Complex opioid-driven modulation of glutamatergic and cholinergic neurotransmission in a GABAergic brain nucleus associated with emotion, reward, and addiction

Substance use disorders (SUDs) are agnostic to demographic group posing a widespread public health crisis impacting many communities (Degenhardt et al., 2018). Drugs of misuse influence synaptic function, resulting in acute and protracted circuit adaptations that can exacerbate compulsive behaviors resulting in dependence (Lüscher and Malenka, 2011; Salmanzadeh et al., 2020). Mu-opioid receptors (mORs) represent the primary molecular substrate responsible for both the clinically efficacious (i.e. pain management) and the euphoric effects promoting opioid misuse and addiction (Matthes et al., 1996). Additionally, mainly via liberation of endogenous opioids (Trigo et al., 2010), mORs also facilitate the addictive propensity of other substances (Trigo et al., 2010; Darcq and Kieffer, 2018; Le Merrer et al., 2009) for example alcohol and nicotine, which, together with opioids, comprise the three most prevalent drugs of abuse worldwide (Degenhardt et al., 2018). mORs are expressed by varied neural cell types resident in numerous regions within the brain (Le Merrer et al., 2009; Reeves et al., 2022; Valentino and Volkow, 2018). Thus, detailed investigations as to how mOR activation impacts circuit dynamics constitute an important endeavor critical for identification of potential therapeutic interventions aimed at alleviating the deleterious societal consequences of SUDs.

Here, we focus on a relatively understudied brain circuit comprising the medial habenula (mHb) and interpeduncular nucleus (IPN). The mHb is a bilateral epithalamic structure receiving significant synaptic input from, for example, the diagonal band and various septal regions (Chung et al., 2023; Qin and Luo, 2009; Otsu et al., 2018). The efferent output of the mHb is predominantly mediated by two distinct populations, substance P (SP) and cholinergic neurons, that primarily impinge on the IPN (Qin and Luo, 2009; Ables et al., 2023). This latter structure in the ventral midbrain is mainly populated with GABAergic neurons that influence activity in downstream brain regions such as the raphe nuclei and ventral tegmental area (McLaughlin et al., 2017). Thus, the mHb/IPN axis is anatomically positioned to integrate incoming limbic forebrain signals to ultimately control the function of these midbrain monoaminergic nuclei. It is therefore unsurprising that perturbation/modulation of the synaptic recruitment or activity of IPN GABAergic neurons precipitates varied emotion, reward, and addiction phenotypes (Ables et al., 2023; McLaughlin et al., 2017; Ables et al., 2017; Molas et al., 2017; Klenowski et al., 2022; Tuesta et al., 2017; Zhao-Shea et al., 2013; Wolfman et al., 2018; Souter et al., 2022; Liang et al., 2024).

The mHb/IPN axes house one of the highest densities of mORs in the central nervous system (Gardon et al., 2014; Bailly et al., 2020), thus representing a prominent brain locus for opioid action. Despite this, descriptions regarding the role of mHb/IPN mORs in shaping behavior are still in their relative infancy (Allain et al., 2022; Boulos et al., 2020). mOR activation and subsequent G-protein signaling (Gi/o) classically produces an acute direct inhibitory effect in many neuronal types typified by membrane potential hyperpolarization via activation of inward rectifying potassium (GIRK) channels and/or reduced function of voltage-gated calcium channels that are coupled to neurotransmitter release (Reeves et al., 2022; Law et al., 2000). Strikingly, there is a dearth of information concerning the cellular effects and subsequent circuit influence of mOR signaling in the mHb/IPN, rendering the neural correlates underlying the behavioral outcomes elicited by opioids unclear at present. Interestingly, direct excitatory effects of mOR activation are apparent in certain areas of the nervous system either under basal conditions or following chronic drug regimens (Margolis et al., 2014; Hashimoto et al., 2009; Crain and Shen, 1990; Madhavan et al., 2010). Furthermore, within the IPN itself, GABA_B-receptor activation (another Gi-linked receptor) produces an increase in synaptic transmission (Koppensteiner et al., 2024; Zhang et al., 2016; Bhandari et al., 2021), questioning whether this surprising effect is generalized to other members of this receptor family expressed within this region such as mORs.

The central role of the mHb/IPN circuitry underlying numerous behavioral aspects relating to emotion and the cycle of addiction, the conspicuous expression of mORs and a clear precedent for non-canonical effects of Gi-linked receptors together formed the impetus for the current work. Employing cell-type conditional optogenetics in combination with slice electrophysiology and pharmacogenetics, we systematically dissect the effects of opioids on the mHb/IPN circuitry with a particular focus on synaptic signaling between these structures.

