# Nicotinic Receptors in the Medial Habenula to Interpeduncular Nucleus Pathway: Modulators of Reward, Aversion and Emotion

Maria Ciscato, Mathilde Chouvaeff, Alexandre Mourot

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Maria Ciscato, Mathilde Chouvaeff, Alexandre Mourot. Nicotinic Receptors in the Medial Habenula to Interpeduncular Nucleus Pathway: Modulators of Reward, Aversion and Emotion. European Journal of Neuroscience, 2025, 62 (11), pp.e70352. 10.1111/ejn.70352. hal-05516815

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1 Nicotinic receptors in the medial habenula to interpeduncular nucleus pathway: modulators

2 of reward, aversion and emotion

3

4

5 Running title: nicotinic modulation of the MHb-IPN axis

6

7 Maria Ciscato¹*, Mathilde Chouvaeff¹* and Alexandre Mourot¹*

8

9 1. Brain Plasticity Unit, CNRS, ESPCI Paris, PSL Research University, 75005 Paris, France

10 * These authors contributed equally

11

12 Corresponding author: alexandre.mourot@espci.fr

13

14 Total number of pages: 53

15 Total number of figures: 4

16 Total number of tables: 1

17 Total number of words in the whole manuscript: 15503

18 Total number of words in the whole abstract: 144

19

20 Keywords: nicotine addiction; acetylcholine; anxiety; cholinergic synapse; avoidance

21 behavior; laterodorsal tegmentum

22
23 **Abstract:**

The medial habenula - interpeduncular nucleus pathway is a highly conserved and densely
innervated brain circuit known for its unique cholinergic transmission and exceptional
expression of nicotinic acetylcholine receptors. This pathway plays a critical role in regulating
motivational and emotional processes, particularly those related to nicotine consumption,
avoidance behaviors, and negative emotional states. Recent advances have revealed the
intricate cellular architecture and receptor diversity of this system, highlighting how specific
subunits of acetylcholine receptors influence both the rewarding and aversive properties of
nicotine. Genetic and functional studies in rodents and humans point to this pathway as a key
regulator of nicotine intake, with potential implications for addiction treatment. In this review,
we examine the organization and molecular composition of nicotinic receptors within this
pathway, describe their functional and behavioral roles, and explore how cholinergic signaling
contributes to nicotine dependence, stress responses, and affective states.
## 1. Introduction

The medial habenula (MHb) and its primary downstream target, the interpeduncular nucleus (IPN), form a phylogenetically conserved pathway that plays a central role in regulating affective and motivational states (Viswanath et al., 2013; McLaughlin et al., 2017; Molas, DeGroot, et al., 2017; Ables et al., 2023). Situated between limbic forebrain regions and midbrain neuromodulatory centers, the MHb-IPN axis is uniquely positioned to integrate emotional and contextual inputs and shape downstream neuromodulatory tone. Accordingly, this pathway has been implicated in a wide array of behaviors, including aversion, reward processing, stress responses, social interactions and nicotine dependence (Fowler & Kenny, 2014; Velasquez et al., 2014; Antolin-Fontes et al., 2015; Molas, DeGroot, et al., 2017; Ables et al., 2023).

The MHb-IPN circuit stands out in the central nervous system (CNS) for its exceptional density and diversity of nicotinic acetylcholine receptors (nAChRs) (Zoli et al., 2015). These pentameric ligand-gated ion channels are critical for synaptic transmission and modulation within this pathway. Among the predominant subtypes expressed are heteromeric α3β4-containing receptors, often in combination with accessory subunits such as α5. Strikingly, the genes encoding these subunits—*CHRNA5*, *CHRNA3*, and *CHRNB4*—are part of a gene cluster in the chromosome 15q25.1 region, a locus strongly associated with genetic risk for tobacco dependence and smoking-related pathologies such as lung cancer (Mathis & Kenny, 2019; Wills et al., 2022). Functionally, nicotinic modulation appears to play a key role in regulating the activity and output of the MHb-IPN pathway, although the precise mechanisms and behavioral implications of this influence remain to be fully elucidated.
In this review, we focus on the nicotinic regulation of the MHb-IPN pathway. We will detail the molecular composition and organization of nAChRs in this circuit, explore mechanisms by which cholinergic and nicotinic signaling influence MHb-IPN function, and examine the behavioral and physiological consequences of such modulation. We will discuss emerging evidence linking this pathway to both the rewarding and aversive effects of nicotine, its broader roles in stress and emotional regulation, and its relevance to psychiatric and substance use disorders. Finally, we will highlight recent findings, including novel functional links to the ventral tegmental area (VTA), and identify key gaps in knowledge that remain to be addressed.

## 2. Anatomy of the MHb-IPN Pathway

The habenula is a highly conserved epithalamic structure of the diencephalon located adjacent to the third ventricle which, in mammals, is subdivided into the medial habenula (MHb) and lateral habenula (LHb) (Figure 1A) (Herkenham & Nauta, 1977; Aizawa et al., 2012; Quina et al., 2017). The LHb has been extensively reviewed elsewhere recently (Mondoloni et al., 2022; Ables et al., 2023) and will not be covered here.

The MHb is subdivided into two dorsal (dMHb) and three ventral (vMHb) subnuclei (Figure 1A) (Aizawa et al., 2012; Wagner et al., 2014). Glutamatergic neurons are ubiquitous throughout the MHb, but the dMHb predominantly contains substance P-expressing neurons, whereas the vMHb is composed of cholinergic neurons (Ren et al., 2011; Aizawa et al., 2012; Hsu et al., 2014; Frahm et al., 2015). The posterior septum represents the main source of excitatory drive to the MHb (Herkenham & Nauta, 1977; Qin & Luo, 2009; Aizawa et al., 2012; Yamaguchi et al., 2013; Otsu et al., 2018) and also sends GABAergic projections (Figure 1B) (Herkenham &
Nauta, 1977; Qin & Luo, 2009; Vickstrom et al., 2021). However, because of the atypical chloride gradient in MHb neurons, GABAergic transmission is functionally excitatory in this region (Kim & Chung, 2007; Choi et al., 2016). Beyond septal inputs, the MHb receives more modest projections from subcortical regions, including the VTA, the locus coeruleus, and the raphe nuclei (Antolin-Fontes et al., 2015; McLaughlin et al., 2017).

The interpeduncular nucleus (IPN) is an unpaired midline structure located in the ventral midbrain and subdivided into seven nuclei (Figure 1A) (Herkenham & Nauta, 1977; Lenn & Hamill, 1984; Antolin-Fontes et al., 2015). It consists predominantly of GABAergic projection neurons and interneurons, with relatively sparse glutamatergic neurons that are localized mainly in the rostral (IPR), lateral (IPL) and caudal (IPC) parts of the IPN (Hsu et al., 2013; Lima et al., 2017; Quina et al., 2017). The IPN receives inputs from pontine nuclei including the median (MnR) and dorsal (DR) raphe, the laterodorsal tegmental (LDTg) and the dorsal tegmental (DTg) nuclei (Lima et al., 2017; Quina et al., 2017) (Figure 1B). Additional inputs arise from several forebrain and midbrain regions, such as the septum, the medial preoptic area, the ventral thalamus, the central and ventral hypothalamus, the central gray, the periaqueductal gray and the locus coeruleus (Lima et al., 2017; Quina et al., 2017). While several studies have reported functional dopaminergic inputs from the VTA (Zhao-Shea et al., 2015; Molas, Zhao-Shea, et al., 2017; DeGroot et al., 2020; Molas et al., 2022), the existence of a direct anatomical projection remains debated (Nasirova et al., 2021). Efferently, the IPN projects to two major neuromodulatory systems, the serotonergic raphe nucleus (MnR and DR) and the pontine cholinergic system via the LDTg (Figure 1B) (Hsu et al., 2013; Lima et al., 2017; Quina et al., 2017), and exerts inhibitory control over the nucleus incertus (NI), the central gray, and the medial part of the LHb (Lima et al., 2017; Quina et al., 2017).
105 The MHb is exceptional within the CNS in that it projects almost exclusively to a single target—
106 the IPN—via the fasciculus retroflexus (FR), a phylogenetically conserved fiber bundle
107 (Herkenham & Nauta, 1977). These projections follow a highly precise topographical and
108 neurochemical organization (Figure 1A): cholinergic neurons from the vMHb project
109 bilaterally, with their axons crossing the midline multiple times at both the IPR and IPC levels
110 (Quina et al., 2017), while substance P-expressing neurons from the dMHb project exclusively
111 ipsilaterally and terminate primarily in the IPL (Molas, DeGroot, et al., 2017).
112

112

113 **3. The MHb-IPN Pathway Expresses a high Density and Diversity of Nicotinic Receptors**

114 Neuronal nAChRs are ligand-gated cation channels of the Cys-loop receptor superfamily that
115 are activated by the endogenous neurotransmitter ACh, as well as by nicotine, the addictive
116 alkaloid found in tobacco (Taly et al., 2009; Nemecz et al., 2016; Gharpure et al., 2020). In the
117 rodent CNS, 11 nAChR genes encode for eight α subunits (α2-α7, α9, α10) and three β
118 subunits (β2-β4) (Gotti et al., 2006). These subunits assemble into pentameric structures
119 surrounding a central pore that is permeable to sodium, potassium, and calcium ions (Figure
120 2A). Upon binding of agonists, the receptor undergoes a conformational change that opens
121 the ion channel, allowing cation influx (Nemecz et al., 2016). Prolonged exposure to agonists
122 leads to a transition into a high-affinity, non-conducting desensitized state (Taly et al., 2009;
123 Nemecz et al., 2016).

