneously, gamma and beta oscillatory rhythms undergo potentiation of spectral power. Using reversible, cell type-specific chemogenetic and optogenetic manipulations in conjunction with behavior, we demonstrate that this potentiation of cholinergic activity in the NBM and its projections to the prefrontal cortex suppresses nociceptive hypersensitivity in both inflammatory and neuropathic pain conditions, thus paving the way for novel therapeutic strategies specifically targeting these cholinergic cell groups.

Oscillatory rhythmic activity in the NBM in nociception and inflammatory pain
Intraplantar hindlimb injection of capsaicin in wild-type mice, which acutely induces strong, tonic pain, led to a robust increase in expression of the activity-dependent immediate early gene product, Fos, in the NBM (schematically shown in Fig. 1a), including cholinergic neurons as seen via co-labelling for the marker choline acetyltransferase (ChAT; Fig. 1b, d). We next targeted this area in electrophysiology experiments in awake, behaving mice to directly study changes in NBM activity, both at the level of field potentials and single cells using tetrodes.
Application of mechanical force via von Frey filaments was associated with an increase in activity across all frequency bands over baseline (pre-stimulus) activity levels (Fig. 1d, e; also see Suppl. Fig. 1a). Across multiple trials and animals, the increase was statistically significant in the power of beta oscillations (14-30 Hz) when stimuli at and above the nociceptive thresholds were applied (von Frey force of 0.6-1.0 g) as well as with low intensity, non-noxious tactile stimuli (0.07-0.16 g), whereas power of gamma oscillations (30-100 Hz) selectively increased with nociceptive strength stimulation (Fig. 1f). This finding is particularly interesting because gamma oscillations in cortical areas have been functionally linked with nociception in both human and rodent studies 8,9,10 , and are known to be associated with synchronization of activity via GABAergic interneurons 11 . Noxious mechanical stimulation-induced increase in gamma activity was seen to reach statistically significant levels prior to the behavioral nocifensive response and was maintained for 2 seconds after application of the stimulus (Fig.   1g).

6
We next sought to test the potential significance of the NBM in the progression of nociception to hypersensitivity that is characteristic to persistent inflammatory pain. Indeed, mice with unilateral hindpaw inflammation induced by injection of Complete Freund's Adjuvant (CFA) demonstrated enhanced Fos expression in cholinergic neurons in the NBM (Fig. 2a, b). In a longitudinal study design, we then compared oscillatory activity between naïve conditions and after CFA-induced hypersensitivity was established (Suppl. Fig. 1b). At the time of peak mechanical hypersensitivity, paw stimulation elicited a significantly larger increase in the power of beta and gamma rhythms (Fig. 2c, d), but not of alpha and theta activity (Suppl. Fig.   1c) in the NBM. An interesting finding was that the inflammatory pain-associated increase in gamma and beta power was seen with low intensities of mechanical stimulation, which are typically non-noxious in physiological conditions but are perceived as noxious in inflammatory pain (Fig. 2e). Taken together, these findings indicate that the NBM is recruited during nociception and shows facilitation of its responsivity over the transition to hypersensitivity in inflammatory pain.