The two major neuronal populations of the mHb, substance P (SP) and cholinergic subtypes, can be distinguished by virtue of Tac1 and Chat gene expression. Leveraging the Allen Brain Institute’s publicly available whole mouse brain 10X single-cell RNAseq dataset (https://alleninstitute.github.io/abc_atlas_access/intro.html) (Yao et al., 2023), it is evident that these two neuronal classes within the mHb cluster (Subclass 145 MH) can be parsed based on their transcriptomic profiles (Figure 1a). Spatially, the SP and cholinergic neurons largely segregate to the dorsal versus ventral mHb, respectively (Figure 1b, c). The projection pattern as delineated by use of specific Cre transgenic mouse lines (i.e ChatCre and Tac1Cre) when crossed to a conditional TdTomato reporter (Ai9) reveals a predominant innervation of IPL by SP neurons, whereas cholinergic neurons impinge on the IPR/IPC subdivisions (Figure 1c). Together, these distinct neuronal classes provide much of the afferent input to the IPN via the fasciculus retroflexus (fr) axonal tracts (Figure 1c) ultimately dictating IPN neuronal recruitment. At the mRNA level, two of the designated three Chat neuronal Supertypes and the single Tac1 Supertype demonstrate significant expression of Oprm1 (mean expression levels including zero values = 0.33, 6.8, and 4.1 in Chat Supertypes 0632, 0634, and 0635, respectively, and 8.7 in the Tac1 Supertype 0633; Figure 1d). At the protein level, and as previously reported (Gardon et al., 2014), we confirm that mOR expression is notable in the mHb, the fasciculus retroflexus axonal tracts, and particularly prevalent in the IPN (Figure 1e). Within this latter structure, high-resolution microscopy clearly shows mOR expression is densest in the lateral IPN (IPL) with intermediate and relatively lower levels found in the rostral IPN (IPR) and central IPN (IPC) subregions, respectively (Figure 1e, f).

Figure 1

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Selective stimulation of each of these mHb neuronal populations in isolation is achieved using Cre-mediated conditional expression of channel rhodopsin by crossing Ai32 mice with either ChatCre or Tac1Cre mice; see methods for details. Adopting this optogenetic approach, we investigate how mOR receptor activation modulates habenulo-interpeduncular synaptic dialog imparted by these parallel yet distinct afferent systems in adult mice (p > 40). Both SP and cholinergic neurons express the glutamate vesicular transporter, VGluT1 (Slc17a7; Figure 1d) and competently release this excitatory neurotransmitter (Souter et al., 2022; Ren et al., 2011; Singhal et al., 2025).

mOR activation reduces substance P neuronal-mediated glutamatergic transmission onto IPL GABAergic neurons

Since the highest expression of mOR was found in the IPL (Figure 1e, f), we initially probed the effect of a selective mOR agonist (DAMGO, 500 nM) on AMPA receptor-mediated transmission mediated by SP neurons that prominently innervate this subregion (Figure 1c). DAMGO application results in a significant decrease in light-driven (470 nM; typically, 10–50% arbitrary LED power equating to approximately 0.4–3.4 mW/mm²; CoolLED illumination system) AMPAR-mediated EPSCs in Tac1Cre:Ai32 mice that is partially reversed upon washout of the agonist (Figure 2a, b). This is accompanied by an increase in S2/S1 paired pulse ratio (PPR) suggesting the modulation is driven by presynaptic changes in release probability (Figure 2c). Thus, neurotransmitter release by this specific afferent input to the IPL is directly, negatively modulated like that seen in many other neural circuits within the brain (Reeves et al., 2022).

Figure 2

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mOR activation augments the strength and fidelity of glutamatergic transmission mediated by cholinergic neurons, resulting in enhanced excitation-spike coupling in IPR GABAergic neurons

We next examined how mORs modulate mHb-IPN neurotransmission that occurs via the other major input mediated by cholinergic neurons. To this end, we employed ChatCre:Ai32 mice and focused on the IPR (Figure 3a) due to the relatively high levels of combined mOR expression and cholinergic neuronal innervation (Figure 1c, e, f) in this subregion. Under our basal experimental conditions and in agreement with previous studies (Souter et al., 2022; Singhal et al., 2025; Ren et al., 2022), light-evoked postsynaptic responses in the IPN mediated by mHb cholinergic neurons in response to either brief single or paired pulse stimulation are solely mediated by AMPARs as evidenced by complete pharmacological block by DNQX (refer to Figure 5—figure supplement 1a and Figure 7—figure supplement 1a–c). Remarkably, DAMGO application elicited a significant robust and reversible potentiation of light-evoked (470 nM; typically, 10–100% arbitrary LED power equating to approximately 0.4–6.9 mW/mm²; CoolLED illumination system) AMPAR EPSC amplitude (Figure 3a, b) in stark contrast to that seen with mHb Tac1-IPL glutamatergic signaling described previously (Figure 2a, b). Notably, the accompanying PPR changes were not consistent with an increase in release probability (Figure 3c), raising uncertainty as to the synaptic locus of the mOR effect.

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In these experiments, we were ‘blind’ to the molecular identity of the postsynaptic IPR neuron. Since a major population of neurons in this IPN subregion is of the somatostatin (SST) GABAergic subtype (Ables et al., 2017; Hsu et al., 2013), it is likely that most of our recordings are from this subtype. Nevertheless, we also employed Chat-ChR2 transgenic mice containing fluorescently reported SST neurons (i.e. Chat-ChR2:SSTCre:Ai9; Figure 3d) to allow targeted recordings specifically from these neurons. Our data clearly show that DAMGO robustly increases AMPAR EPSCs impinging on IPR RFP+ SST neurons (Figure 3d, e) accompanied by a trending increase in PPR (Figure 3f). We also performed a series of additional experiments agnostic to the postsynaptic neuronal identity in IPC subdivision. Despite the relatively low expression levels of mOR in this subdivision of IPN (Figure 1e, f), we observed a robust increase in AMPAR EPSC amplitude at a similar extent to that seen in IPR (Figure 3—figure supplement 1), thus extending this remarkable potentiation of glutamatergic synaptic transmission to all IPN subfields where cholinergic neuronal terminals reside.