124 Typically, α and β subunits co-assemble to form heteromeric receptors, although some α
125 subunits (e.g., α7 and α9) can form functional homopentamers (Taly *et al.*, 2009). This
126 combinatorial flexibility gives rise to a wide diversity of receptor subtypes (Figure 2B). Some
127 combinations (e.g., α4β2, α4α6β2, α3β4, α3β4β3, α3β4α5...) seem to preferentially occur in
neurons, but the molecular determinants of preferential subunit assembly remain poorly understood (Gotti et al., 2006; Zoli et al., 2015). Subunit composition governs key pharmacological and biophysical features such as affinities for agonists, desensitization rates and ion permeability (Fucile, 2004; Gotti et al., 2006; Taly et al., 2009; Zoli et al., 2015). Affinity for ACh and nicotine is primarily governed by the α subunit (α4 > α3 > α7), and to a lesser extent by the β subunit (β2 > β4) (Gotti et al., 2006). Stoichiometry also matters: heteromers with three β subunits generally display higher agonist affinity than those with three α subunits (Moroni, 2006). Desensitization kinetics differ across subtypes: β4* nAChRs desensitize less and recover faster than β2* receptors, while α7 nAChRs desensitize within milliseconds (Quick & Lester, 2002; Gotti et al., 2009). Ion permeability depends on pore-lining residues of all subunits: Homomeric α7 nAChRs exhibit exceptionally high calcium permeability, comparable to NMDA receptors (Seguela et al., 1993; Fucile, 2004), whereas heteromeric α4β2 and α3β4 receptors have moderate calcium permeability, enhanced in receptors containing three alpha subunits (Tapia et al., 2007) or upon incorporation of α5 (Kuryatov et al., 2010).

The MHb-IPN pathway is unique not only in its expression of a large variety of nAChR subunits, and notably rare ones such as α3 and β4 that are scarcely found elsewhere in the brain, but also in exhibiting one of the highest densities of nAChRs in the CNS (Gotti et al., 2009; Zoli et al., 2015). Table 1 and Figure 2C summarize current knowledge on the expression of nAChR subunits within the MHb and IPN. In the MHb, highest nAChR expression is found in the vMHb and in the FR, with the α3, β3 and β4 being the most enriched subunits (Grady et al., 2009; Shih et al., 2014; Elayouby et al., 2021). The α7 subunit does not seem to be expressed in the MHb (Grady et al., 2009; Ables et al., 2017; Correa et al., 2019), but is present at low levels in a subset of GABAergic cells of the IPL (Jin & Drenan, 2022). In the IPN, the α3 and β4 subunits
151 are highly expressed, followed by α4, α5, β2 and β3. Consistent with their role in mediating 152 cholinergic signaling, nAChRs are highly enriched in the IPR and IPC, which receive dense 153 cholinergic innervation from the vMHb, and are expressed at lower levels in the lateral 154 subnuclei (IPL, IPDL), where cholinergic input is sparse.

155 The MHb and IPN display substantial cellular heterogeneity, with emerging transcriptomic and 156 proteomic studies beginning to map nAChR expression to distinct cell populations (Hashikawa 157 et al., 2020; Wallace et al., 2020; García-Guillén et al., 2021; Sylwestrak et al., 2022). For 158 instance, *CHRNA3* is often co-expressed with G protein-coupled receptor 151 (GPR151) and 159 *CHRNB4* with choline transporter 1 (CHT1) in cholinergic MHb neurons, while *CHRNA5* is 160 enriched in subsets co-expressing somatostatin (SST) in the IPN, although precise cell-type 161 resolution of nAChR subunit distribution remains incomplete (Ables et al., 2017, 2023; Duncan 162 et al., 2019; Caligiuri et al., 2022). Further research is needed to fully delineate these 163 relationships and their functional significance.

164

165 **4. Nicotinic Transmission along the MHb-IPN Pathway**

166 A. <u>The source of ACh in the MHb remains elusive</u>

167 Functional nAChRs —mostly α3β4*— are densely expressed in MHb cholinergic cells (Grady 168 et al., 2009), within both somatic and dendritic compartments (Görlich et al., 2013; Shih et al., 169 2014; Banala et al., 2018; Morton et al., 2018; Passlick et al., 2018; Arvin et al., 2019; Elayouby 170 et al., 2021; Jehl et al., 2025), strongly suggesting these neurons receive cholinergic 171 innervation releasing ACh within the nucleus. Interestingly, MHb neuronal activity follows 172 circadian rhythms, showing higher spontaneous activity during the light phase (Zhao & Rusak, 173 2005), further hinting that ACh signaling might modulate MHb activity.
Despite this, the origin of cholinergic input to the MHb remains unknown, as tracing studies have failed to identify external cholinergic afferents (Chung et al., 2023). Septal inputs to the MHb were proposed to be a source of cholinergic innervation, yet these projections appear to act predominantly through muscarinic and not nicotinic receptors (Mu et al., 2022). A recent study showed that optogenetic stimulation of cholinergic neurons in the MHb increases the firing rate of neighboring, Channelrhodopsin2-negative cholinergic neurons, an effect reduced in presence of nAChR antagonists, suggesting a feedforward mechanism by which ACh facilitates local excitation through nAChRs (Chung et al., 2023).

Moreover, functional α3β4 nAChRs have been detected along the FR (Passlick et al., 2018; Tsuzuki et al., 2024), raising additional questions regarding the source of ACh that activates these receptors and their roles in these axons.

## B. The MHb-IPN cholinergic synapse

It has long been known that ACh and nicotine potently activate IPN neurons and modulate presynaptic release of both glutamate and GABA, indicating that nAChRs are expressed both on postsynaptic IPN neurons as well as on MHb terminals and other afferents (Mulle, Choquet, et al., 1991; Mulle, Vidal, et al., 1991; Léna et al., 1993; McGehee et al., 1995; Girod et al., 2000; Zhao-Shea et al., 2013; Arvin et al., 2019; Mondoloni et al., 2023). vMHb cholinergic terminals coexpress vesicular transporters for both ACh and glutamate and corelease the two neurotransmitters (Figure 3) (Ren et al., 2011; Frahm et al., 2015). Two modes of neurotransmission have been proposed: fast, point-to-point synaptic signaling for glutamate, and slower, diffuse (volume) transmission for ACh. Indeed, brief optogenetic stimulation of MHb afferents evokes rapid AMPA receptor-mediated responses, while sustained, high-frequency stimulation is usually required to trigger slow inward currents mediated by nAChRs
197 (Ren et al., 2011; Chittajallu et al., 2025). Given that ACh and glutamate are released from the
198 same synaptic vesicles (Frahm et al., 2015), the slower time course of nicotinic currents
199 suggests that nAChRs are predominantly extrasynaptic (Figure 3). However, other studies
200 indicate that ACh can also mediate fast, time-locked nicotinic post-synaptic currents in IPN
201 neurons (Soria-Gómez et al., 2015; Zhang et al., 2016), consistent with classical synaptic
202 transmission. This apparent discrepancy may reflect the engagement of distinct IPN neuronal
203 subtypes, or the recruitment of other signaling molecules (see section 4.C), highlighting the
204 need for cell-type-resolved investigations of cholinergic transmission within this pathway.

205 Presynaptic nAChRs can mediate cross-transmitter modulation (Picciotto et al., 2012), as ACh
206 is not only co-released with glutamate but also enhances the release of both
207 neurotransmitters (Girod et al., 2000; Girod & Role, 2001; Frahm et al., 2015). Studies on IPN
208 synaptosomes suggest that only the α3β4* and α3β3β4* nAChRs mediate ACh release (Grady
209 et al., 2009), consistent with their enrichment in MHb axon terminals (Tsuzuki et al., 2024).
210 Given their slow desensitization and sustained activation, α3β4* nAChRs may act as
211 autoreceptors engaging a positive feedback loop that amplifies MHb activity.

212 The MHb-IPN synapse exhibits a distinctive architecture that may restrict ACh diffusion via
213 specialized IPN dendrites (Parajuli et al., 2020). At the ultrastructural level, IPN dendrites form
214 U-shaped crests that tightly wrap around MHb axons without glial separation, forming paired,
215 asymmetric en passant synapses on opposite sides of the same dendrite (Lenn & Hamill, 1984;
216 Parajuli et al., 2020). A single MHb axon can form multiple contacts, creating a repetitive,
217 localized excitatory pattern. This morphology may enhance synaptic efficacy by expanding
218 contact area and locally concentrating neurotransmitters within crest compartments.

219 C. Modulation and plasticity of the MHb-IPN cholinergic synapse
220 Cholinergic transmission at the MHb-IPN synapse is dynamically tuned by several pre- and
221 post-synaptic modulators. For instance, blocking cannabinoid CB1 receptors in the vMHb
222 increases ACh —but not glutamate— release in the medial IPN, indicating that
223 endocannabinoids selectively inhibit cholinergic signaling (Figure 3) (Soria-Gómez *et al.*, 2015).

224 Similarly, increased nitric oxide or SST levels suppress excitatory responses in the IPN following
225 MHb terminal stimulation, suggesting strong presynaptic inhibition of ACh/glutamate release
226 via SST receptors and cGMP-dependent mechanisms (Ables *et al.*, 2017).

227 Paradoxically, some classically inhibitory receptors promote excitation at this synapse.
228 Activation of GABA<sub>B</sub> receptors, which are typically Gi-coupled and inhibitory, actually
229 potentiates MHb-IPN transmission by increasing presynaptic Ca<sup>2+</sup> influx through R-type
230 voltage-gated calcium channels (Cav2.3) and AMPA receptors (Zhang *et al.*, 2016;
231 Koppensteiner *et al.*, 2024). This leads to enhanced co-release of glutamate, ACh, and
232 neurokinin B (Zhang *et al.*, 2016), suggesting that GABA may act as a retrograde messenger to
233 amplify excitatory drive. Notably, Cav2.3 channels localize presynaptically in MHb terminals
234 (Figure 3), a rare feature compared to their typical postsynaptic localization elsewhere
235 (Parajuli *et al.*, 2020).