Single cell analysis of NBM activity in nociception and inflammatory pain
Analyzing activity at the single cell level via spike sorting led to interesting insights into the cellular nature of NBM responsivity and plasticity in pain. Amongst the 221 units recorded under naïve conditions, 14 % showed a consistent increase in firing rate in withdrawal trials upon applying 20 mechanical paw stimulations with either the weak or strong filament pair, and 11 % of units were consistently inhibited in activity by paw stimulation (Fig. 3a, b); Example traces and average Z-scores (denoting number of standard deviations for data points above or below mean) are shown in Fig. 3a and unit proportions in Fig. 3b. In mice with inflammatory pain, the proportion of units responding to mechanical stimulation did not change significantly during strong hypersensitivity over the first 4 days after CFA injection (Fig. 3b).
Over this period however, maximal z-score values increased significantly in neurons excited by noxious intensities of mechanical stimulation (Fig. 3c), but not in neurons inhibited by mechanical stimulation (Suppl. Fig. 2a), thus corresponding to the overall increase in the power of oscillatory activity which we observed at the LFP level. Furthermore, by analyzing the shape of the spike wave-form, we then classified units into Class 1 and Class 2 with broad or narrow spike wave-forms, respectively 12 ; fast-spiking classes of GABAergic projection neurons and interneurons are represented within class 2 units 13 . Interestingly, although maximal z-score values appeared to be elevated in both classes post-CFA, the change was only robust and statistically significant for Class 2 neurons (Fig. 3d). These data suggest that NBM neurons, which are excited by mechanical stimuli, undergo facilitation over the manifestation of inflammatory nociceptive hypersensitivity and further that fast-spiking, Class 2 GABAergic neurons in the NBM particularly contribute to these changes. This finding is noteworthy, since in the mouse NBM, 92 % of ChAT-expressing cholinergic neurons are known to be GABAergic 14 . At late time points after CFA injection (7-14 days), after normal nociceptive sensitivity is recovered, we observed that patterns of oscillatory and single cell activity in the NBM not only normalize, but partly even fall below baseline values (Suppl. Fig. 2; Suppl. Fig.   3).

Optogenetic stimulation of NBM acutely suppresses hypersensitivity
To directly uncover the significance of our findings, we employed a cell-specific optogenetic approach by targeting the blue light-gated cation channel Channelrhodopsin to ChATexpressing neurons. Recombinant adenoassociated virions (AAV) were stereotactically injected to express yellow fluorescent protein-tagged Channelrhodopsin in a Cre-dependent manner (rAAV-Dio-ChR2-YFP) unilaterally in the NBM of ChAT-Cre transgenic mice ( Fig.   8 4a, b). Cre-negative mice subjected to the same treatments served as controls. Delivery of blue light to the NBM via chronically implanted optic fibers significantly increased Fos expression in cholinergic neurons, thereby establishing in vivo validation of the approach (Fig. 4c, d).
Upon blue light stimulation, baseline sensitivity to mechanical stimuli remained unchanged ( Fig. 4e, baseline); however, the left-ward and up-ward shift in the von Frey stimulus-response function, representing the manifestation of inflammatory hypersensitivity, was significantly lowered in Cre+ mice as compared to Cre-controls when tested at peak sensitization on day 2 post-CFA (Fig. 4e, middle panel). Likewise, the mechanical withdrawal threshold was significantly increased in mice with CFA upon blue light stimulation in Cre+, but not in control Cre-mice (Fig. 4f). Overall, the magnitude of mechanical hypersensitivity over baseline values was decreased and the return to baseline sensitivity was faster upon optogenetic stimulation of NBM cholinergic neurons (Fig. 4f). However, CFA-induced heat hyperalgesia was not significantly altered in magnitude or duration (Fig. 4g).