We considered non-specific actions of DAMGO such as those that could be mediated by a possible direct modulation of ChR2 itself to explain this non-canonical observation. However, our previous data demonstrating the reduction of glutamatergic neurotransmission mediated by SP neurons by DAMGO described (Figure 2) renders this possibility unlikely. Furthermore, a similar potentiation of cholinergic neuronal-mediated glutamatergic transmission in the IPN upon activation of GABA_B receptors has demonstrated a propensity for this circuit to undergo such modulation in response to a Gi-linked receptor (Koppensteiner et al., 2024; Zhang et al., 2016; Bhandari et al., 2021). Nevertheless, despite this precedent, we employed a pharmacological approach to further validate this unexpected result. We show that lower concentrations of DAMGO (100 nM) and alternative mOR agonists, Met-enkephalin (Met-enk, 3 µM) and morphine (10 µM), all induce potentiation of ChAT neuronal glutamatergic transmission to similar extents (Figure 3g). In addition, pre-treatment with the mOR selective antagonist (CTAP, 1 µM) completely prevents the DAMGO-induced response (Figure 3g). Together, the pharmacological battery of tests employing an experimental, an endogenous, and clinical/recreational mOR agonists and selective mOR antagonism clearly implicate this opioid receptor in mediating a non-canonical potentiation of AMPAR EPSC amplitude elicited by mHb cholinergic neurons in IPN.

In several recordings from both ‘blind’ patching of IPR neurons and in directly identified SST neurons, no measurable postsynaptic AMPA receptor EPSC could be elicited following light-evoked stimulation of cholinergic axons even with the maximal possible light intensity available (i.e. 100% arbitrary LED power; approximately 6.9 mW/mm²), and thus further interrogation was typically not performed. However, in a few instances, we nevertheless proceeded with DAMGO or Met-enk application, and surprisingly, in a subset of these recordings (9/15 cells tested), a robust and reversible emergence of a significant AMPAR-mediated EPSC was observed (Figure 3h, i). Thus, these data clearly reveal that mOR activation invokes a synaptic mechanism that, at its most extreme, results in an ‘un-silencing’ of ChAT neuronal glutamatergic transmission in IPN including in directly identified SST GABAergic neurons. Since PPR measurements are only valid if the same population of release sites are assayed before and after experimental manipulation, the additional engagement of a putative reluctant vesicular pool (Koppensteiner et al., 2024) by mOR activation could explain the discrepancy between the augmentation of EPSC amplitude and observed changes in PPR (Figure 3b, c, e, f). Future interrogation is warranted for definitive identification of the relative contributions of pre- and postsynaptic loci to the potentiation observed (but see Singhal et al., 2025).

To date, we have examined the effect of mOR on synaptic transmission during paired pulse light activation delivered at 20 Hz. Although mHb cholinergic neurons are capable of burst firing at much higher frequencies in response to afferent stimulation in vivo (Otsu et al., 2018), they typically exhibit low frequency (i.e. <10 Hz) intrinsic (i.e. independent of synaptic input) firing in vitro (Chung et al., 2023; Arvin et al., 2019; Görlich et al., 2013; Cho et al., 2020). In agreement with these previous studies, cholinergic neurons in ventral mHb (identified in ChatCre:Ai9 mice; Figure 1c) spontaneously elicit action potentials measured in cell-attached recordings at an average of ~5 Hz (range 2–10 Hz; Figure 4a). Therefore, we assessed the role of mOR activation on glutamatergic transmission mediated by cholinergic neurons elicited by light stimulation trains delivered at this frequency. Interestingly, even with this relatively low frequency paradigm, glutamatergic transmission is extremely labile in nature as evidenced by the large and rapid depression of AMPAR EPSC during the stimulus train (Figure 4b, c). Remarkably, activation of mORs essentially eliminates activity-dependent depression at this synapse (Figure 4b, c). This switch in transmission dynamics greatly facilitates the probability of excitatory postsynaptic potential-spike coupling (ES coupling) in response to each stimulus within the train (Figure 4d, e). Thus, these data clearly demonstrate that at physiologically relevant stimulus patterns, mORs serve to dramatically increase the fidelity of glutamatergic transmission in the IPR mediated by cholinergic neurons culminating in an enhanced recruitment of postsynaptic IPR neurons.