236 A similar paradox is observed with mu opioid receptors (MORs). Although MORs canonically
237 inhibit neuronal activity, their activation at presynaptic cholinergic MHb terminals enhances
238 synaptic output (Figure 3), particularly within the IPR (Chittajallu et al., 2025; Singhal et al.,
239 2025). This is especially noteworthy given the abundant MOR expression in the MHb, one of
240 the highest in the brain (Gardon et al., 2014). Importantly, nicotinic transmission becomes
241 unmasked only when Kv1.2 potassium channels are inhibited, suggesting that voltage-gated
242 K<sup>+</sup> channels normally suppress nAChR-mediated responses under baseline conditions
243 (Chittajallu et al., 2025). However, the physiological regulation of Kv1.2 activity remains
244 unresolved.

245 Together, these mechanisms may underlie activity-dependent plasticity at MHb-IPN
246 synapses. High-frequency stimulation of IPN neurons induces AMPA receptor-dependent
247 GABA release, which retrogradely activates presynaptic GABA<sub>B</sub> receptors, leading to long-
248 lasting facilitation of glutamate release (Koppensteiner et al., 2017). GABA<sub>B</sub> receptor
249 activation also triggers short-term potentiation by shifting MHb synapses from tonic to phasic
250 release, increasing the pool of readily releasable vesicles (Koppensteiner et al., 2024).
251 Whether long-term potentiation or depression occurs at these synapses —and whether
252 nAChRs contribute to such plasticity— remains to be explored, particularly in the context of
253 nicotine addiction and negative emotional state regulation.

254

255 **5. Molecular and Cellular Effects of Nicotine on the MHb-IPN axis**

256 **A. Acute effects**

257 Nicotine increases the spontaneous firing of MHb neurons in slice preparations (Banala et al.,
258 2018), an effect primarily mediated by α3β4* nAChRs (Görlich et al., 2013; Shih et al., 2014;
259 Elayouby et al., 2021; Jehl et al., 2025) but also involving β2* (Elayouby et al., 2021) and α4*
260 nAChRs (Shih et al., 2014) but not α5* nAChRs (Morton et al., 2018). Activation of α5* nAChRs
261 by nicotine increases excitability indirectly, through the release of substance P and neurokinin
262 B (Dao et al., 2014). Nicotine's effects on MHb firing are regionally specific: rapid, transient
263 firing in vIMHb neurons; slower, sustained firing in vmMHb neurons; and no effect in the
264 vcMHb (Shih et al., 2014). This heterogeneity likely reflects differences in nAChR subunit
265 composition: fast-desensitizing α4 predominates in the vIMHb, whereas α6 subunits are
266 enriched in the vmMHb (Table 1) (Shih et al., 2015). Finally, nicotine also enhances glutamate
267 release at MHb-IPN terminals, a process regulated by presynaptic nAChRs, GPR151, and ACh
268 co-release (Girod & Role, 2001; Frahm et al., 2015; Arvin et al., 2019; Antolin-Fontes et al.,
269 2020). Importantly, all of these findings come from slice recordings, where MHb inputs are
270 severed. In vivo studies with calcium indicators have shown bulk activation of the MHb by
271 nicotine (Caligiuri et al., 2022), while single unit electrophysiology recordings have revealed
272 dose-dependent effects: low nicotine doses activate MHb neurons, while higher doses
273 suppress activity, likely through depolarization block mechanisms involving calcium-activated
274 chloride channels (Kawai et al., 2025).

275 In the IPN, neurons within the IPR also exhibit increased activity in response to acute nicotine
276 application in brain slices, an effect mainly mediated by α5* (Morton et al., 2018) and β4*
277 nAChRs (Mondoloni et al., 2023; Jehl et al., 2025). However, in vivo responses are more
278 heterogeneous. Electrophysiological studies have identified two populations of IPN neurons:
279 those activated and those inhibited by nicotine, intermixed within the IPR and IPC without
280 strict regional segregation (Mondoloni et al., 2023; Jehl et al., 2025). The activation response
281 is primarily mediated by β4* nAChRs, while the inhibition response likely involves other nAChR
282 subtypes acting through yet-undefined circuit mechanism (Mondoloni et al., 2023; Jehl et al.,
283 2025).

284

285 **<u>B. Chronic effects</u>**

286 Nicotine intake perturbs endogenous cholinergic signaling due to its longer half-life compared
287 to ACh —approximately two hours in humans and six minutes in mice (Matta et al., 2006). As
288 a result, chronic nicotine promotes prolonged desensitization of nAChRs (Pidoplichko et al.,
289 1997) and induces neuroadaptations that alter network excitability, including changes in
290 nAChR expression levels (i.e. upregulation), subunit stoichiometry and trafficking (Nashmi et al., 2007; Govind et al., 2009; Wills et al., 2022).

292 At the MHb level, chronic nicotine has dual effects: it reduces the acute response to nicotine, 293 likely through nAChR desensitization (Dao et al., 2014), but simultaneously increases 294 spontaneous firing, alters action potential waveforms, and enhances nicotine-evoked 295 glutamate release from MHb axon terminals (Arvin et al., 2019), likely through functional 296 upregulation of nAChRs on the soma (Jin et al., 2020), dendrites (Banala et al., 2018; Jin et al., 297 298 299 300 301 302 303 304 305 306

307 At the IPN level, chronic nicotine induces nAChR upregulation in mice (Zhao-Shea et al., 2013; 308 Arvin et al., 2019) and rats (Tapia et al., 2022), but in a subtype- and cell-specific manner. For 309 example, the α7 subunit is selectively upregulated in SST-negative cells while β3 and β4 310 subunits are upregulated in SST-positive cells (Zhao-Shea et al., 2013). Sex-dependent 311 differences have also been reported in rats: α7 upregulation predominates in males, while α5 312 increases in females (Correa et al., 2019). By contrast, α4* nAChRs are downregulated
following chronic nicotine exposure (Nashmi et al., 2007). Functionally, these changes translate into potentiated nicotine responses in specific IPN neuron populations, particularly within the IPR (Banala et al., 2018; Arvin et al., 2019) and in SST-positive neurons (Zhao-Shea et al., 2013). However, opposite findings have also been reported: prolonged nicotine exposure can lead to downregulation and/or desensitization of β4* nAChRs in IPN neurons, resulting in reduced nicotine responsiveness both in brain slices and in vivo—an effect absent in β4 KO mice (Mondoloni et al., 2023). Discrepancies may stem from the recording context: persistent nicotine exposure in in vivo recordings maintains receptors in a desensitized state, whereas withdrawal conditions occurring in brain slices allow partial resensitization.

322

## 6. Behavioral Effects of Nicotine

A large body of human genetic studies has established a strong link between nicotinic signaling in the MHb-IPN axis and nicotine dependence. Genome-wide association studies have identified allelic variation in the *CHRNA5/A3/B4* gene cluster—encoding the α5/α3/β4 nAChR subunits that are highly enriched in this pathway—as a major risk for tobacco dependence and smoking-related diseases (Berrettini et al., 2008; Bierut et al., 2008). In particular, the α5 D398N polymorphism, which reduces receptor function (Kuryatov et al., 2010), is associated with reduced aversive responses to nicotine (Jensen et al., 2015) and increased likelihood that light smokers progress to heavy smoking (Saccone et al., 2007; Bierut et al., 2008).

Furthermore, allelic variations in Hedgehog-interacting protein (HHIP), an endogenous inhibitor of Hedgehog signaling, are associated with smoking-related lung diseases. HHIP is enriched in MHb neurons where it modulates nAChR function through cholesterol-dependent mechanisms and influences nicotine aversion (Caligiuri et al., 2022).
336 The MHb-IPN axis also appears vulnerable to neurodegenerative effects of high-dose nicotine, 337 which can lead to degeneration of the FR (Carlson et al., 2000, 2001; Ciani et al., 2005), 338 possibly through desensitization-resistant α3β4* nAChRs. This raises the possibility that 339 nicotine levels reached in habitual smokers might damage MHb axons, particularly those 340 involved in nicotine aversion, potentially contributing to the transition toward compulsive 341 tobacco use. 342

342 Finally, acute nicotine elevates blood glucose via a polysynaptic pathway from the MHb to the 343 pancreas, while chronic nicotine increases circulating glucagon and insulin levels— 344 mechanisms that may link cigarette smoking to an elevated risk of type 2 diabetes (Duncan et 345 al., 2019). Together, these findings underscore the broad physiological and behavioral roles 346 of the MHb-IPN pathway in nicotine addiction. In the following sections, we review rodent 347 studies demonstrating that this pathway regulates both the aversive and rewarding properties 348 of nicotine, thereby determining overall intake, and discuss its involvement in nicotine 349 withdrawal and relapse. 350

351 A. Nicotine aversion

352 In both humans and rodents, nicotine intake is thought to be titrated to maximize rewarding 353 effects while avoiding aversive ones (Fowler & Kenny, 2014; Wills et al., 2022). This balance is 354 strongly dose-dependent (Risinger & Oakes, 1995; Matta et al., 2006; Liu et al., 2022): low 355 doses preferentially activate VTA dopaminergic neurons via high-affinity α4β2* nAChRs, often 356 incorporating α6, promoting reinforcement (Tapper et al., 2004; Maskos et al., 2005; Pons et 357 al., 2008; Tolu et al., 2013; Cuttoli et al., 2018; Nguyen et al., 2021). At higher doses, nicotine 358 produces satiety and aversion, leading to reduced intake (Fowler et al., 2011; Frahm et al.,
359 2011; Liu et al., 2022; Mondoloni et al., 2023). While aversion to nicotine can be mediated in 360 part by VTA circuitry (Laviolette et al., 2002; Tolu et al., 2013; Liu et al., 2022; Wills et al., 361 2022), compelling evidence supports a central role for the MHb-IPN pathway, particularly at 362 high doses (Antolin-Fontes et al., 2015; Wills et al., 2022).