Dissecting the contribution of NBM cholinergic-GABAergic projections to the medial prefrontal cortex
Because the NBM projects to a large number of neocortical targets, many of which affect pain and hypersensitivity in multiple ways, we then sought to dissect the significance of NBM neuronal projections to the medial prefrontal cortex (mPFC), which represents a key hub in brain circuits underlying pain. The mPFC undergoes marked plasticity in several human clinical chronic pain conditions and particularly, a major focus has emerged on its deactivation observed in chronic pain patients 15 , a finding that is also reported in animal models 16,17,18,19 .
We therefore expressed ChR2-YFP in ChAT neurons of the NBM and placed the optic fiber for blue light illumination in the prelimbic cortex (PL), the mouse counterpart of the human mPFC (Fig. 5a). Selectively activating NBM cholinergic projections to the PL did not influence baseline mechanical sensitivity, but had an even stronger analgesic effect than direct activation of NBM neurons on CFA-induced mechanical hypersensitivity, which was completely reversed to baseline values (Fig. 5b). Furthermore, thermal hypersensitivity was robustly suppressed over a long period post-CFA in mice with optogenetic stimulation of the NBM-PL projections (Fig. 5c).
Electrophysiological and modelling studies indicate that cholinergic inputs from the basal forebrain exert direct excitatory effects via receptor-mediated signaling on cortical pyramidal neurons and also have the ability to evoke either inhibition or dis-inhibition of pyramidal neurons via signaling on different classes of local neocortical GABAergic interneurons or via signaling through different types of nicotinic and muscarinic receptors 14,20 . Interestingly, recent studies also indicate that GABA is co-released from cholinergic projections originating from the basal forebrain nuclei and can thus disinhibit neocortical pyramidal neurons by suppressing local inhibitory interneurons 14,21 (schematic in Fig. 5d). Both mechanisms have been suggested to act towards enhancing cortical signal-to-noise processing of sensory inputs, e.g., to visual inputs in the visual cortex and tactile inputs in the somatosensory cortex 21 . To address how NBM-PL cholinergic-GABAergic projections affect the PL, we performed viral tracing and c-Fos mapping across layers in conjunction with optogenetics. Interestingly, while NBM projections to most of the neocortical mantle diffusely span all cortical layers, our tracing analyses revealed that projections from the NBM to PL terminate in layer 5 in a particularly abundant manner as compared to other layers ( Fig. 5e; compare with neighboring motor cortex M2 and cingulate cortical domains). In both baseline conditions (naïve mice) and mice with inflammatory pain, optogenetic stimulation of NBM-PL projections led to a significant increase in Fos levels throughout the PL (Fig. 5f). Recent studies have shown that layer 5 pyramidal neurons in the PL project to the periaqueductal grey and thereby link to descending nociceptive modulatory systems 22 . We observed that optogenetically activating NBM-PL connections enhances the activity in layer 5 neurons, beyond the increase induced by inflammatory pain (Fig. 5g). We therefore selectively stimulated layer 5 neurons chemogenetically by virally directing hM3D(Gq) expression 23

Targeting the NBM in chronic pain
From the view point of translational relevance, it is important to address whether targeting the NBM cholinergic system is also beneficial in other forms of pain, particularly neuropathic pain.
Hence, we addressed whether the cholinergic NBM system is recruited in the neuropathic pain state and is related to mechanical allodynia by studying Fos expression in the absence of or upon plantar application of low intensity mechanical von Frey force, which is innocuous under baseline conditions. Fos expression in the NBM was significantly elevated in neuropathic mice as compared to sham-injured mice and showed a further increase upon paw stimulation associated with mechanical allodynia (Fig. 6a, b). Importantly, this was also reflected in ChATexpressing cholinergic neurons (Fig. 6a, b), which suggests increased recruitment by stimuli that are innocuous in baseline conditions but perceived as noxious in neuropathic pain conditions, thus showing parallels to our findings in electrophysiological experiments in the inflammatory pain model described above.
To test the functional significance of these findings in the context of neuropathic pain conditions, we employed a chemogenetic approach to activate cholinergic neurons of the basal forebrain, which provided two advantages: one, it enabled targeting a larger area than optogenetic stimulation (which is limited owing to the maximum area that can be sufficiently illuminated), and second, it permitted achieving more long-lasting activation of cholinergic neurons. ChAT-Cre transgenics were injected with rAAV expressing either the excitatory chemogenetic actuator (mcherry-tagged hM3D(Gq)) or control (mCherry) protein in a Credependent manner and treated with Clozapine-N-Oxide, which enables inducible activation of hM3D(Gq) 23  We then employed the chronic constriction injury (CCI) model involving unilateral loose ligation of the sciatic nerve, leading to local inflammation and swelling of the nerve, neuropathy and nociceptive hypersensitivity lasting up to a month 24 . CCI-induced mechanical hypersensitivity was markedly reduced in hM3D(Gq)-expressing mice in comparison to mCherry-expressing mice starting from day 4 after CCI surgery (Fig. 6f). At day 11, mechanical responses in hM3D(Gq)-expressing mice were indistinguishable from baseline sensitivity, while mCherry-expressing mice continued to show mechanical hypersensitivity and regained baseline values only on day 28 (Fig. 6f). Similarly, heat hypersensitivity was robustly reduced in hM3D(Gq)-expressing mice as compared to mCherry-expressing mice until day 14 post-CCI (Fig. 6g). These data show that activation of the NBM robustly suppresses neuropathic hypersensitivity over a long duration.