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mORs constitute a molecular switch to alter the salience of glutamatergic transmission mediated by cholinergic and substance P neurons in the IPR

An intriguing inconsistency was observed during the conduction of our experiments regarding the DAMGO effect on light-evoked and spontaneous EPSCs (sEPSCs) impinging on IPR neurons. Under the experimental conditions employed, sEPSC events were exclusively mediated by AMPARs as evidenced by their virtually complete cessation upon DNQX application (Figure 5—figure supplement 1a). Remarkably, in contrast to the previously described robust potentiation of light-evoked AMPAR EPSCs, activation of mORs significantly attenuates sEPSC frequency in agreement with a recent study (Singhal et al., 2025), with no effect on amplitude (Figure 5—figure supplement 1b–d). Thus, in a single IPR postsynaptic neuron, opposing effects on light-evoked and spontaneous AMPAR EPSCs are evident (Figure 5—figure supplement 1e). Although these data do not directly identify the origin and neuronal subtype(s) responsible for the sEPSCs measured, the incongruent effect of mOR activation on evoked versus spontaneous events led us to consider the possible existence of an additional afferent system impinging on IPR neurons. Prevailing circuit schemas based on axonal arborization patterns portray a mutually exclusive, non-overlapping afferent input to IPN mediated by cholinergic and SP neurons to IPR/IPC versus IPL, respectively. However, in the current study by employing high-resolution imaging in Tac1Cre:Ai32 mice, a clear presence of synaptic bouton-like structures in the IPR is noted (Figure 5a, b). These structures do not co-localize with endogenous ChAT, but do express the glutamate vesicular transporter, VGluT1 (Figure 5a, b). This presence of putative presynaptic anatomical substrates points to the existence of a secondary input to IPR mediated by SP neurons distinct to the well-established one originating from mHb cholinergic neurons. Indeed, significant light-evoked AMPAR EPSCs in IPR neurons can be elicited in Tac1Cre:Ai32 mice with essentially similar amplitudes across all LED powers employed to that seen in ChatCre:Ai32 mice (Figure 5c). Thus, in contrast to recent ultrastructural EM analyses (Koppensteiner et al., 2024), these data indicate a previously unidentified functional glutamatergic input to IPR mediated by SP neurons. Interestingly, DAMGO application significantly depresses the SP neuronal glutamatergic output in IPR and increases PPR (Figure 5d–f) to a similar extent to that observed in IPL (Figure 2). This is in direct contrast to the role of mORs in positively modulating the ChAT neuronal glutamatergic transmission in the IPR (Figure 3a, b, d, e). Thus, for the first time, we demonstrate both SP and cholinergic input to the same subregion of IPN and that mOR activation elicits diametrically opposing effects on transmission mediated by these respective presynaptic neuronal populations.

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Dynamic regulation of the mOR elicited afferent specific switch in salience of the glutamatergic transmission to the IPR during adolescence

Our functional studies thus far have been restricted to sexually mature, adult mice (>p40). mOR expression emerges in many brain structures during prenatal development largely overlapping with their final adult distribution profile by mid/late gestation (Zhu et al., 1998). In agreement, we reveal that mOR protein expression in IPR is present at appreciable levels from early postnatal stages (i.e. from P10 onwards; Figure 6a). This begs the question of whether modulation of habenulo-interpeduncular synaptic transmission described during these critical early epochs mirrors that seen in the adult. To examine this, we extended our analyses to earlier time points (P15–P40) and found that mOR modulation of glutamatergic transmission mediated by cholinergic neurons in the IPR undergoes a remarkable developmental regulation. This is characterized by an initial inhibitory effect on light-evoked AMPAR EPSCs transitioning to the previously described augmentation (Figure 6b, c, f). In contrast, the glutamatergic input to the IPL and the newly discovered input to the IPR elicited by SP neurons is not developmentally regulated with synaptic transmission being consistently inhibited by DAMGO application at all ages tested (Figure 6d–f). Thus, despite essentially similar levels of IPR mOR expression at the ages assayed (Figure 6a), at earlier development stages, opioids result in overall inhibition of afferent input to the IPR agnostic to afferent input with the emergence of the previously highlighted differential regulation of mHb cholinergic versus substance P neuronal output emerging during the late adolescent stages (Figure 6f).

Figure 6

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mOR-induced potentiation of nicotinic receptor signaling in IPR is conditional on the removal of a molecular brake mediated by Kv1 channel function

In addition to mORs, this circuitry contains dense expression of nicotinic receptors located at pre- and postsynaptic sites (Chung et al., 2023; McGehee et al., 1995; Shih et al., 2014). However, the ability to reliably evoke postsynaptic nicotinic receptor (nAChR) EPSCs in response to physiologically appropriate stimuli has been challenging. Indeed, many studies have resorted to high frequency and prolonged stimulation (Ren et al., 2011) or bath/puff applications of nicotine to investigate nAChR function in this circuit (Zhao-Shea et al., 2013; Hsu et al., 2013; Arvin et al., 2019; Wei et al., 2022; Elayouby et al., 2021; Mondoloni et al., 2023). Despite the faithful co-expression of endogenous ChAT in ChatCre:Ai32 expressing terminals located in the IPR (Figure 7a), evoked transmission by brief light pulses (1–5 ms) from cholinergic neurons predominantly elicits pure AMPAR EPSC in postsynaptic IPR neurons under our basal conditions as described above and in agreement with previous observations (Souter et al., 2022; Ren et al., 2011; Singhal et al., 2025; Hsu et al., 2013) (see Figure 5—figure supplement 1a and Figure 7—figure supplement 1a-e). Additionally, DAMGO, at concentrations that strongly potentiate glutamatergic transmission, does not result in a measurable postsynaptic nAChR EPSC after AMPARs are pharmacologically silenced (Figure 7—figure supplement 1a–c). Furthermore, repeated light stimulation at either 25 or 50 Hz fails to elicit the emergence of any nAChR-mediated EPSCs in the presence of DNQX (Figure 7—figure supplement 1d, e). Finally, the potentiated ESPC following mOR activation is purely AMPAR mediated, as evidenced by complete block by DNQX (Figure 7—figure supplement 1f–h). Together, our functional analyses demonstrate that brief pulses of light (1–5 ms) in ChatCre:Ai32 mice do not result in sufficient ACh release, if any, to elicit measurable postsynaptic nAChR responses in response to high frequency stimulus regimens and even under conditions where mOR activation strongly enhances glutamatergic transmission.