363 Initial insights came from studies using *CHRNA5* knockout (α5 KO) mice, which display normal 364 nicotine intake at low doses but consume significantly more at higher doses (Fowler et al. 365 2011; Bagdas et al. 2019). This excessive intake phenotype is reproduced by downregulating 366 α5* nAChRs specifically in the MHb, and reversed by region-specific rescue of α5 expression 367 (Fowler et al., 2011). Likewise, genetically-modified rats carrying the α5 D398N polymorphism, 368 which causes a partial loss of receptor function, exhibit greater nicotine self-administration at 369 high doses (Forget et al., 2018). Wild-type (WT) mice typically regulate their intake, whereas 370 α5 KO mice show a dose-dependent escalation in consumption (Fowler et al., 2011), 371 consistent with a reduction in aversive signaling (Fowler & Kenny, 2014; Grieder et al., 2017). 372 However, α5 is also expressed in the VTA, where its deletion diminishes the rewarding effect 373 of nicotine as well. Specifically, VTA dopaminergic neurons in α5 KO mice fail to respond to 374 low nicotine concentrations, preventing self-administration at those doses (Morel et al., 375 2014). Thus, α5 deletion (or loss of function) has additive effects, dampening both the 376 rewarding and aversive effects of nicotine, which disrupts titration mechanisms and promotes 377 overconsumption.

378 Complementary evidence indicates that loss or reduction of α5, α3, or β4 nAChR subunits 379 increases nicotine intake, while their over-expression intensifies aversion (Figure 4) (Wills et 380 al., 2022). For example, TABAC mice, which overexpress β4 at endogenous sites, exhibit 381 reduced intake and conditioned place aversion (Frahm et al., 2011; Husson et al., 2020).
Similarly, local overexpression of β4 or β4 gain-of-function variants in the MHb induces aversion (Ślimak et al., 2014). Conversely, antagonizing IPN α3β4* nAChRs with 18-Methoxycoronaridine (18-MC) increases nicotine self-administration (Glick et al., 2011). *CHRNB4* knock-out (β4 KO) mice show greater nicotine intake, particularly at high doses (Husson et al., 2020; Mondoloni et al., 2023), though some studies report divergent effects (Harrington et al., 2016). Local re-expression of β4 in either the MHb or IPN restores normal intake (Husson et al., 2020; Mondoloni et al., 2023). Likewise, local knockdown of α3, or pharmacological blockade of IPN α3β4* nAChRs, increases nicotine self-administration (Elayouby et al., 2021).

Nicotine consumption varies markedly among individuals and is influenced by prior exposure (Fowler & Kenny, 2014; Wills et al., 2022). Similar variability is observed in isogenic WT mice: roughly half avoid high nicotine concentrations, while others persist in consuming “aversive” doses (Mondoloni et al. 2023). Notably, nicotine intake correlates inversely with IPN neuron responsivity: animals with larger nicotine-evoked responses in the IPN show stronger aversion, while those with blunted responses consume more. Accordingly, β4 KO mice exhibit drastically reduced IPN responses and universally lack aversion (Mondoloni et al., 2023).

More broadly, increased IPN activity drives aversion (Figure 4): optogenetic activation of IPN neurons induces place avoidance (Wolfman et al., 2018), and stimulation of α5-expressing IPN neurons projecting to the raphe and tegmentum produces aversion, yet only after prior nicotine exposure (Morton et al., 2018), suggesting that exposure to nicotine experience triggers rapid adaptations in this pathway. Similarly, optogenetic activation of nucleus tractus solitarius neurons in the brainstem, which release glucagon-like peptide-1 (GLP-1) onto IPN neurons, increases excitatory currents and elicits avoidance (Tuesta et al., 2017). Given that
405 GLP-1 neurons regulate satiety and inhibit food consumption (Bloemendaal et al., 2014), and
406 GLP-1 receptors are enriched in α5-positive IPN neurons, nicotine may engage these circuits
407 to evoke satiety-like signals that limit nicotine intake (Tuesta et al., 2017).

408 Conversely, inhibiting the IPN decreases aversion to the drug and promotes intake (Figure 4).
409 α5 KO mice show attenuated IPN responses to nicotine (Morton et al., 2018), consistent with
410 reduced aversive behavior (Fowler et al., 2011; Grieder et al., 2017). Local infusion of lidocaine
411 (Fowler et al., 2011), or genetic deletion of the GPR151 receptor, which decreases MHb to IPN
412 transmission (Antolin-Fontes et al., 2020), raises the threshold for aversion and increases
413 intake at high doses. On a circuit level, the LDTg has emerged as a downstream target:
414 optogenetic silencing of IPN terminals in the LDTg blocks nicotine-induced place aversion
415 (Wolfman et al., 2018). Finally, chronic treatment with nicotine decreases IPN sensitivity to
416 the drug, producing tolerance to its aversive effects (Mondoloni et al., 2023). This creates a
417 vicious cycle in which reduced IPN sensitivity promotes escalating intake, reinforcing
418 addiction—a process likely mediated by adaptive changes in nAChR function and circuit
419 dynamics within the MHb-IPN axis.

420

421 B. Nicotine reward

422 Emerging evidence indicates that the MHb-IPN axis not only mediates nicotine aversion but
423 also influences its motivational properties. For instance, silencing amigo1-positive IPN
424 neurons, which co-express α5* nAChRs, SST and Nos1, abolishes nicotine-induced conditioned
425 place preference (Ables et al., 2017). Similarly, selective knock down of ChAT in the MHb
426 reduces glutamate co-release in the IPN and eliminates nicotine preference (Frahm et al.,
427 2015). These findings highlight that proper nicotinic transmission within the MHb-IPN circuit
428 is required for nicotine to acquire reinforcing value.

429 Conversely, reduced activity in the IPN can alter the balance toward excessive reward, as
430 shown using a chemogenetic method enabling local, pharmacologically specific and sustained
431 inhibition of β4* nAChRs. Blocking IPN β4* nAChRs enhances the dopaminergic response to
432 nicotine in the VTA and induces preference for doses that are normally subthreshold for
433 reward (Jehl et al., 2025), demonstrating that the IPN exerts inhibitory control over the
434 dopaminergic reward system (Henderson, 2025; Jehl et al., 2025). These findings align with
435 previous studies showing that β4 subunit deletion (Harrington et al., 2016) or pharmacological
436 blockade of α3β4* nAChRs in the MHb (McCallum et al., 2012) disrupts dopamine release in
437 the nucleus accumbens. Notably, VTA dopaminergic neurons from β4 KO mice show elevated
438 sensitivity to nicotine and altered conditioning (Harrington et al., 2016). At the circuit level,
439 this IPN-mediated brake involves projections to the LDTg (Jehl et al., 2025). Together, these
440 findings suggest that nicotine reward is not solely a function of VTA dopaminergic activation,
441 but is finely tuned by inhibitory signals originating in the IPN and relayed through the LDTg
442 (Figure 4). This modulatory mechanism likely serves to balance nicotine's rewarding and
443 aversive effects across different doses and contexts.

444

445 C. Nicotine withdrawal and relapse

446 The MHb-IPN pathway is critically involved in both the emotional and somatic components of
447 nicotine withdrawal. When nicotine levels drop, neuroadaptations induced by chronic
448 exposure —such as nAChR desensitization, receptor upregulation, and network
449 reorganization— unmask a state of cholinergic imbalance. This imbalance contributes to the
450 emergence of somatic, affective, and motivational withdrawal symptoms that drive the urge to relapse. In rodents, nicotine withdrawal induces a broad spectrum of symptoms, including aversion, anhedonia, anxiety and fear-like behavior, as well as somatic signs such as body and head shakes, paw tremors, scratching, grooming, and hyperalgesia (Koob & Moal, 1997; Salas et al., 2009; Zhao-Shea et al., 2013; Molas, DeGroot, et al., 2017; Wills et al., 2022).

455 Withdrawal can be precipitated either by nicotine cessation or systemic administration of non-selective nAChR antagonists such as mecamylamine (Watkins et al., 2000). Notably, local microinjections of mecamylamine into the habenula or IPN - not into the cortex, VTA or hippocampus - are sufficient to drive withdrawal, demonstrating the central role for the MHb-IPN axis (Salas et al., 2009).

460 During chronic nicotine exposure, the IPI shows upregulation of corticotropin releasing factor receptor-1 (CRF1), a key mediator of stress responses (Zhao-Shea et al., 2015). Upon withdrawal, CRF released from the VTA activates IPI neurons through increased glutamatergic transmission from the MHb (Zhao-Shea et al., 2015), contributing to aversion and anxiety during withdrawal (Figure 4) (Grieder et al., 2014). Sex differences are notable: females show stronger IPN activation during withdrawal, and CRF1 antagonism increases glutamate release only in females (Carcoba et al., 2022).

467 β4* nAChRs play a key role in the somatic manifestations of withdrawal: mice lacking β4 exhibit reduced withdrawal symptoms (Salas et al., 2004). Furthermore, MHb pacemaker cholinergic cells, which respond to nicotine via α3β4* nAChRs, become hypersensitive after withdrawal, and inhibition of these cells using HCN channel blockers precipitates somatic withdrawal symptoms (Görlich et al., 2013). At the IPN level, withdrawal is associated with increased activity of SST-positive interneurons in the IPR, which upregulates β4* nAChRs
473 during chronic nicotine exposure. Blockade of these receptors precipitates somatic
474 withdrawal signs and induces c-fos expression in SST-negative projection neurons (Zhao-Shea
475 et al., 2013). These findings suggest that β4* nAChRs in SST-positive interneurons act as a
476 brake on withdrawal, and that their inhibition disinhibits projection neurons, driving somatic
477 withdrawal behaviors.

478 Distinct nAChR subunits contribute differentially to withdrawal phenotypes. Both male and
479 female rats show upregulation of IPN β3 and β4 subunits during nicotine withdrawal, while
480 α2* and α3* nAChRs are selectively increased in males (Correa et al., 2019). The β4 subunit is
481 primarily involved in somatic signs but not in affective components, as blocking α3β4 nAChRs
482 in the MHb fails to alleviate withdrawal-induced anxiety (Pang et al., 2016). In contrast,
483 pharmacological blockade of α4β2* or α6β2* nAChRs in the MHb reduces anxiety-like
484 behavior in nicotine-withdrawn mice (Pang et al., 2016). α5* nAChRs modulate withdrawal via
485 neurokinin signaling: blocking neurokinin receptors in the MHb precipitates somatic
486 withdrawal signs (Dao et al., 2014).

