Functional α7 nicotinic acetylcholine receptors in GABAergic neurons of the interpeduncular nucleus

Abstract

The interpeduncular nucleus (IPN) plays a key role in nicotine dependence and is involved in regulation of fear responses, affective states, and novelty processing. IPN neurons express nicotinic acetylcholine receptors (nAChR) and receive strong cholinergic innervation from the ventral medial habenula. Dorsal medial habenula neurons are primarily peptidergic, releasing substance P (SP) mainly onto IPN neurons in the lateral subnucleus (IPL). IPL neurons are sensitive to SP, but it is not known if they are involved in cholinergic transmission like other IPN neurons. We examined nAChR subunit gene expression in IPL neurons, revealing that Chrna7 (α7 nAChR subunit) is expressed in a subset of GABAergic IPL neurons. In patch-clamp recordings from IPL neurons, ACh-evoked inward currents were attenuated by methyllycaconitine (α7 nAChR antagonist) and potentiated by NS1738 (α7 Type I positive allosteric modulator). We confirmed α7 functional expression in IPL neurons by also showing that ACh-evoked currents were potentiated by PNU-120596 (Type II positive allosteric modulator). Additional pharmacological experiments show that IPN neurons expressing α7 nAChRs also express α3β4 nAChRs. Finally, we used 2-photon laser scanning microscopy and nicotine uncaging to directly examine the morphology of IPL neurons that express α7 nAChRs. These results highlight a novel aspect of α7 nAChR neurobiology, adding to the complexity of cholinergic modulation by nAChRs in the IPN.

1. Introduction

The habenulo-interpeduncular pathway, consisting of the medial habenula (MHb) and its projection to the interpeduncular nucleus (IPN), plays an important role in nicotine dependence and other motivated behaviors. The ventral portion of the MHb (vMHb) is comprised of dual-transmitting cholinergic/glutamatergic neurons (Aizawa et al., 2012; Ren et al., 2011) whose axons project to the IPN via the fasciculus retroflexus, a prominent descending fiber bundle. vMHb fibers are found in the rostral (IPR), central (IPC), dorsolateral (IPDL), and intermediate (IPI) subnuclei of the IPN, but they are conspicuously absent from the lateral (IPL) subnucleus of the IPN (Contestabile et al., 1987). vMHb neurons express high levels of a variety of nicotinic acetylcholine receptor (nAChR) subunits (Shih et al., 2014), resulting in strong functional expression of nAChRs in vMHb somata (Quick et al., 1999; Shih et al., 2014; Shih et al., 2015), axons (Arvin et al., 2019), and axon terminals in IPN (Arvin et al., 2019; Grady et al., 2009). Among the nAChRs expressed in this pathway are those that contain the α5 subunit, which may play a role in regulating nicotine intake and aversion to high nicotine concentrations (Fowler et al., 2011; Frahm et al., 2011; Zhao-Shea et al., 2013). IPN neurons receiving input from the vMHb connect to several hindbrain structures, including the Raphe (Hsu et al., 2016; Hsu et al., 2013; Morton et al., 2018; Quina et al., 2017) and the laterodorsal tegmental nucleus (LDTg) (Wolfman et al., 2018).

Neurons in the dorsal aspect of the MHb (dMHb) are neurochemically distinct from those in the vMHb. In particular, dMHb neurons exhibit strong and specific expression of substance P (Contestabile et al., 1987; Kawaja et al., 1991). These peptidergic dMHb neurons, which are also glutamatergic (Aizawa et al., 2012), project selectively to the IPL via the fasciculus retroflexus and are involved in voluntary exercise, sucrose preference, mood regulation, and intrinsic reward modulation (Hsu et al., 2016; Hsu et al., 2014). IPL neurons project to specific areas within the central gray nucleus of the pons (Quina et al., 2017).