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Interestingly, cholinergic transmission at the neuromuscular junction can be boosted via potassium channel blockade with fampridine (4-aminopyridine) and has been clinically indicated in disorders such as myasthenia gravis and multiple sclerosis (Kostadinova and Danchev, 2019). Furthermore, low micromolar 4-AP in slices amplifies acetylcholine release, as assessed by Grab-ACh-mediated fluorescence in the IPN (Jing et al., 2018; Jing et al., 2020). Experimentally, these concentrations are relatively selective in inhibiting K⁺-channels of the Kv1 family. Probing the publicly available 10X single-cell RNAseq database provided by the Allen Brain Institute reveals that Kcna2 (the gene encoding Kv1.2) is the most prevalently expressed in the designated Chat Supertypes (Figure 1d and Figure 7b; mean expression levels including zero values = 9.1, 6.8, and 4.1 in Supertypes 0632, 0634, and 0635, respectively). Furthermore, spatial interrogation via in situ hybridization (Allen Brain; https://mouse.brain-map.org/gene/show/16263) demonstrates the presence of appreciable Kcna2 transcript levels in ventral habenula (Figure 7c) a region that is enriched in cholinergic neurons (Figure 1b, c). We therefore speculated that Kv1.2 block could result in enhancement of ACh release to perhaps reveal postsynaptic nAChR EPSCs in IPR neurons. Indeed, following the block of glutamatergic transmission (DNQX + APV), subsequent application of 4-AP (50–100 µM) or the more selective Kv1 channel antagonist, dendrotoxin-α (100 nM), results in the emergence of an EPSC elicited by brief single light pulses (Figure 7d) and in response to trains of 5 Hz stimulation (Figure 7e). This emergent EPSC is mediated by nAChRs as evidenced by ~80–90% block by the nicotinic receptor antagonist, DHβE (Mulle et al., 1991; Morton et al., 2018) (2 µM; Figure 7f).

Interestingly, this experimental approach allows for the calculation of nAChR;AMPAR peak amplitude ratio enabling an assessment of fast excitatory cholinergic transmission normalized to stimulus intensity required to produce a given AMPA EPSC response in an individual postsynaptic IPR neuron. Thus, the size of the nAChR response can be directly compared across multiple recordings from differing slices, mice, and experimental conditions. We first assessed the extent to which both 4-AP and DTX-α reveal light-evoked nAChR EPSCs, finding no significant difference in the nAChR:AMPAR ratio, demonstrating that the specific Kv1.1/Kv1.2 channel antagonist unmasks nAChR-mediated synaptic transmission in a similar manner to low concentration 4-AP (Figure 7g). This, in combination with the predominant expression of Kcna2 (Figure 7b and c) and relative lack of other Kcna transcripts (Figure 7b) in mHb ChAT neurons, confirms our hypothesis that Kv1.2 plays a central role in unmasking nAChR-mediated synaptic transmission. In addition to ChatCre:Ai32 mice, we also employed the Chat-ChR2 mouse. Not surprisingly, nAChR EPSCs could also be elicited in this mouse line following 4-AP application (Figure 7g). However, the measured nAChR:AMPAR ratio is significantly higher than that seen in ChatCre:Ai32 mice (Figure 7g), indicating a skew toward more prominent cholinergic transmission when compared to that mediated by glutamate. It is unclear as to the exact reason for this divergence, but it may be related to increased levels of the vesicular Ach transporter (vAChT) expression observed in Chat:ChR2 mice (Kolisnyk et al., 2013). Although Tac1 mHb neurons also express Kcna2 transcript, albeit lower than in the ChAT neurons (data not shown), 4-AP application following block of glutamatergic transmission does not result in emergence of any additional EPSC in response to single stimuli in Tac1Cre:Ai32 mice (Figure 7g). Finally, application of a cholinesterase inhibitor (ambenonium, 50 nM) markedly prolongs the EPSC waveform with minimal effect on amplitude (Figure 7h). Thus, together, these results corroborate the role of Kv1.2 as a molecular brake, removal of which results in the emergence of habenulo-interpeduncular synaptic transmission via postsynaptic nAChR signaling on IPR GABAergic neurons.