Potential contributions of modulation of anxiety, attention and motor function
Activity of NBM neurons has the potential to affect pain processing directly via cholinergic signaling in neocortical targets that are important in pain networks, as indicated by our observations above on prefrontal layer 5 neurons. However, because the NBM is known to be a key modulator of circuits underlying arousal and attention 20 , there is also a possibility that the observed antihyperalgesic effects are related to attention and expectancy. We therefore also addressed whether optogenetic stimulation of NBM cholinergic neurons affects attention behavior using the widely accepted five choice serial reaction task test (center-to-margin ratio was unchanged). In contrast, optogenetically stimulating NBM-PL projections reduced the center-to-margin ratio in naïve mice, suggesting anxiolytic effects (Fig.   7f). Finally, locomotion was unchanged in all of the groups involving optogenetic or chemogenetic modulation of the NBM or NBM-PL circuits, suggesting a lack of confounding effects on motor function in behavioral analyses ( Fig. 7d-f).

Discussion
Literature on the basal forebrain cholinergic nucleus and pain perception is surprisingly scarce.
To date, fewer than a handful of studies have tested the activity of the basal forebrain cholinergic nucleus upon noxious stimulation 26,27 . In this study, we now report the precise nature of oscillatory rhythms in the NBM as well as a detailed analysis at a single cell level in vivo, showing that the NBM not only responds to noxious stimuli, but also undergoes dynamic changes during the transition to chronic pain. The most interesting observation was that the power of gamma oscillatory activity in the NBM is specifically enhanced in conjunction with noxious stimulation prior to the behavioral response. Gamma oscillations in the S1 have been functionally linked to nociceptive modulation in both human and rodent systems in vivo 28,29 , and these pain-related alterations in gamma activity have only been recently extended to other neocortices, such as the prefrontal and insular cortices 9,30,31 . This study, to the best of our knowledge, represents the first report linking gamma rhythms in a sub-cortical structure to nociceptive sensitivity. These have been likely missed in human studies owing to technical limitations from scalp recordings.
Importantly, our observation that in inflammatory pain, the power of gamma activity is potentiated in response to non-noxious tactile stimulation correlates with the manifestation of mechanical allodynia and mimics similar observations made in the S1 cortex 29 . Taken together with current knowledge, a tantalizing implication of our findings is that gamma activity in the NBM is functionally linked via cholinergic pathways to neocortical gamma oscillations during nociceptive processing. This is supported by several conceptual points and experimental observations. First, a recent study in rats reported hemodynamic blood flow changes in the NBM following noxious stimulation and demonstrated that cerebral blood flow changes in the ipsilateral S1 cortex evoked by noxious stimulation were significantly reduced upon lesioning the NBM 32 , thus suggesting importance of the NBM in the full manifestation of pain-related responses in the somatosensory cortex. Second, in both S1 and the prefrontal cortex, cholinergic signaling facilitates or even directly elicits gamma band oscillatory activity via modulation of local GABAergic interneurons 33,34 , thereby enhancing acuity of stimulus processing in sensory and attentional networks, although these phenomena have not been addressed in the context of pain so far. Moreover, gamma oscillatory activity has been proposed to coordinate and link activity states across distant sites in the brain, which is a particularly noteworthy concept in the context of pain 8 , since pain is essentially a network function 35,36 .
A salient role in the emergence of gamma oscillatory activity is attributed to fast-spiking GABAergic interneurons, which are extensively interconnected via gap junctions; they not only streamline and synchronize excitatory output within a region, but are also capable of doing so at distant sites via long range GABAergic projections, which typically synapse on GABAergic neurons thereby leading to disinhibition 8,11 . Importantly, here, we observed that while different sets of NBM neurons showed excitation or inhibition upon nociceptive stimulation, the NBM neurons undergoing significant changes during the transition to nociceptive hypersensitivity were derived from waveform analysis to be fast-spiking GABAergic neurons. In the NBM, an overwhelmingly large majority of cholinergic neurons are GABAergic 11 and these comprise long range projections to the neocortical mantle, thus providing further credence to the association between the origins of gamma oscillatory activity in the NBM and cortical modulation of pain. We also observed changes in the beta frequency range of oscillatory activity; however, much less is known about its cellular origins and functional significance to pain. Studies in healthy subjects have reported that activity in the low frequency bands, particularly in the alpha and beta ranges, is suppressed in the S1, prefrontal and insular cortices in correlation to subjective pain ratings 9, 37 . More work will be needed to unravel the significance of beta rhythm changes in the NBM in pain states and whether and how this contributes to changes in beta oscillations in the neocortex. The results of this study suggest that neuronal depletion in the NBM will likely lead to a loss of central antinociceptive modulatory effects, thereby contributing to pain disorders that are frequently associated with these states. Our observation of reduction in NBM activity over very late stages after paw inflammation are also interesting in this regard. The ongoing development and testing of deep brain stimulation of the NBM 54, 56 thus holds promise in not only reducing cognitive decline, but also suppressing pain and restoring normal sleep in these disorders.