487 Overall, nicotine withdrawal increases the frequency of excitatory inputs and enhances IPN
488 activity (Zhao-Shea et al., 2013; Klenowski et al., 2022), particularly within the IPI (Zhao-Shea
489 et al., 2015). However, IPN firing transiently decreases during the expression of somatic
490 withdrawal behaviors, possibly reflecting a coping mechanism to buffer aversive states
491 (Klenowski et al., 2022). Optogenetic activation of IPN neurons precipitates both somatic
492 withdrawal signs (Zhao-Shea et al., 2013) and anxiety-like behavior (Klenowski et al., 2022),
493 whereas suppression of activity with optogenetics (Klenowski et al., 2022; Monical &
494 Mcgehee, 2025) or NMDA receptor antagonists (Zhao-Shea et al., 2013) alleviates somatic
495 withdrawal symptoms (Figure 4). At the circuit level, mecamylamine is thought to precipitate
496 withdrawal symptoms by inhibiting GABAergic interneurons in the IPN, thereby disinhibiting
497 projection neurons (Zhao-Shea et al., 2013), which likely target to the LDTg. Accordingly,
498 during nicotine withdrawal, LDTg activity is suppressed and dopaminergic activity is decreased
499 (Monical & Mcgehee, 2025), supporting the existence of a shared IPN-LDTg-VTA pathway for
500 both the positive (Jehl et al., 2025) and negative motivational aspects of nicotine dependence.

501 Beyond withdrawal, nicotinic signaling within the IPN also contributes to relapse-like
502 behaviors. KI rats carrying the human α5 D398N polymorphism exhibit enhanced nicotine-
503 induced reinstatement of nicotine-seeking behavior (Forget et al., 2018). This relapse-like
504 behavior is associated with reduced neuronal activity in the IPN, and the degree of
505 reinstatement negatively correlates with IPN responsivity (Figure 4). Brain slice recordings
506 from these rats revealed a deficit in IPN neuron responses to nicotine, suggesting that blunted
507 IPN activity facilitates relapse-like behaviors, and highlight a critical role for IPN α5* nAChRs
508 in controlling relapse vulnerability.

509

510 **7. Role of Endogenous Nicotinic Modulation in Negative Emotional States**

511 The MHb-IPN pathway plays a critical role in processing negative emotional states, such as
512 stress, aversion, fear and anxiety (McLaughlin et al., 2017; Molas, DeGroot, et al., 2017; Ables
513 et al., 2023). This pathway integrates exteroceptive and interoceptive aversive cues, acting as
514 a hub for modulating avoidance learning, defensive behavior, coping strategies, and emotional
515 homeostasis. Notably, MHb neurons possess primary cilia (Foll & French, 2018), which may
516 enable them to sense cues from the cerebrospinal fluid and contribute to the integration of
517 internal states. Although nAChRs within the MHb-IPN axis are known to mediate nicotine
518 aversion and anxiety during withdrawal, the potential involvement of endogenous habenular
519 ACh in modulating emotional processes remains largely underexplored; this possibility will be
520 examined in the sections below.

521

522 A. <u>Stress and aversion</u>

523 The MHb-IPN axis is robustly activated by stressful and aversive experiences such as foot
524 shocks, tail lifts or physical restraints (Hashikawa et al., 2020; Klenowski et al., 2023; Liang et
525 al., 2024; Williams et al., 2025). Optogenetic activation of MHb (Bailly et al., 2023) or IPN
526 neurons (Wolfman et al., 2018), or blockade of MORs in the MHb with naloxone (Boulos et al.,
527 2020), induces robust avoidance behavior (Figure 4). Importantly, visceral stressors such as
528 lithium chloride injection also engages this pathway to drive aversion (Otsu et al., 2019),
529 pointing to its sensitivity to interoceptive negative states. From a circuit point of view, the
530 projections from the IPN to the NI were shown to amplify aversion (Liang et al., 2024), while
531 those to the LDTg can directly elicit avoidance (Wolfman et al., 2018).

532 Several lines of evidence suggest that cholinergic signals within the MHb-IPN axis regulates
533 behavioral avoidance of negative stimuli. Nicotine itself produces aversion by activating
534 nAChRs within this pathway (Wills et al., 2022), suggesting that endogenous ACh may exert
535 similar effects. The finding that optogenetic activation of the pathway induces aversion
536 supports the idea that nicotinic excitation encodes aversive valence (Fowler et al., 2011;
537 Frahm et al., 2011; Wolfman et al., 2018). In addition, cholinergic projections from the MS to
538 the MHb regulate the generalization of visually conditioned aversion, though this process
539 involves M1 muscarinic receptors rather than nAChRs on MHb cholinergic neurons (Mu et al.,
540 2022).

541
542 B. <u>Anxiety</u>

543 The MHb-IPN pathway also contributes to anxiety-like behavior, with strong evidence
544 implicating nicotinic signaling. MHb cholinergic neurons show higher activity in anxiogenic
545 contexts, such as the open arms of the elevated plus maze (Sylwestrak et al., 2022).

546 Accordingly, in most studies, activation of MHb-IPN transmission enhances anxiety-like
547 responses, such as avoidance and reduced exploration (Figure 4), in paradigms such as the
548 elevated plus maze and forced swim test (Seigneur et al., 2018; Klenowski et al., 2023).

549 Conversely, silencing this pathway reduces passive coping strategies and anxiety-related
550 responses (Figure 4) (Yamaguchi et al., 2013; Klenowski et al., 2023), with projections from
551 the TS to the vMHb and central IPN implicated in these effects (Yamaguchi et al., 2013).

552 However, opposite results have also been reported (Cho et al., 2019), notably in the dMHb-
553 lateral IPN pathway, which is substance Pergic rather than cholinergic (Handa et al., 2025;
554 Wang et al., 2025). Additionally, dopamine release from VTA neurons onto the IPN may also
555 participate in anxiety regulation (DeGroot et al., 2020). A subset of habenula-IPN neurons
556 expressing the transcription factor Otx2 appears particularly sensitive to chronic stress
557 exposure during the peripubertal period, a developmental window that may confer increased
558 vulnerability to anxiety-related behaviors later in life (Rakotobe et al., 2024).

559 Nicotinic receptors are key mediators of anxiety encoding in this circuit. Blocking nAChRs in
560 the IPN induces nicotine withdrawal-like symptoms in nicotine-dependent mice, including
561 anxiety (Zhao-Shea et al., 2015). Intriguingly, deleting the β4 subunit has the opposite effect:
562 it reduces sensitivity to anxiety paradigms and blunts aversive reactivity (Salas et al., 2003;
563 Semenova et al., 2012; Husson et al., 2020). Conversely, boosting vMHb cholinergic signaling
564 with a hypersensitive α4* nAChR increases anxiety (Pang et al., 2016). This paradox—where
565 β4* nAChRs are required for withdrawal symptoms, yet their pharmacological inhibition
566 alleviates anxiety and their activation exacerbates it—underscores the complex, bidirectional
567 role of nicotinic signaling in the MHb-IPN axis and highlights key gaps in our understanding of
568 its regulation of anxiety.

569

570 C. Fear and defensive learning

571 IPN GABAergic neurons are also recruited by potential threats such as an approaching
572 predator, and their silencing reduces defensive responses (Williams *et al.*, 2025). Threat-
573 evoked responses decline with repeated exposure, suggesting that the IPN undergoes
574 adaptive changes associated with defensive learning. Distinct subcircuits within the MHb-IPN
575 pathway appear to contribute to different components of fear processing: IPN GABAergic
576 neurons projecting to the LDTg are involved in threat learning, whereas IPN SST neurons
577 mediate more generalized avoidance (Williams *et al.*, 2025). In parallel, substance P-
578 expressing neurons in the dMHb, which project to the IPL, participate in contextual fear, and
579 ablation of their afferents from the BAC impairs this function (Yamaguchi *et al.*, 2013).

580 Synaptic mechanisms implicate nAChRs in fear modulation. Deleting or blocking CB1 receptors
581 at MHb-IPN synapses increases ACh transmission and reduces freezing responses in
582 contextual fear conditioning experiments, while intra IPN infusion of the non-specific nAChR
583 antagonist mecamylamine – but not of glutamate receptor antagonists - in these mice restores
584 aversive behavior (Soria-Gómez *et al.*, 2015). Conversely, inactivating GABA<sub>B</sub> receptors at the
585 MHb-IPN synapses, or ablating MHb cholinergic neurons, suppresses neurotransmission and
586 enhances the expression of aversive memories, and intra-IPN infusion of ACh reduces fear
587 memory expression (Zhang *et al.*, 2016). This plasticity is restored during extinction
588 (Koppensteiner et al., 2016). Together, these results show that increased IPN ACh levels
589 facilitate fear extinction.

590 D. <u>Depression and stress adaptation</u>

591 The MHb-IPN pathway is also implicated in mood regulation and depressive-like states,
592 particularly anhedonia. In a model of stress-enhanced sucrose seeking, optogenetic inhibition
593 of IPN GABAergic neurons reduces stress-induced motivation, while their photoactivation —
594 or increased excitatory input from the MHb, potentially through nAChRs— potentiates
595 reward-seeking behavior (Klenowski et al., 2023). These neurons express SST and appear to
596 gate a behavioral shift toward positive reinforcement under stress. Mice lacking the β4
597 subunit display reduced depression-like behavior (Semenova et al., 2012), suggesting a role of
598 MHb-IPN nAChRs in these processes.

599 Consistent with this role in stress adaptation, alterations in the MHb-IPN axis have been
600 observed in models of chronic stress and depression, and are associated with depression-like
601 behaviors, including anhedonia (Xu et al., 2018). At the molecular level, stress remodels the
602 MHb transcriptome, modifying nAChR subunit expression (e.g., α5, β4) and glutamate
603 transporter levels (Yoo et al., 2022), likely altering MHb-IPN excitability.