Having established the ability to reliably assess an otherwise reluctant cholinergic transmission, we next investigated how nAChR-mediated signaling in the IPR is impacted by mOR activation. As with AMPAR-mediated EPSCs, DAMGO produces a marked potentiation of light-evoked nAChR EPSC amplitude in IPR, with the augmented EPSC being completely blocked by DHβE (Figure 8a, b). The mOR-mediated potentiation of both the glutamatergic and cholinergic output is essentially similar (Figure 8i). Till now, we have utilized optogenetic approaches to assay synaptic transmission in the IPN. Interestingly, following acute block of Kv1, nAChR EPSCs could also be elicited via electrical stimulation (Figure 8c). Further, DAMGO reliably potentiated both electrical and light-evoked nAChR EPSCs in a single IPR neuron to similar extents, a response that is effectively blocked by DHβE (Figure 8c–e). Finally, we tested the molecular specificity of the DAMGO response employing Chat-ChR2:global Oprm1 knockout mice. In these mice, DAMGO application is ineffective in augmenting glutamatergic or cholinergic-mediated neurotransmission in IPR, while the potentiation upon GABA_B receptor activation previously described (Koppensteiner et al., 2024; Bhandari et al., 2021; Melani et al., 2019) remains intact (Figure 8f–h; note that in these experiments, CGP55845A, which is routinely included in all other experiments, was omitted). This genetic approach complements our previous pharmacological data (Figure 3g) to directly implicate mORs in the potentiation of mHb-IPN transmission. The overall effects of mOR activation on synaptic transmission mediated by the distinct mHb afferent systems in the various subdivisions of IPN tested are summarized in Figure 8i.

Figure 8

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In the current study, we reveal a remarkable augmentation of glutamatergic/cholinergic co-transmission by mHb cholinergic neurons following mOR activation in the IPN. However, the underlying cellular/network mechanisms responsible remain unclear. The most parsimonious explanation is that mORs in proximity to the release machinery of cholinergic nerve terminals (Gardon et al., 2014) mediate this effect. However, one must consolidate the fact that a Gi-linked receptor, which typically serves to directly inhibit release, results in a seemingly paradoxical potentiation. Functional and mechanistic studies have elegantly demonstrated that GABA_B receptors (another member of the Gi-linked subfamily) trigger acute molecular and structural adaptations resulting in enhanced Ca²⁺ influx and a switch of transmission modes within single presynaptic terminals to ultimately increase neurotransmitter release (Koppensteiner et al., 2024; Zhang et al., 2016). Although these observations set a precedent for potentiation by Gi-linked receptor activation in the IPN, similar experimental approaches to that employed in the examination of the GABA_B-receptor-mediated potentiation (Koppensteiner et al., 2024; Zhang et al., 2016; Jing et al., 2020) are required to definitively implicate overlapping mechanisms. In other brain regions, a canonical inhibitory influence of mORs elicits overall network excitation via disinhibition (Chen et al., 2015; McQuiston and Saggau, 2003; Johnson and North, 1992; Lau et al., 2020). Furthermore, numerous studies have revealed an intricate interplay involving diverse neuromodulatory components intrinsic to the IPN that serve to regulate synaptic transmission (Ables et al., 2017; Melani et al., 2019; Koppensteiner et al., 2017). Thus, taking the results of the current study in isolation, we cannot discount the possibility that the potentiation observed here may result via the activation of mORs residing on other neural elements within the IPN microcircuitry such as postsynaptic neurons or glia, for instance. However, it must be noted that a recent study employing a viral strategy to elicit conditional knockout of mORs in Oprm1-expressing mHb neurons specifically prevents the DAMGO-mediated potentiation of glutamatergic transmission onto the IPN, indicating a presynaptic locus (Singhal et al., 2025). Regardless of the exact underlying cellular and/or network mechanisms, our data extend the previously described Gi-linked receptor potentiation of neurotransmission in the IPN (Koppensteiner et al., 2024; Zhang et al., 2016; Jing et al., 2020) to mORs, a predominant target of a societally relevant and prevalently misused class of drugs. It would be of interest to determine whether GABA_B receptor activation exerts a similar inhibitory influence as mORs on the newly discovered SP neuronal-mediated transmission to the IPR. In addition, does the modulation of ChAT neuronal glutamatergic output by GABA_B receptors undergo a similar developmental regulation to that observed with mORs? These additional functional comparisons of the synaptic influences of these two distinct Gi-linked receptors may shed light as to the similarity, or lack thereof, regarding the respective underlying cellular mechanisms.