Competing interests
The authors declare no competing interests.

Methods: Animals
Experiments were performed in male and female two to eight-month old heterozygous Chat-IRES-Cre mice (B6;129S6-Chat tm2(cre)Lowl//Uhg ; 57 ) with a C57BL/6 background, and referred to here as ChAT-Cre mice. Cre-ve littermates were used for control experiments. Two-to four-  For the chronic constriction injury (CCI, 24 ) mice were placed under isoflurane anesthesia (2%) and the fur of the right thigh was shaved. An incision was made to the lateral skin surface of the thigh and through the biceps femoris muscle to expose the sciatic nerve just above it branches into sural, common peroneal and tibial nerves. Four loose ligatures were placed around the sciatic nerve using cat gut surgical sutures, the muscle and skin subsequently sutured close, and animals left to recover in a heated cage for 24 h. Behavioral testing commenced from day 2 after the operation. The same surgery was performed without placing the sciatic nerve ligatures on sham control animals.

Optical stimulation
Mice were restrained securely in a soft cotton cloth in order to attach the optical patch cables

Thermal sensitivity
The Hargreaves plantar test setup (Ugo Basile Inc., Italy) with an infra-red heat source (Model 37370-001, Ugo Basile) was used to test thermal withdrawal thresholds by applying radiant heat to the plantar surface of the hind paw. The intensity level was set to 25 and the cut-off time to 30 s. A heat stimulus was applied only during the quite wake phase and the withdrawal latency from stimulus onset was recorded. Six trials were performed per treatment condition and animal on a test day, using a minimum inter-trial interval of 2 min. Laser ON trials were randomly interspersed with laser OFF trials during an optogenetic Hargreaves test session. The laser was turned on 20 s before initiating the thermal stimulus and switched off 5 s after a paw withdrawal response.