Motivation for the present work stems from several observations and knowledge gaps in previously published work. First, we previously characterized the expression of numerous nAChRs in the MHb->IPN pathway using GFP knock-in mice (Shih et al., 2014), but did not have the opportunity to study the α7 subunit with this approach. Second, classical studies using radiolabeled α7 nAChR ligands suggest that α7 binding sites are found in IPL and IPR, and that those sites are postsynaptic and not on fibers from the MHb (Arimatsu et al., 1981; Rotter and Jacobowitz, 1984; Whiteaker et al., 1999). Third, we noted that McGehee, Role, and colleagues reported α-bungarotoxin (αBgTx)-sensitive (typically indicating α7 nAChRs) glutamate release between co-cultured IPN and MHb neurons (McGehee et al., 1995), yet there is no evidence for α7 nAChR expression in MHb neurons according to the Allen Brain Atlas. Fourth, and consistent with the previous observation, Grady and colleagues found no evidence for α7-modulated acetylcholine release from cholinergic MHb terminals in IPN (Grady et al., 2009). A synthesis of these results and literature gaps suggests that IPL neurons may express functional α7 nAChRs, which prompted the present study.

2. Materials and Methods

2.1. Materials –

Picrotoxin (PTX), atropine sulfate (atropine), acetylcholine chloride, and N-(5-Chloro-2-hydroxyphenyl)-N’-[2-chloro-5-(trifluoromethyl)phenyl]urea (NS1738) were obtained from Sigma. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), N-(5-chloro2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea (PNU-120596), methyllycaconitine (MLA), and D-(−)-2-Amino-5-phosphonopentanoic acid (D-AP5) were obtained from Tocris. QX314 chloride (QX314) was from EMD-Millipore. Nicotine hydrogen tartrate salt was obtained from Glentham Life Sciences. PA-Nic was synthesized as previously described (Banala et al., 2018) and was a generous gift from Dr. Luke Lavis (Janelia Research Campus, Howard Hughes Medical Institute).

2.2. Mice –

All experimental protocols involving mice were reviewed and approved by the Wake Forest University School of Medicine Animal Care and Use Committee. Procedures also followed the Guide for the Care and Use of Laboratory Animals provided by the National Institutes of Health Office of Laboratory Animal Welfare. All efforts were made to minimize animal distress and suffering during experimental procedures, including during the use of anesthesia. Mice were housed at 22°C on a 12-hour light/dark cycle with food and water ad libitum. A total of n=41 (male, n = 36; female, n = 4; unknown sex, n = 1) mice were used. The number of mice used in each figure is as follows, Figure 1: 4 mice (2 male, 1 female, 1 unknown sex), Figure 2: 16 male mice, Figure 3: 7 male mice, Figure 4: 5 mice (2 male, 3 female), Figure 5: 9 male mice.

Figure 1 – Chrna7 mRNA expression in lateral IPN. A.

Fluorescence in situ hybridization (FISH) in IPN with probes for Chrna7, Gad2, and Chrna5. B. The boxed area in the merge panel in (A) is shown enlarged, with Chrna7 and Gad2 signals indicated. C. Scatterplot of Gad2 (abscissa) versus Chrna7 (ordinate) normalized % coverage for all nuclei in IPL FISH images. At right, a pie graph shows the fraction of α7⁺ and α7⁻ Gad2⁺ (top) and Gad2⁻ cells. D. Scatterplot of Chrna7 (abscissa) versus Mgat5 (ordinate) normalized % coverage for all nuclei in IPL FISH images. At top right, a pie graph shows the fraction of Mgat5⁺ and Mgat5⁻ Chrna7⁺ cells. E. Scatterplot of Chrna7 (abscissa) versus Vat1l (ordinate) normalized % coverage for all nuclei in IPL FISH images. At top right, a pie graph shows the fraction of Vat1l⁺ and Vat1l⁻ Chrna7⁺ cells.

Figure 2 – Functional α7 nAChRs in IPL neurons.

A. Patch clamp recordings were made in IPL neurons and ACh (1 mM) was applied locally to stimulate nAChR currents. B. nAChR currents in IPL neurons are antagonized by methyllycaconitine (MLA, 10 nM). Example ACh-evoked responses are shown for an IPL neuron before and after bath application of MLA. Inset: Summary before/after plot showing ACh-evoked currents following MLA. p value: paired permutation test. Scale bars: 20 pA, 0.8 s C. nAChR currents in IPL neurons are potentiated by NS1738 (10 μM). Example ACh-evoked responses are shown for an IPL neuron before and after bath application of NS1738. Inset: Summary before/after plot showing ACh-evoked currents following NS1738. p value: paired permutation test. Scale bars: 20 pA, 1 s D. and E. Summary before/after plot showing ACh-evoked currents in IPR (D) and IPC (E) following NS1738. p value: paired permutation test.