Recent generation of transgenic mice (Bailly et al., 2020; Weibel et al., 2013; Mengaziol et al., 2022) has permitted manipulation of mOR-expressing neurons and mOR receptors including those expressed specifically within the mHb/IPN. Emerging behavioral studies adopting such conditional genetic approaches have highlighted an important role of mORs within this circuitry in mediating varying aspects of reward and aversion (Allain et al., 2022; Boulos et al., 2020). Unsurprisingly, it has been concluded that the observed behavioral effects of such manipulations are due to perturbations of a generalized inhibition mediated by mORs within the mHb/IPN circuit. Here, we reveal a functional dichotomy characterized by an inhibitory yet excitatory influence on neurotransmission mediated by SP and cholinergic neurons, respectively. These distinct neuronal populations participate in and drive specific behavioral facets demonstrating a division of labor. For example, SP neuronal activity positively correlates with reward outcome, history, and hedonic value (Sylwestrak et al., 2022; Hsu et al., 2014), whereas that of cholinergic neurons is reduced during behaviors associated with reward (Sylwestrak et al., 2022). mHb cholinergic neurons have also been linked to negative affect including anxiety and depression (Cho et al., 2020; Seigneur et al., 2018; Pang et al., 2016) that promote drug-seeking behaviors during withdrawal (Batalla et al., 2017). We reveal for the first time that the IPR subdivision receives functional glutamatergic afferent input from SP neurons that, based on previous anatomical interrogation, were considered to exclusively target the IPL (Quina et al., 2017). Interestingly, the novel opposing effect on transmission extends to the IPR, thus demonstrating a role for mORs in altering the relative salience of these distinct afferent systems with regard to the recruitment of common downstream IPR neurons. The IPR houses SST GABAergic neurons whose afferents impinge on the raphe nucleus and lateral dorsal tegmental nucleus, the latter influencing the VTA and, in turn, its downstream structures (Ables et al., 2017; Hsu et al., 2013; Monical and McGehee, 2025). Thus, careful consideration regarding future examination of behavioral outcomes resulting from the described contrasting effects of mOR activation on the parallel processing of reward (mHb SP) and anti-reward (mHb ChAT) in this circuit is essential.

A limitation of the current study arises from the sole utilization of a transgenic approach to selectively assay light-evoked synaptic transmission from ChAT and SP neuronal populations, respectively. Although the mHb provides most of the afferent input to the IPN, this approach does not exclude possible activation of additional inputs from other brain regions (Liang et al., 2024; Lima et al., 2017; Bueno et al., 2019). This caveat can be circumvented by use of stereotaxic viral delivery to express ChR2 solely in the mHb (Souter et al., 2022; Singhal et al., 2025; Hsu et al., 2013). However, it is unclear if neuronal inputs from these possible alternate sources (Liang et al., 2024; Lima et al., 2017; Bueno et al., 2019) are glutamatergic in nature and mediated by a Tac1/Oprm1-expressing neuronal population. Nevertheless, to definitively identify the described novel input as one that originates from mHb SP neurons will require the future use of such viral strategies.

Throughout this study, we contextualize the effect of mOR activation in the IPN primarily through the lens of exogenously introduced opioids. However, physiological activation of mORs can be mediated to differing extents by endogenous ligands such as dynorphin or enkephalin. Indeed, we demonstrate that met-enkephalin effectively boosts co-transmission from cholinergic neurons in a similar manner to morphine. Interestingly, the IPN is home to a population of pro-enkephalin (PENK)-expressing neurons and detectable met-enkephalin immunoreactivity (Gardon et al., 2014; Mahalik and Finger, 1986). In other brain regions, manipulation of PENK neuronal activity and/or ablation of PENK itself elicits mOR-mediated circuit modulation and behavioral alterations (You et al., 2023; Castro et al., 2021; Leroy et al., 2022). It is anticipated that activation of mORs by local endogenous opioid release yields similar complex effects to those following exogenous application of mOR agonists described here. Thus, this physiological route will have major implications concerning the role of mHB/IPN mORs in mediating the positive reinforcing effects of non-opioid drugs of abuse (e.g. nicotine, alcohol, amphetamines) and other ‘natural’ rewarding stimuli (e.g. such as those associated with social interaction, exercise, and food intake), thus extending the relevance of our study to these additional modalities.

Another novel finding in the current study relates to the identification of a ‘molecular brake’ that exerts strong control over nAChR signaling impinging on postsynaptic IPN neurons. Specifically, compromising the function of delayed rectifying K⁺-channels containing Kv1.2 subunits results in the emergence of nAChR EPSCs in response to light and electrical stimuli delivered with physiological paradigms likely through increased axonal/bouton excitability and calcium influx via action potential waveform modulation. Kv1.2 function can be bidirectionally impacted by use-dependent mechanisms or through secondary activity-mediated cascades resulting in post-translational modifications (e.g. phosphorylation state) that impact its function and/or trafficking (Baronas et al., 2015; Thayer et al., 2016; Yang et al., 2007). These plausible endogenous mechanistic routes could serve to titer the strength nAChR-mediated transmission in the IPR.

The resident nAChRs in the mHb/IPN are primarily encoded by the CHRNA3/B4/A5 gene cluster and the role of this circuitry in nicotine use is well characterized. For example, increased propensity for nicotine abuse in humans is associated with dysregulation of nAChR function precipitated by single nucleotide polymorphisms of this cluster (Amos et al., 2008; Improgo et al., 2010; Brynildsen and Blendy, 2021). Experimental manipulation of nAChR signaling in the mHb/IPN generates phenotypes associated with various aspects of nicotine consumption in mouse models (Morton et al., 2018; Antolin-Fontes et al., 2015; Frahm et al., 2015; Fowler et al., 2011; Mathis and Kenny, 2019). Furthermore, chronic nicotine results in adaptations in nAChR expression and function that can exacerbate continued nicotine consumption (Arvin et al., 2019; Mondoloni et al., 2023; Govind et al., 2009; Jin et al., 2020). Together, these studies establish a link between nAChR-mediated signaling within this circuitry and prevalence of nicotine misuse. Here we highlight an intriguing interplay between opioid and cholinergic systems within the mHb/IPN axes. Our data clearly demonstrate that mORs and Kv1.2 together comprise synergistic molecular targets that, in addition to others previously identified (Bhandari et al., 2021), when leveraged in tandem can manipulate nAChR-mediated signaling to yield potential interventions for nicotine overuse.