Open field test
The open field test was performed in a square box (40 x 40 cm, 38 cm in height) with a USB camera fixed above the box in order to track animal movement patterns and record experimental parameters using ANY-maze software (Stoelting Co., Ireland). The animals were not acclimatized to this setup so that they encountered a novel arena to explore. The box was divided into three zones for analysis. A 3-cm-wide border along the walls of the box was defined as the thigmotaxic zone. A square zone of 20 x 20 cm in the geometric center of the box was defined as the center zone. The remaining area was defined as the marginal zone. Each mouse was placed in the center of the box and allowed to explore the entire field freely for an 8 min period. The 8 min test was divided into 30 s periods with the laser turned ON and OFF alternately in a random tact so that each mouse received 4 min illumination overall. During the test, the locomotion parameters (distance and mean speed) within each segment were recorded.
The ratio of the time spent in the center versus the thigmotaxic zones was used to assess anxiety-like behavior. Motor function was assessed from the total distance moved in all three zones.

5-Choice serial reaction time (CSRT) test
Two cohorts of ChAT-Cre mice either expressing the hM3(Gq) DRADD (n = 10) or the Optical patch cords were connected daily already during the later training phase of the optogenetic cohort without turning the laser on. Upon reaching the performance criterion with cues displayed for 1.8 s, animals were tested using a cue presentation period of 1.6 s with the laser turned ON at the start of each trial and OFF after collection of the water reward, or immediately after the 5 s extended time window if no correct touch response was detected.

Histology and immunohistochemistry
At the end of the experiment, mice were killed with an overdose of carbon dioxide and transcardially perfused with phosphate-buffered saline (PBS) followed by 10 % formalin

Electrophysiology
After one week of recovery from the implantation surgery, tetrodes were lowered down by 0.5 mm on average into the region of interest and remained unchanged until the end of the experiment. Mice were allowed 2 days to habituate to the elevated grid of the von Frey test recording setup. Naïve mechanical sensitivity tests were performed with weak (0.07 g and 0.6 g) and strong (0.6 g and 1.0 g) filaments for 4 days. Each filament was applied 10 times on the planter surface of the right hind paw with a minimal 60 s interval between stimulation trials.
Chronic inflammatory pain was induced by injecting CFA solution (25μl, Complete Freund's Adjuvant, Sigma) subcutaneously on the plantar side of right hind paw. Mechanical nociception tests were performed on days 1, 2, 3, 4, 7, 9, 12, 14 after CFA injection with the same filaments used for the baseline tests. At the end of the behavioral experiments, mice were deeply anesthetized with 2 % isoflurane, the location of each tetrode tip labelled by applying electrical current to induce a small lesion, and the animals perfused transcardially to fix the brain tissue.
Neural signals were acquired via a HS-18-MM headstage using Digital Lynx 4SX system and cheetah data acquisition software (Neuralynx). The raw data was acquired at 32 kHz with a bandpass filer (1-6000 Hz). The von Frey stimulation was recorded by a custom-made piezo transducer (Piezo ceramic element, part #717770, Conrad), which transduced the pressure of von Frey stimulation into an analog signal bandpass filtered at 1-2000 Hz 29 . In addition, videos of mechanical stimulation events were recorded by a USB camera (20 frames/second), synchronized via a keyboard-generated event signal to the piezo signal. Stimulation onset was defined as the time of contact of the von Frey filament with the hind paw corresponding with an initial deflection of the piezo signal by visually inspecting the video and piezo recordings, respectively.

Analysis of electrophysiology data
Local field potential (LFP) and single unit activity were analyzed with custom-written scripts using MATLAB (The Mathworks Inc, Version R2014a). Statistical analysis and post-hoc tests were performed in Graphpad Prism (version 9).