604 Moreover, this pathway interacts with the hypothalamic-pituitary-adrenal (HPA) axis, further
605 reinforcing its involvement in the broader physiological stress response. Chemogenetic
606 manipulation of MHb neurons alters corticosterone release after stress (Hsu et al., 2014),
607 underscoring its function at the interface of emotional and neuroendocrine responses.
608 Additionally, CRF release into the IPN contributes to nicotine withdrawal-induced anxiety
609 (Zhao-Shea et al., 2015). As the MHb-IPN axis is heavily cholinergic and nAChR-driven, these
610 findings support a broader nicotinic control over neuroendocrine stress physiology.
611

612

## 8. Role of Endogenous Nicotinic Modulation in Social Interactions

613

614

615

The MHb-IPN pathway is emerging as a key regulator of social affective behaviors by integrating cholinergic, glutamatergic, and dopaminergic signals to shape processes such as social interaction, conflict resolution and motivation (Ables et al., 2023).

616

617

618

619

620

621

622

623

624

625

626

627

628

In mice, recent studies have provided direct evidence linking this pathway to social novelty exploration. IPN neuronal activity increases during social interactions, yet only after repeated social exposure, as the stimuli become familiar (Molas, Zhao-Shea, et al., 2017). Optogenetic manipulation of IPN neurons bidirectionally shifts novelty preference (Figure 4), suggesting that the IPN modulates the motivational salience of familiar cues. Dopamine signals within the IPN increase during novel social interaction but not during familiar encounters, implicating the VTA-IPN circuit, and particularly its projections to the LDTg, in motivated exploratory behavior (Molas et al., 2024). Furthermore, the IPN-LDTg projection contributes to the processing of novel social olfactory cues during nicotine withdrawal (Monical & McGehee, 2025), reinforcing the idea that this circuit integrates motivational and affective information to regulate social behavior. Mice lacking the β4 subunit exhibit altered interactions with familiar conspecifics (Salas et al., 2013) and enhanced novelty-induced activity (Husson et al., 2020), suggesting that nicotinic signaling contributes to social engagement and salience attribution.

629

630

631

632

633

Beyond novelty, the MHb-IPN circuit appears to regulate affiliative and hierarchical social behaviors. In zebrafish, the dorsal habenula (homologous to rodent MHb) modulates social defeat and conflict resolution via α7 nAChR activation (Kinoshita & Okamoto, 2023). Moreover, two subregions of the dorsal habenula exert opposite effects on social outcome: silencing either side biases the fish toward winning or losing in social conflict (Chou et al.,
634 2016). These findings suggest a finely tuned control of social strategy via habenular outputs.
635 In mice, similar principles apply. Postnatal ablation of MHb neurons markedly reduces ACh
636 levels in the IPN and produces behavioral impairments including hyperactivity, impulsivity,
637 maladaptation, and cognitive inflexibility (Kobayashi et al., 2013). Optogenetic activation of
638 the MHb-IPN cholinergic pathway promotes social submission by inhibiting serotonergic
639 neurons in the MnR, whereas disrupting cholinergic transmission enhances dominance
640 behavior (Matsumata et al., 2025).

641 Together, these findings identify the MHb-IPN pathway as a conserved circuit for regulating
642 social behaviors, from novelty detection to social conflict resolution. Nicotinic signaling within
643 this circuit may serve as a crucial modulator of social motivation, behavioral flexibility, and
644 hierarchy-related responses.

645

646 **9. Future Directions and Open Questions**

647 Despite major advances, fundamental questions remain about the nicotinic modulation of the
648 MHb-IPN pathway. While the anatomical and functional complexity of this circuit is
649 increasingly recognized, we are only beginning to resolve how nAChR subunit composition and
650 function vary across specific neuronal populations —and how these properties change with
651 context, physiological state, or experience. The MHb-IPN pathway expresses one of the
652 densest and most diverse arrays of nAChRs in the brain, yet the precise localization of these
653 subtypes, their cell-specific functional roles, and their regulation under physiological and
654 pathological conditions remain incompletely understood. Although α3β4* and α5* nAChRs
655 have been clearly implicated in nicotine aversion, the roles of less-characterized subunits such
656 as α2 or β3 remain largely undefined. Moreover, the endogenous source of ACh within the
657 MHb is still unclear, particularly in light of the presence of functional nAChRs on MHb somata
658 and axons in the absence of known cholinergic inputs (Passlick et al., 2018). Recent findings
659 of feedforward cholinergic excitation suggest a form of intra-habenular transmission (Chung
660 et al., 2023), but direct evidence for this mechanism remains sparse. Addressing these
661 knowledge gaps will require both anatomical and functional refinement. Mapping receptor
662 subunit localization at single-cell resolution using spatial transcriptomics and multiplexed in
663 situ hybridization should be complemented by functional profiling of receptor subtypes in
664 genetically defined neuronal populations.

665 A major open question concerns how distinct IPN output pathways mediate diverse behavioral
666 outcomes. The IPN projects to several neuromodulatory and behavioral control centers,
667 including the LDTg, raphe nuclei, and NI, each implicated in specific processes. Notably, IPN-
668 LDTg projections have been linked to nicotine aversion (Wolfman et al., 2018), reward (Jehl et
669 al., 2025) and withdrawal symptoms (Monical & Mcgehee, 2025), as well as to anxiety and
670 coping (Klenowski et al., 2023), fear learning (Williams et al., 2025) and social novelty
671 preference (Molas et al., 2024). Determining how these parallel subcircuits are organized, and
672 how their nicotinic modulation shapes target activity, will be critical to understand how the
673 IPN gates different motivational and emotional states. Combining cell-type-resolved tracing,
674 functional imaging, and projection-specific manipulations will be essential to map how
675 nicotinic signaling influences information flow from the IPN to its downstream targets.

676 Addressing these questions will benefit from newly developed methods that allow precise
677 manipulation of nAChRs with unprecedented pharmacological and spatial control (Kramer et
678 al., 2013; Mondoloni et al., 2019; Paoletti et al., 2019; Colleoni et al., 2025). Caged nicotinic
679 agonists (Banala et al., 2018, 2024; Passlick et al., 2018) and antagonists —particularly those
sensitive to two-photon excitation (Warther et al., 2010) —allow subcellular mapping of functional nAChRs in situ. When applied in behaving animals (Paoletti et al., 2019; Cuttoli et al., 2020; Ma et al., 2023; McClain et al., 2023), these photochemical tools can bridge receptor-level activity to circuit function and behavior. Complementary chemogenetic strategies permit manipulation of nAChRs with both cell-type and subunit specificity. These include hypersensitive nAChRs, carrying single-point mutations in the channel pore that increase agonist sensitivity by over 100-fold, enabling selective activation of α4* or α6* nAChRs in knock-in mice (Tapper et al., 2004; Drenan et al., 2008; Fonck et al., 2009). In parallel, cysteine-substituted subunits permit covalent tethering of agonists or antagonists—some of which are photoresponsive—near the ACh-binding site, allowing for either rapid and reversible receptor manipulation (Tochitsky et al., 2012; Cuttoli et al., 2018) or sustained antagonism in darkness (Jehl et al., 2025). These engineered subunits can be delivered virally to specific populations (Cuttoli et al., 2018; Mondoloni et al., 2019), or expressed from knock-in alleles (e.g., β2*, β4*) to enable endogenous targeting (Jehl et al., 2025). Collectively, these tools offer unprecedented temporal, spatial, and molecular resolution for dissecting nAChR function in vivo. Finally, the recent development of optical ACh sensors (Jing et al., 2018, 2020; Mineur & Picciotto, 2023; Favier et al., 2024) provides the means to monitor real-time fluctuations in cholinergic signaling during behavior or pharmacological manipulation, a critical step for understanding endogenous ACh dynamics across MHb-IPN subregions and behavioral states.

Beyond basic neuroscience, this pathway holds significant therapeutic potential. The α5, α3, and β4 nAChR subunits regulate both the rewarding and aversive properties of nicotine (Wills et al., 2022; Jehl et al., 2025), positioning the MHb-IPN axis as a critical regulator of nicotine
703 intake. Strategies targeting these subtypes—whether via selective agonists, antagonists, or
704 allosteric modulators—could inspire new smoking cessation therapies that reduce craving
705 while preserving or enhancing aversive responses to the drug. Moreover, given the emerging
706 roles of the MHb-IPN circuit in anxiety, depression, and social behavior regulation, modulating
707 its nicotinic tone may also be beneficial in affective and neuropsychiatric disorders,
708 particularly in individuals carrying polymorphisms in nicotinic receptor genes.

709 Importantly, the MHb-IPN pathway may serve as a shared substrate for multiple psychiatric
710 conditions that frequently co-occur with nicotine dependence. For instance, smoking and
711 opioid use disorders exhibit strong comorbidity (Rajabi et al., 2019), potentially reflecting the
712 high density of MORs in this circuit and their ability to modulate MHb-IPN transmission.
713 Similarly, the elevated prevalence of smoking in individuals with depression and generalized
714 anxiety disorder (Molas, DeGroot, et al., 2017) aligns with the MHb-IPN pathway's well-
715 documented role in anxiety and aversion encoding. These links underscore the broader
716 therapeutic potential of targeting nicotinic signaling in this pathway—not only for treating
717 nicotine addiction, but also for addressing a spectrum of comorbid psychiatric conditions.

718 In summary, a deeper understanding of the cell-type-specific roles of nAChRs in the MHb-IPN
719 axis will be essential to elucidate how this circuit processes aversive and emotional states, and
720 how its dysfunction contributes to psychiatric and substance use disorders. Continued
721 integration of transcriptomic, optical, and behavioral methods will be essential to fully unravel
722 the complexity—and therapeutic potential—of this ancient cholinergic circuit.
723 Acknowledgements: the authors would like to thank Sarah Mondoloni (Inmed, Marseille) for critically reading the manuscript and for the initial draft of the figures. This work has received support under the Major Research Program of PSL Research University "PSL-Neuro" launched by PSL Research University and implemented by ANR (ANR-10-IDEX-0001). Funding was provided by the Agence Nationale de la Recherche (ANR-21-CE16-0012 CHOLHAB and ANR-21-CE37-0026 NICOPTOTOUCH to A.M.); the Human Frontier Science Program (RGP0035/2020 to A.M.); National Institutes of Health (NS111600 to A.M.); and the Fondation pour la Recherche Médicale: EquipeFRM grant (EQU201903007961 to A.M.) and fourth year PhD fellowship to Maria Ciscato (FDT202404018105).