Figure 3 – IPL α7 nAChRs are potentiated by PNU-120596.

A. Example ACh-evoked responses are shown for an IPL neuron before and after bath application of PNU-120596 (2 μM). Scale bars: 50 pA, 0.8 s B. ACh-evoked current amplitude in IPL neurons before and after PNU-120596 application. Black symbols denote cells that did not show an appreciable change in amplitude after PNU-120596, whereas green symbols denote PNU-120596-response cells. At right, the amplitude data after PNU-120596 application are shown normalized to the control (pre PNU-120596) amplitude. p value: paired permutation test.

Figure 4 – α3β4 nAChRs mediate non-α7 nicotinic responses in IPL neurons.

A. Example ACh-evoked current from an IPL neuron before drug application (control), after MLA (10 nM) application, and after MLA + SR16584 (20 μM) application. Scale bars: 25 pA, 1 s. B. Summary before/after plot showing all ACh-evoked current amplitude values in IPL neurons following the indicated drug treatments. p value: paired permutation test.

Figure 5 – Morphology of IPL neurons with functional α7 nAChRs.

A. Nicotinic current amplitude following nicotine uncaging in IPL neurons from PNU-120596-treated (2 μM) slices vs. control slices. p value: unpaired permutation test. B. Nicotinic current 10–90% decay time following nicotine uncaging in IPL neurons from PNU-120596-treated slices vs. control slices. Data in (B) were derived from the same cells and nicotinic responses represented in (A). p value: unpaired permutation test. C-D. IPL neuron morphology. 2PLSM images of a subset of control (C) and PNU-120596-treated (D) IPL neurons represented in (A) and (B). E. PNU-120596-treated IPL neuron and uncaging current. A 2PLSM image is shown for an IPL neuron exhibiting a large (250 pA) nicotineuncaging current and a long (~19 s) current decay time from a PNU-120596-treated slice. Scale bars: 50 pA, 10 s.

2.3. mRNA In situ Hybridization and Expression Analysis –

Mice were deeply anesthetized with Euthasol and decapitated. Brains were quickly removed on ice, snap frozen, and embedded in cryo-embedding medium (OCT). Brains were sectioned on a cryostat (CM3050; Leica) into 20 μm sections, sections were adhered to Superfrost^® Plus slides, and kept at −20°C to dry for 60 min and stored at −80°C until use. Sections were fixed with 4% paraformaldehyde and processed for RNAscope (Advanced Cell Diagnostics) multichannel fluorescent in situ hybridization (FISH) according to the manufacturer manual for Multiplex assays. Sections were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific; Waltham, MA). Probes for detection of specific targets (Chrna5, Chrna7, Gad2, mGat5, Vat1l) were purchased from Advanced Cell Diagnostics (ACD, http://acdbio.com).

Sections were imaged on a Nikon A1 confocal microscope according to the following parameters: 1024 × 1024 pixels, ~200 nm/pixel, 20× 0.75 NA objective. Nikon system images were processed with custom scripts in ImageJ (NIH). All images to be used for FISH quantification were acquired and processed in the same manner. FISH quantification employed the “fluorescence coverage (%)” method, which reports the fraction of fluorescent pixels to total pixels in a cellular region of interest (ROI). An ImageJ script used DAPI staining to locate nuclei for automated and unbiased creation of cellular ROIs. The DAPI image was filtered with a Gaussian blur filter (sigma = 3), thresholded (ImageJ “default” threshold), and the thresholded nuclei were dilated slightly (MorphoLibJ dilation filter; disc, radius = 2) to allow capture of RNA fluorescence signal just outside the nucleus but still presumably within the cell. A watershed algorithm was then applied to the filtered, binary DAPI image to isolate/separate adjacent nuclei. Finally, ROIs were detected in ImageJ (Analyze Particles algorithm; size = 20 to infinity, circularity = 0.5 to 1.0) and saved for application to fluorescence channel images. FISH channel images were each processed as follows: Gaussian blur filter (sigma = 1), Mexican Hat filter (radius = 2), threshold (Otsu algorithm). ROIs from the DAPI image routine were then applied to the filtered/thresholded FISH channel and a raw “% coverage” value was derived for each ROI. For each channel, these raw “% coverage” values for each ROI were then scaled to the single ROI in the data set with the highest “% coverage”. This normalization step accounted for differences in probe performance and target gene expression levels. Using these transformed/normalized data sets, a cutoff value of 10% “normalized % coverage” was uniformly assigned to each distribution. A scatter plot shows this cutoff for each channel as a horizontal and vertical line at 10% “normalized % coverage”. Exactly n = 3 mice were sampled for each triple hybridization shown in Figure 1 (n = 4 total mice used for RNAscope), and 2 images of the IPL were analyzed per mouse.