Power spectrogram analysis
For the spectrogram analysis of the LFP activity, 3s before and 3s after the onset of the von Frey filament application for withdrawal trials was extracted from the raw data. One channel of a tetrode was analyzed per animal. Raw data episodes were filtered with 3 rd -order lowpass Chebyshev type I filter with 0.5 dB ripples in the passband and a passband edge frequency of 200 Hz and down sampled to 1000 Hz. Power spectrograms were generated with the Morlet wavelets function, setting the central frequency to 0.8125 Hz, frequency accuracy at 0.5 Hz, and the time resolution to 1 ms. The 1 s-baseline period before the stimulation onset was used to normalize each 0.5 Hz frequency segment by the respective mean, and expressed as % deviations from the pre-stimulation baseline. The normalized power spectrograms of individual trials were then averaged for weak and strong filaments for each mouse and day. Grand mean averages of these normalized spectrograms for all animals are shown in Fig. 1e, 2c, and Suppl. For the quantitative analysis, averages of four frequency bands, including theta (4-8 Hz), alpha (8)(9)(10)(11)(12)(13)(14), beta (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30), and gamma (30-

Single unit analysis:
Spike sorting was performed with Kilosort2 61 to isolate single units. Raw data was preprocessed with a bandpass filter from 300 to 6000 Hz. Drift correction, unit clustering, and template matching was automatically performed based on the template matching method.
Automatically clustered units were manually curated in Phy (version 2.0; https://github.com/cortex-lab/phy) using waveform similarity and cluster features, firing rate, as well as cross-correlation and auto-correlation features.

Detecting stimulation responsive units:
For the analysis of evoked activity changes in the single unit data, firing activity of each withdrawal trial was aligned to the stimulation onset for the withdrawal trials of either all filaments, or separately for weak and strong filament groups. The firing rate across trials was calculated for 250 ms bins and z-scores computed based on the mean and standard deviation of the 3 s pre-stimulation baseline activities 42 . Units showing significantly increased or decreased activity were identified if at least one of the normalized bins in the 3 s poststimulation period exceeded 3.09 or -3.09, respectively, corresponding to a significance level of p < 0.001. Otherwise, the unit was classified as an unresponsive unit. Units were excluded from this analysis if the mean firing rate was smaller than 1 Hz or the number of withdrawal trials less than 3. In order to compare the magnitude of stimulation-evoked responses, the maximal and minimal z-score was extracted for all units with significantly increased or decreased firing rates within the 3 s post-stimulation periods, respectively.

Unit type classification:
For the single unit classification, various parameters were calculated, including: firing rate, coefficient of variation of the inter-spike intervals, peak-peak amplitude of the waveform, time between early and late waveform peaks, time from waveform trough to the return to baseline, and waveform asymmetry (the quotient of the difference between the baseline to early peak, and late peak to return of baseline times, to the sum of these two times). These multidimensional parameters were projected into two dimensions using the t-SNE (t-distributed stochastic neighbor embedding) Matlab function 62 . Then k-means algorithm was applied to cluster these units into two clusters. Based on cluster separation, the best unit classification was achieved using just two waveform parameters: the asymmetry parameter and the time from trough to the return to baseline. We did not attempt to distinguish if the units we classified were excitatory or inhibitory neurons, nor can we distinguish between projection neurons and interneurons.

Statistical analysis
All data are expressed as mean ± S.E.M. unless stated otherwise. Prism (version 9) was used for the statistical analysis of all behavioral data and for performing post-hoc comparison tests of electrophysiological data sets. A one-sample t-test was performed to detect if specific frequency bands of the LFP power spectrogram of withdrawal trials deviated significantly from the pre-stimulation baseline. A repeated measures one-way ANOVA with Fischer's LSD test was used for the time-course analysis in Fig. 1g. All grouped data sets were analyzed with a two-way ANOVA using Sidak's test for multiple comparisons for relevant treatment combinations that had significant main group effects. The unpaired Student's t-test was used to test for treatment effects compared to a control group. The Chi-square contingency test for the unit response types (Suppl. Fig. 2b) was applied for all time periods, as well as for pairwise time period combinations to detect the deviating data set. In all tests, a p value of < 0.05 was considered significant.          Resolving changes in NBM activity during acute nociception and in ammatory at the single cell level. Figure 4 Optogenetic activation of the NBM cholinergic neurons attenuates in ammatory mechanical, but not thermal, hypersensitivity.

Supplementary Files
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