732

733 Competing interest: none

734

735 Author contribution

736 All authors wrote the manuscript. AM prepared the illustrations.

737

738 Abbreviations

739 18-MC: 18-Methoxycoronaridine

740 ACh: acetylcholine

741 AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

742 BAC: bed nucleus of the anterior commissure

743 CB1 receptor: cannabinoid receptor 1

744 ChAT: choline acetyl transferase

745 CHT1: choline transporter 1

746 CNS: central nervous system
747 CRF: corticotropin releasing factor

748 CRF1: corticotropin releasing factor receptor-1

749 dMHb: dorsal MHb

750 dIMHb: dorsolateral MHb

751 dmMHb: dorsomedial MHb

752 DR: dorsal raphe

753 DTg: dorsal tegmental nucleus

754 FR: fasciculus retroflexus

755 GABA: γ-amino butyric acid

756 GABAB receptor: GABA type B receptor

757 GLP-1: glucagon-like peptide-1

758 GPR151: G protein-coupled receptor 151

759 HCN: Hyperpolarization-activated cyclic nucleotide-gated channels

760 HHIP: Hedgehog-interacting protein

761 IPA: apical IPN

762 IPC: caudal IPN

763 IPDL: dorsolateral IPN

764 IPDM: dorsomedial IPN

765 IPI: intermediate IPN

766 IPL: lateral IPN

767 IPN: interpeduncular nucleus

768 IPR: rostral IPN

769 KI: knock-in

770 KO: knock-out
771 LDTg: laterodorsal tegmental nucleus

772 LHb: lateral habenula

773 MHb: medial habenula

774 MnR: median raphe nucleus

775 MOR: mu opioid receptor

776 MS: medial septum

777 nAChR: nicotinic acetylcholine receptor

778 NDB: nucleus of the diagonal band

779 NI: nucleus incertus

780 Nos1: nitric oxide synthase 1

781 SFi: septofimbrial nucleus

782 SST: somatostatin

783 TS: triangular septal nucleus

784 vcMHb: ventrocentral MHb

785 VGluT: vesicular glutamate transporters

786 vIMHb: ventrolateral MHb

787 vMHb: ventral MHb

788 vmMHb: ventromedial MHb

789 VTA: ventral tegmental area

790 WT: Wild-type
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1314 Xu, C., Sun, Y., Cai, X., You, T., Zhao, H., Li, Y., & Zhao, H. (2018) Medial Habenula-

1315 Interpeduncular Nucleus Circuit Contributes to Anhedonia-Like Behavior in a Rat Model of

1316 Depression. *Front. Behav. Neurosci.*, **12**, 238.

1317 Yamaguchi, T., Danjo, T., Pastan, I., Hikida, T., & Nakanishi, S. (2013) Distinct Roles of

1318 Segregated Transmission of the Septo-Habenular Pathway in Anxiety and Fear. *Neuron*,

1319 **78**, 537–544.

1320 Yoo, H., Kim, H.J., Yang, S.H., Son, G.H., Gim, J.-A., Lee, H.W., & Kim, H. (2022) Gene

1321 Expression Profiling of the Habenula in Rats Exposed to Chronic Restraint Stress. *Mol.*

1322 *Cells*, **45**, 306–316.

1323 Zhang, J., Tan, L., Ren, Y., Liang, J., Lin, R., Feng, Q., Cell, J.Z., Hu, F., Ren, J., Wei, C., Yu, T.,

1324 Zhuang, Y., Bettler, B., Wang, F., & Luo, M. (2016) Presynaptic excitation via GABAB

1325 receptors in habenula cholinergic neurons regulates fear memory expression. *Cell*, **166**,

1326 716–728.

1327 Zhao, H. & Rusak, B. (2005) Circadian firing-rate rhythms and light responses of rat habenular nucleus neurons in vivo and in vitro. *Neuroscience*, **132**, 519–528.

1328

1329 Zhao-Shea, R., DeGroot, S.R., Liu, L., Vallaster, M., Pang, X., Su, Q., Gao, G., Rando, O.J.,

1330 Martin, G.E., George, O., Gardner, P.D., & Tapper, A.R. (2015) Increased CRF signalling in a

1331 ventral tegmental area-interpeduncular nucleus-medial habenula circuit induces anxiety

1332 during nicotine withdrawal. *Nature Communications*, **6**, 1–13.
1333 Zhao-Shea, R., Liu, L., Pang, X., Gardner, P.D., & Tapper, A.R. (2013) Activation of GABAergic

1334 Neurons in the Interpeduncular Nucleus Triggers Physical Nicotine Withdrawal

1335 Symptoms. *Curr Biol*, **23**, 2327-2335.

1336 Zoli, M., Pistillo, F., & Gotti, C. (2015) Diversity of native nicotinic receptor subtypes in

1337 mammalian brain. *Neuropharmacology*, **96**, 302-311.

1338

1339

1340

1341

1342

1343 Tables

1344

1345 Table 1: nAChR subunit expression patterns in the MHb-IPN pathway. αCtxMII: alpha-

1346 Conotoxin MII (α3β2* and β3* nAChR antagonist); IHC: immunohistochemistry; IP:

1347 immunoprecipitation; ISH: in situ hybridization; MLA: Methyllycaconitine (selective α7

1348 antagonist); PC: patch-clamp electrophysiology; PNU-120596: positive allosteric modulator of

1349 α7 nAChRs; RNAseq: RNA sequencing; RT PCR: reverse transcriptase PCR; scRNA-seq: Single-

1350 cell RNA sequencing; SR16584: specific α3β4 antagonist; TABAC: transgenic α3β4α5 cluster;

1351 TRAP: Translating Ribosome Affinity Purification

1352

1353 Figures legends

1354

1355 **Figure 1: Anatomy of the MHb-IPN pathway.** A. Different subnuclei of the MHb and IPN. The

1356 ventral MHb (vMHb) includes the ventrolateral (vIMHb), ventrocentral (vcMHb) and

1357 ventromedial (vmMHb; also called ventroinferior in some studies) subnuclei, while the dorsal

1358 MHb (dMHb) includes the dorsolateral (dlMHb) and dorsomedial (dmMHb) subnuclei. The IPN

1359 is subdivided into three unpaired subnuclei that lie along the midline and include the apical

1360 (IPA), rostral (IPR), and caudal (IPC) subnuclei, and four paired subnuclei that are found
1361 bilaterally and comprise the dorsomedial (IPDM), dorsolateral (IPDL), intermediate (IPI), and lateral (IPL) subnuclei. The IPA is more caudal and not shown here. dMHb neurons are mostly substance P positive and project to the lateral subnuclei (IPL and IPDL) of the IPN, whereas vMHb neurons are mostly cholinergic and they project mostly on the medial (IPR and IPC) subnuclei of the IPN. B. Major inputs of the MHb and outputs of the IPN. 3V: third ventricle; BAC: bed nucleus of the anterior commissure; ChAT: choline acetyltransferase; dl: dorsolateral; dm: dorsomedial; DR: dorsal raphe nucleus; DTg: dorsal tegmental nucleus; FR: fasciculus retroflexus; IPC: central IPN; IPDL: dorsolateral IPN; IPDM: dorsomedial IPN; IPI: intermediate IPN; IPL: lateral IPN; IPN: interpeduncular nucleus; IPR: rostral IPN; LDTg: laterodorsal tegmental nucleus; LHb: lateral habenula; MHb: medial habenula; MnR: median raphe nucleus; MS: medial septum; NDB: nucleus of the diagonal band; NI: nucleus incertus; SFi: septofimbrial nucleus; SP: substance P; TS: triangular septal nucleus; vc: ventrocentral; vl: ventrolateral; vm: ventromedial; VTA: ventral tegmental area.

1374

1375 **Figure 2: Structure, assembly and expression patterns of neuronal nAChRs in the MHb-IPN axis.** A. Various assemblies of α and β subunits into hetero- and homo-pentameric structures with two, three or five ACh binding pockets. B Structure of the two α, three β stoichiometry of human α4β2 nAChR in complex with ACh (PDB ID 8ST0 (Mazzaferro et al., 2024)). Left, top view; right, side-view. C. Patterns of expression of the different nAChR subunits in the MHb (left) and IPN (right). Font sizes indicate expression levels. Further details can be found in Table 1.

1382

1383 **Figure 3: The MHb-IPN cholinergic synapse.** MHb cholinergic neurons co-release ACh and glutamate onto IPN neurons. Vesicular transporters for both neurotransmitters (VAChT and
1385 VGlut) are co-expressed on the same synaptic vesicles. nAChRs are present on MHb
1386 presynaptic terminals (α3β4* subtype), as well as on both synaptic and extra-synaptic sites on
1387 IPN neurons. MORs and GABA<sub>B</sub> receptors facilitate neurotransmission, whereas CB1
1388 receptors, SST and nitric oxide act to suppress it.

1389

1390 **Figure 4: Receptors and circuits of the MHb-IPN axis involved in nicotine effects, emotional regulation and social behaviors.** Stimuli or conditions that affect neuronal excitability or synaptic transmission are shown in blue, whereas those influencing nicotine responses are in red. Overall, increased neurotransmission (top) promotes aversion, threat learning, anxiety, thereby suppressing nicotine intake. Conversely, decreased neurotransmission (bottom) relieves aversion and anxiety, alters coping strategies and enhances nicotine consumption.
1391
1392
1393
1394
1395
1396 Question marks denote IPN outputs for which the functional target remains unknown. lof: loss of function; gof: gain of function.