2.4. Brain Slice Preparation and Recording Solutions –

Mice were anesthetized with isoflurane before trans-cardiac perfusion with oxygenated (95% O₂/5% CO₂), 4°C N-methyl-D-glucamine (NMDG)-based recovery solution that contains (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO₄·7H₂O, and 0.5 CaCl₂·2H₂O; 300–310 mOsm; pH 7.3–7.4. Brains were immediately dissected after the perfusion and held in oxygenated, 4°C recovery solution for one minute before cutting a brain block containing the IPN and sectioning the brain with a vibratome (VT1200S; Leica). Coronal slices (200 μm, bregma −3.5 to −3.9 mm) were sectioned through the IPN and transferred to oxygenated, 33°C recovery solution for 12 min. Slices were then kept in holding solution containing in mM: 92 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 30 NaHCO₃, 20 HEPES, 25 glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 2 MgSO₄·7H₂O, and 2 CaCl₂·2H₂O; 300–310 mOsm; pH 7.3–7.4 for 60 min or more before recordings. Brain slices were transferred to a recording chamber (1 mL volume), being continuously superfused at a rate of 1.5–2.0 mL/min with oxygenated 32°C recording solution. For our recording chamber and solution flow rate, we estimate that complete solution exchange occurs in 5 to 8 min. The recording solution contained (in mM): 124 NaCl, 2.5 KCl, 1.2 NaH₂PO₄, 24 NaHCO₃, 12.5 glucose, 2 MgSO₄·7H₂O, 2 CaCl₂·2H₂O, 0.01 CNQX, 0.03 D-AP5, and 0.1 picrotoxin; 300–310 mOsm; pH 7.3–7.4). For puffer experiments, the recording solution was supplemented with 1 μM atropine. Patch pipettes were pulled from borosilicate glass capillary tubes (1B150F-4; World Precision Instruments) using a programmable microelectrode puller (P-97; Sutter Instrument). Tip resistance ranged from 5.0 to 7.0 MΩ when filled with internal solution. A potassium gluconate-based internal solution was used for recordings (in mM): 135 potassium gluconate, 5 EGTA, 0.5 CaCl₂, 2 MgCl₂, 10 HEPES, 2 MgATP, and 0.1 GTP; pH adjusted to 7.25 with Tris base; osmolarity adjusted to 290 mOsm with sucrose. The internal solution contained QX314 (2 mM) for improved voltage control.

2.5. Patch Clamp Electrophysiology –

Electrophysiology experiments were conducted using a Nikon Eclipse FN-1 upright microscope equipped with a 40x (0.8 NA) water-dipping (3.3 mm working distance) objective. Neurons in the rostral, central, or lateral IPN (IPR, IPC, IPL) were targeted for recording. Neurons within brain slices were first visualized with infrared or visible differential interference contrast (DIC) optics. A computer running pCLAMP 10 software was used to acquire whole-cell recordings along with a Multiclamp 700B amplifier and a Digidata 1550A A/D converter (all from Molecular Devices Inc.). Data were sampled at 10 kHz and low pass filtered at 1 kHz. Immediately prior to giga seal formation, the junction potential between the patch pipette and the superfusion medium was nulled. Series resistance was uncompensated. To record physiological events following local application of drugs, a drug-filled pipette was moved to within 20–40 μm of the recorded neuron using a second micromanipulator. A Picospritzer (General Valve) dispensed drug (dissolved in recording solution) onto the recorded neuron via a pressure ejection (10 psi, 20–300 ms). Pipette location relative to the recorded cell, along with ejection pressure, were held constant throughout the recording. Puff duration needed to be varied (minimum 20 ms, maximum 300 ms) to allow for a workable current amplitude (usually 50 to 200 pA baseline current amplitude) to be evoked in any particular cell. Because we identified a correlation between the puff duration (in ms) and the ACh-evoked current decay kinetics, we did not report these decay current values. Note that all puff experiments utilized 300 μM ACh, a saturating concentration for neuronal nAChRs. Drugs were superfused for 10–20 min prior to testing the effect of the drug on ACh-evoked nAChR currents.