Adolescence is a critical period for many aspects of brain and social development and represents a high-risk demographic group for drug use developing into long-term addiction (Ahmadi-Soleimani et al., 2024). Propelled by the societal introduction of highly potent synthetic morphine analogs (e.g. fentanyl), one of the many devastating sequelae of the well-documented opioid crisis is an alarming high rate of overdose deaths not only in adults but also during vulnerable teenage years. Of note is the striking valence conversion from depression to potentiation of mHb ChAT neuronal signaling in the IPR occurring around the late postnatal stages. This is in stark contrast to that seen with the newly described SP neuronal-mediated transmission in this same IPN subdivision where mOR activation is inhibitory at all life stages assayed. Thus, during postnatal development, opioids acting via mORs result in a blanket inhibition of these two distinct inputs prior to the establishment of the afferent specific modulation in the adult. Interestingly, numerous studies have highlighted changes in the role of mORs in reward-associated behaviors coinciding with similar periods (Harda et al., 2023; King’uyu et al., 2024). An attractive hypothesis is that coordinated modifications at the cellular, molecular, and/or network level underlie this dynamic developmental regulation. Thus, our circuit analyses provide insights into the neural correlates for the differing propensity of mORs to regulate not only positive reinforcement but also the negative effects associated with withdrawal at various stages of development (Wang, 2019). Whether these adaptations underlie the increased propensity for substance abuse during development remains to be ascertained. Furthermore, opioid exposure during early developmental epochs imparts long-lasting effects on the brain's reward circuitry (Salmanzadeh et al., 2020). Thus, together, it is imperative to consider the newly described developmentally regulated impact of mORs in the modulation of the mHb/IPN circuitry. This is of particular relevance with respect to the generation of potential preventative treatments for the deleterious consequences of fetal opioid exposure and SUDs in high-risk juveniles.

In addition to a central role in addiction, the mHb/IPN circuitry encodes fear-associated behaviors (Koppensteiner et al., 2024; Zhang et al., 2016; Fernández-Suárez et al., 2021; Soria-Gómez et al., 2015; Roy and Parhar, 2022). Interestingly, prevention of GABA_B-receptor function in mHb cholinergic neurons or increasing IPN neuronal activity facilitates and augments expression of fear extinction, respectively (Koppensteiner et al., 2024; Zhang et al., 2016). Dysregulation of this behavioral aspect is a key underlying cause of post-traumatic stress disorder (PTSD) (Careaga et al., 2016). Based on this previous work, the novel mOR-mediated potentiation of the mHb cholinergic transmission and hence IPN neuronal recruitment described here constitutes a parallel, yet alternative circuit mechanism involved in the extinction of learned fear. Thus, pharmacogenetic manipulation of mOR-mediated signaling specifically in the IPN may constitute a viable approach to alleviate conditions associated with abnormal fear processing such as those seen in PTSD.

In summary, we highlight several previously undescribed and hence unappreciated roles of mORs in the dynamic regulation of synaptic transmission impinging on IPN GABAergic neurons following exposure to exogenous opioids. Although additional studies are needed to reveal the underlying cellular mechanisms responsible for these complex, divergent facets of opioid-mediated effects in the habenulo-interpeduncular axis, the current detailed functional analyses lay out a valuable roadmap. One that necessitates consideration in future interrogation concerning the role of these receptors in this relatively understudied brain circuitry that encodes aspects of emotion, hedonic, and addiction behaviors.

All analyses of data associated with the manuscript have been made available: https://data.mendeley.com/datasets/3dtkmss9xz/2. Adapted Python notebooks employed for analyses and graphical representation of publicly available Allen Brain Cell Atlas mouse whole brain scRNA sequencing data can be accessed via the following link: https://github.com/acaccavano/ABC-Atlas_Chittajallu2025/ (copy archived at Caccavano, 2025).

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Author details

1. Ramesh Chittajallu

   Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   ramesh.chittajallu@nih.gov

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9794-0052

2. Anna Vlachos

   Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4411-9447

3. Adam P Caccavano

   Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Data curation, Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6819-533X

4. Xiaoqing Yuan

   Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Steven Hunt

   Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Resources, Investigation

   Competing interests

   No competing interests declared

6. Daniel Abebe

   Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

7. Edra London

   Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Kenneth A Pelkey

   Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Conceptualization, Supervision

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9731-1336

9. Chris J McBain

   Section on Cellular and Synaptic Physiology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5909-0157

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  citation for Reviewed Preprint v1 https://doi.org/10.7554/eLife.106062.1