1398
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>
A

<figure><img src="image_4.png" /></figure>

Ligand
Binding
Domain

Trans-
membrane
Domain

Intra-
cellular
Domain

<figure><img src="image_5.png" /></figure>

B

Heteromeric nAChRs

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

Homomeric
nAChRs

<figure><img src="image_8.png" /></figure>

C

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

ChAT+ axon terminals

SP+ axon
terminals
Pre-synaptic MHb neuron

<figure><img src="image_11.png" /></figure>

Post-synaptic IPN neuron
Increased transmission

<figure><img src="image_12.png" /></figure>

Optogenetic activation
Nicotine withdrawal (CRF)
Anxiogenic context
CB1R deletion/antagonism
GABA<sub>B</sub>R activation
MOR antagonism
Hypersensitive α4
Lithium

<figure><img src="image_13.png" /></figure>

Avoider mice
High nicotine doses (β4/α5)
β4 over-expression/gof
TABAC mice

<figure><img src="image_14.png" /></figure>

Optogenetic activation
Nicotine withdrawal
Stressors / Threats
Familiar conspecifics
GLP-1

LDTg

↗ Aversion / avoidance

↗ Threat learning

↗ Withdrawal symptoms

↘ Social interactions with novel stimuli

NI

↗ Aversion

MnR

↗ Anxiety

↗ Social submission

↘ Nicotine intake

Nicotine satiety

↘ Fear and

<figure><img src="image_15.png" /></figure>

Fear extinction

?

Decreased transmission

<figure><img src="image_16.png" /></figure>

Cholinergic neuron ablation
β2 antagonism
GABA<sub>B</sub> R inactivation
GPR151 KO

<figure><img src="image_17.png" /></figure>

Chronic nicotine
Non-avoider mice
β4/α5/α3 antagonism/deletion/lof

<figure><img src="image_18.png" /></figure>

Optogenetic inhibition
Nicotine relapse
Social novelty
Lidocaine
NMDAR antagonism

LDTg

↗ Social interaction with familiar stimuli

↘ Aversion

VTA

<figure><img src="image_19.png" /></figure>

Reward

↘ Anxiety

↘ Depression

↘ Coping strategies / defensive responses

↘ Withdrawal symptoms

↘ Stress-induced motivation

?

↗ Reward-seeking

↗ Nicotine intake

↗ Nicotine relapse

↗ Expression of fear memory

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>

For Peer Review
<table><thead><tr><td>Subunit</td><td>MHb expression</td><td>IPN expression</td><td>Localization</td><td>Method</td><td>Reference</td></tr></thead><tbody><tr><td>α2</td><td>No - low</td><td>Low - high</td><td>MHb: vMHb<br>IPN: mostly IPR, but also found everywhere IPN</td><td>RNAseq<br>RTqPCR<br>ISH<br>Single-cell RT PCR<br>IP</td><td>(Ishii et al., 2005; Grady et al., 2009; Scholze et al., 2012; Ables et al., 2017; Correa et al., 2019)</td></tr><tr><td>α3</td><td>High</td><td>Moderate - high</td><td>MHb: entire vMHb, lateral region of dMHb and FR CHT1+ neurons<br>ChAT+ and ChAT- neurons<br>Cytoplasm and extrasynaptic sites<br>GPR151+ axons<br>IPN: IPR<br>VGluT2- neurons (i.e., GABAergic)</td><td>RNAseq<br>RTqPCR<br>ISH<br>TRAP profiling<br>Single-cell RT PCR<br>IP<br>IHC<br>Autoradiograms<br>GFP-KI mice<br>PC + TABAC mice<br>PC + SR16584</td><td>(Zoli et al., 1998; Sheffield et al., 2000; Salas et al., 2003; Salas, Cook, et al., 2004; Grady et al., 2009; Quina et al., 2009; Frahm et al., 2011; Scholze et al., 2012; Shih et al., 2014; Ables et al., 2017; Correa et al., 2019; Tsuzuki et al., 2024)</td></tr><tr><td>α4</td><td>Moderate</td><td>Moderate - high</td><td>MHb: some areas vMHb (vIMHb) and FR ChAT+ and ChAT- neurons<br>IPN:</td><td>RNAseq<br>RTqPCR<br>Single-cell RT PCR<br>IP<br>GFP-KI mice<br>PC + Hypersensitive KI mice</td><td>(Sheffield et al., 2000; Fonck et al., 2009; Grady et al., 2009; Scholze et al., 2012; Shih et al., 2014, 2015; Ables et al., 2017; Correa et al., 2019)</td></tr><tr><td>α5</td><td>Low</td><td>Moderate - high</td><td>MHb: some areas vMHb (vIMHb and VcMHb) and some areas dMHb (dIMHb)</td><td>ISH<br>RNAseq<br>TRAP profiling<br>Single-cell RT PCR<br>IP</td><td>(Sheffield et al., 2000; Salas, Cook, et al., 2004; Grady et al., 2009; Fowler et al., 2011; Scholze et al., 2012; Hsu et al., 2013; Ables et al., 2017; Correa et al., 2019)</td></tr></tbody></table>
<table><tr><td></td><td></td><td></td><td>IPN: IPR, IPC/IPI<br>Projection neurons (to MnR, DTg/DRI)<br>SST+ neurons<br>GABAergic neurons</td><td>GFP-KI mice<br>CHRNAS<sup>cre</sup> mice<br>PC + KO mice<br>RTqPCR<br><sup>86</sup>Rb<sup>+</sup> efflux + KO mice<br>shRNA knock-down</td><td>Ables et al., 2017; Forget et al., 2018; Morton et al., 2018; Correa et al., 2019; Jin & Drenan, 2022)</td></tr><tr><td>α6</td><td>Low</td><td>Low</td><td>MHb: some areas vMHb (vmMHb) and some areas dMHb (dmMHb)<br>ChAT+ and ChAT- neurons<br>IPN: IPR</td><td>RNAseq<br>ISH<br>Single-cell RT PCR<br>IP<br>GFP-KI mice<br>PC + Hypersensitive KI mice<br>PC + αCtxMII</td><td>(Novere et al., 1996; Sheffield et al., 2000; Grady et al., 2009; Scholze et al., 2012; Shih et al., 2014, 2015; Ables et al., 2017)</td></tr><tr><td>α7</td><td>No</td><td>Very low - low</td><td>IPN: mainly IPL<br>GABAergic neurons</td><td>RNAseq<br>RTqPCR<br>Single-cell RT PCR<br>ISH<br>PC + MLA/PNU-120596</td><td>(Sheffield et al., 2000; Ables et al., 2017; Correa et al., 2019; Jin & Drenan, 2022)</td></tr><tr><td>β2</td><td>Moderate</td><td>Moderate</td><td>MHb: entire vMHb and dMHb<br>ChAT+ and ChAT- neurons<br>IPN: mainly IPR</td><td>RTqPCR<br>RNAseq<br>Single-cell RT PCR<br>IP<br>GFP-KI mice<br><sup>86</sup>Rb<sup>+</sup> efflux</td><td>(Sheffield et al., 2000; Grady et al., 2009; Scholze et al., 2012; Shih et al., 2014; Ables et al., 2017; Correa et al., 2019)</td></tr><tr><td>β3</td><td>Moderate - high</td><td>low - high</td><td>MHb: entire vMHb, some areas dMHb (dlMHb) and FR<br>ChAT+ and ChAT- neurons<br>IPN: mainly IPR</td><td>RNAseq<br>RTqPCR<br>Single-cell RT PCR<br>IP<br>autoradiography<br>GFP-KI mice</td><td>(Sheffield et al., 2000; Cui et al., 2003; Grady et al., 2009; Shih et al., 2014; Ables et al., 2017; Correa et al., 2019)</td></tr></table>
<table><caption>**Table 1: nAChR subunit expression patterns in the MHb-IPN pathway.** αCtxMII: alpha-Conotoxin MII (α3β2* and β3* nAChR antagonist); IHC: immunohistochemistry; IP: immunoprecipitation; ISH: in situ hybridization; MLA: Methyllycaconitine (selective α7 antagonist); PC: patch-clamp electrophysiology; PNU-120596: positive allosteric modulator of α7 nAChRs; RNAseq: RNA sequencing; RT PCR: reverse transcriptase PCR; scRNA-seq: Single-cell RNA sequencing; SR16584: specific α3β4 antagonist; TABAC: transgenic α3β4α5 cluster; TRAP: Translating Ribosome Affinity Purification</caption><tr><td rowspan="10">β4</td><td rowspan="10">High</td><td rowspan="10">High</td><td><strong>MHb</strong>: entire vMHb and dMHb, FR</td><td>RNAseq</td><td>(Zoli et al., 1998; Sheffield et al., 2000; Salas et al., 2003; Salas, Cook, et al., 2004; Salas, Pieri, et al., 2004; Grady et al., 2009; Quina et al., 2009; Scholze et al., 2012; Beiranvand et al., 2014; Shih et al., 2014; Ables et al., 2017; Mondoloni et al., 2023; Tsuzuki et al., 2024; Jehl et al., 2025)</td></tr><tr><td>VGluT1+ CHT1+ neurons</td><td>RTqPCR</td></tr><tr><td>Neuropil region</td><td>ISH</td></tr><tr><td>Extrasynaptic</td><td>IHC</td></tr><tr><td rowspan="5"><strong>IPN</strong>: IPR</td><td>Single-cell RT PCR</td></tr><tr><td>TRAP profiling</td></tr><tr><td>IP</td></tr><tr><td>Autoradiograms</td></tr><tr><td>VGluT2-negative neurons (i.e., GABAergic)</td><td>GFP-KI mice</td></tr><tr><td></td><td>PC + β4E61C KI mice</td></tr><tr><td></td><td>PC + KO mice</td></tr><tr><td></td><td>PC + SR16584</td></tr><tr><td></td><td>[³H] ACh release + KO mice</td></tr></table>
<figure><img src="image_36.png" /></figure>

Nicotine intake

Aversion

Anxiety

Fear

Stress

Depression

Reward

Social interactions