2.6. 2-Photon Laser Scanning Microscopy (2PLSM), Electrophysiology, and Nicotine Uncaging –

PA-Nic photolysis was performed as previously described (Arvin et al., 2019a; Arvin et al., 2019b; Banala et al., 2018; Yan et al., 2018). A modified Olympus BX51 upright microscope and a 60x (1.0 NA) water-dipping (2 mm working distance) objective was used to visualize cells. Prairie View 5.5 (Bruker Nano) software was used for image acquisition, photostimulation, and electrophysiology acquisition via a Multiclamp 700B patch clamp amplifier. Analog signals were sampled at 1 kHz and an A/D converter (6052; National Instruments) was used for digitization. Patch clamp recordings were carried out using the internal solution mentioned above, except that Alexa 488 (hydrazide salt; 100 μM) was also included in the recording pipette to visualize cells using 2PLSM. After establishing the whole-cell configuration, the internal solution with the Alexa dye was allowed to equilibrate for 15–20 min before imaging was initiated. A Chameleon Ultra I (Coherent Laser Group; Santa Clara, CA) tunable (690–1040 nm) Ti:sapphire laser system tuned to 930 nm (80 MHz pulse repetition frequency and ~140 fs pulse duration) was used to excite Alexa 488. A M350-80-02-BK Pockels cell (ConOptics) was used for power attenuation. The system was equipped with two non-de-scanned detectors (Hamamatsu side-on multi-alkali R3896 photomultiplier tubes) for detection of green and red wavelengths (emission filters: 525/70 nm, 595/50 nm), but only the green channel was used in this study. A 405 nm continuous wave laser (100 mW OBIS LX; Coherent) was used for photostimulation/uncaging via a partially independent light path and a second set of x-y galvanometers incorporated into the scanhead (Cambridge Technologies). Laser power from the imaging and uncaging beams was measured as the beam exited the scanhead at the turndown mirror position, which is above the primary dichroic and microscope objective. An additional ~20% loss of power is expected between the point where we measured the power and the sample. Power was measured using an integrating sphere photodiode power sensor (S142C; Thorlabs). PA-Nic (100 μM) was dissolved in 10 mL of recording solution and the solution was applied to the slice via a recirculation system. The Markpoints module of Prairie View 5.5 software was used to select spots in the field of view (~1 μm diameter) for focal uncaging of nicotine via 405 nm laser light flashes (15 or 50 ms, 3–4 mW). For some recorded cells, a Z-series 2PLSM image of the cellular morphology was acquired after completion of electrophysiological recordings. A maximum intensity projection from such Z-series images was used to display uncaging positions along dendrites.

2.7. Statistics and Data Analysis –

α level was set to 0.05 for all statistical tests, which were conducted with GraphPad Prism 9 (La Jolla, CA) software or via software built into the following website: https://www.estimationstats.com. Null hypothesis statistical testing was employed, where the null hypothesis stated, in general, that treatments have no effect on the physiological measures being taken. Samples from both groups are pooled and a total of 5000 permutations (5000 bootstrap resamples, with replacement) were constructed; each permutation is a re-ordering of the original data into two groups. The mean difference is calculated for each re-ordering, and the reported p value represents the likelihood of observing a mean difference greater than or equal to the one we report if the null hypothesis is true. Paired permutation tests were conducted for data in the following figures: 2B–E, 3B, and 4B. Unpaired permutation tests were conducted for data in Fig. 5A–B. It should be noted that the p values we report from permutation tests are p value estimates. This can result in estimated p values equal to zero, even though an exact p value of zero is impossible. For example, Fig. 2C and Fig. 4B report p = 0.0, which overestimates the exact p value to some degree. Image analysis was performed with ImageJ (NIH). Analysis of electrophysiology data was performed with Clampfit (Molecular Devices) and custom scripts written in MATLAB.

3. Results

To determine whether IPN neurons express functional α7 nAChRs, we first examined Chrna7 gene expression. RNAscope fluorescence in situ hybridization experiments revealed that a subset of neurons in IPL exhibit strong expression of Chrna7 (Fig. 1A). These Chrna7⁺ cells were intermingled with IPL GABAergic neurons expressing Gad2 (Fig. 1B) and were distinct from Chrna5⁺ neurons in the IPR (Fig. 1A). We quantified Chrna7 and Gad2 mRNA expression in IPL neurons, revealing that 28% (54/192) of Gad2⁺ cells were Chrna7⁺ and ~2% (7/379) of Gad2⁻ cells were Chrna7⁺ (Fig. 1C). To further identify Chrna7⁺ IPL neurons, we searched the mouse Allen Brain Atlas for cellular markers with specific expression in IPL. We identified Mgat5 and Vat1l as genes with strong and specific expression in IPL. We then examined co-expression of Chrna7 with Mgat5 and Vat1l. Mgat5 and Vat1l were expressed in ~85% (76/89) and ~83% (69/83) of Chrna7⁺ cells, respectively (Fig. 1D, E).

Our RNAscope results suggest that some IPL neurons may express functional somatodendritic α7 nAChRs. To test this, we cut mouse coronal brain slices containing IPN. We made patch-clamp recordings from IPL neurons while stimulating nAChRs with local application of ACh (1 mM) (Fig. 2A). ACh application evoked modest inward current responses that are characteristic of nAChRs, and methyllycaconitine (MLA, 10 nM) treatment partially inhibited these responses (Fig. 2B; p = 0.002, paired permutation test; paired mean difference = −43.6 pA [95% CI = −82.2, −23.8]). To further probe the idea that IPL neurons express α7 nAChRs, we tested the ability of the α7 Type I positive allosteric modulator (PAM) NS1738 (Timmermann et al., 2007) (10 μM) to potentiate ACh-evoked inward currents. nAChR responses were significantly enhanced by NS1738 (p = 0.0, paired permutation test; paired mean difference = 33.1 pA [95% CI = 26.2, 39.5]), further supporting the conclusion that IPL neurons express functional α7 nAChRs. NS1738 did not potentiate ACh-evoked currents in IPR (p = 0.689, paired permutation test; paired mean difference = −5.76 pA [95% CI = −27.5, 2.19]) or IPC (p = 0.386, paired permutation test; paired mean difference = 5.96 pA [95% CI = −6.89, 19.0]) (Fig. 2D, E), which is consistent with our RNAscope data (Fig. 1A) showing no Chrna7 expression in these areas.

To corroborate our NS1738 data, we determined whether the α7 nAChR Type II PAM, PNU-120596 (2 μM) (Hurst et al., 2005), could similarly potentiate ACh-evoked currents. We found PNU-120596 to be capable of boosting the amplitude of ACh-evoked inward current responses in IPL neurons (example trace: Fig. 3A). PNU-120596 boosted the current amplitude in 7 of 11 cells with a measurable ACh response (Fig. 3B; p = 0.021, paired permutation test; paired mean difference = 35.7 pA [95% CI = 13.1, 69.0]). Although we also noted an increase in the decay kinetics of these responses in the presence of PNU-120596, we identified a bias in these measures that is intrinsic to the method we used to locally apply ACh to neurons within brain slices (see Materials and Methods). As such, we did not report baseline or drug-modified current decay kinetics for ACh-evoked responses.

Our MLA data (Fig. 2) indicates that a residual, MLA-resistant nAChR current is present in IPL neurons. Our prior work with nAChR GFP-knockin mice indicates that α3 and β4 nAChR subunits may be expressed in IPL (Shih et al., 2014), and our more recent Chrnb4 IPN RNAcope data did not rule out expression of these genes in IPL (Arvin et al., 2019). We tested the hypothesis that the MLA-resistant current in α7⁺ IPL neurons is mediated by α3β4* nAChRs. The α3β4 nAChR antagonist SR16584 (20 μM) (Zaveri et al., 2010) eliminated the MLA-resistant nAChR currents in α7⁺ IPL neurons (Fig. 4A,B; p = 0.0, paired permutation test; paired mean difference = −45.9 pA [95% CI = −59.7, −21.0]), confirming our hypothesis.

Finally, we examined the morphology of α7⁺ IPL neurons using 2-photon laser scanning microscopy (2PLSM) during patch-clamp recordings. We used a photoactivatable nicotine (PA-Nic) probe to evoke nAChR currents. This approach involves 405 nm laser-mediated uncaging of nicotine at discrete sites adjacent to neurons during recording and 2PLSM. Similar to our experiments using locally applied ACh (Fig. 2,3), currents evoked by nicotine uncaging were greater in amplitude (Fig. 5A, p = 0.0056, unpaired permutation test; unpaired mean difference = −182 pA [95% CI = −381, −68.7]) and had a longer decay (Fig. 5B, p = 0.0178, unpaired permutation test; unpaired mean difference = −15.1 pA [95% CI = −23.1, −5.42]) in the presence of PNU-120596 compared to control currents without a PAM. IPL neurons from the control group and those treated with PNU were imaged in the x-, y-, and z-dimension via 2PLSM. These neurons typically have an oval-shaped soma and 2–3 primary dendrites with minimal arborization (Fig. 5C,D). An exemplar micrograph and uncaging response from a PNU-treated IPL neuron is shown (Fig. 5E).

4. Discussion

Our data reveal robust functional expression of α7 nAChRs in GABAergic neurons of the lateral mouse IPN, the primary IPN subnucleus receiving input from the peptidergic dorsal MHb. In mouse, IPN Chrna7 expression appears to be exclusive to IPL neurons. There is evidence for species-specific expression of Chrna7, however. For example, an early study using radiolabeled αBgTx in rat brain reported putative α7 binding sites in dorsal aspects of the IPN in addition to the lateral portion that we report on here (Rotter and Jacobowitz, 1984). Such dorsal IPN α7 nAChRs, which were also suggested by data from Whiteaker and colleagues (Whiteaker et al., 1999), may be presynaptic receptors on fibers from α7⁺ IPL neurons, or from another brain area. α7 nAChR distribution and expression in non-human primate differs from rodent; αBgTx binding and Chrna7 mRNA studies in Macaca mulatta provided evidence for Chrna7 mRNA expression in vMHb but little to no evidence of α7 binding sites in IPN (Han et al., 2003).

We used 2-photon laser scanning microscopy and nicotine uncaging to examine the morphology of α7⁺ IPL neurons. Relative to other IPN subnuclei, most notably the IPR (Arvin et al., 2019), we observed that IPL neurons are sparse and their somata are modestly sized. IPL neurons appeared to have relatively low dendritic complexity, similar to Sst⁺ and vGlut2⁺ IPN neurons that we previously imaged (Arvin et al., 2019). Although we previously discovered that some IPN neurons have dendritic spines (Arvin et al., 2019), we did not detect this feature in IPL neurons. Given our data indicating that Chrna7 mRNA expression and functional α7 responses are only found in a fraction of IPL neurons, we speculate that α7 nAChR expression may define a subset of IPL GABA neurons. The broader function of these cells, and their neurochemical and electrical characteristics, remain to be defined.

What is the functional significance of the population of neurons in the IPL, and therefore of α7-mediated cholinergic modulation? Each dMHb hemisphere innervates the IPL in a bilateral manner, and IPL neurons send efferent projections to the central gray nucleus of the pons (CGPn), including the nucleus incertus (Quina et al., 2017). Ablation of the peptidergic dMHb to IPL pathway substantially reduces voluntary exercise and alters affect in several mood assays (Hsu et al., 2016; Hsu et al., 2014). Substance P released in IPN from the dMHb activates IPN neuronal activity and is associated with anhedonia-like behavior (Xu et al., 2018). α7 nAChRs on IPL somata or GABAergic terminals in the CGPn are therefore poised to modulate these behaviors by altering IPL firing and/or GABA release. Moreover, optical activation of the dMHb to IPL pathway is intrinsically rewarding and silencing the pathway is aversive (Hsu et al., 2014). Thus, α7 nAChRs on IPL neurons provide an additional mechanism for cholinergic modulation of motivated behavior.

α7 nAChRs are famous for their extremely rapid desensitization kinetics (Revah et al., 1991), which often makes them difficult to study with patch-clamp electrophysiology and certain drug-application techniques. As such, we were surprised to find that α7 currents in IPL neurons did not show the distinct, rapid kinetics that have previously been reported in other expression systems and in brain slice studies (Wooltorton et al., 2003). However, the kinetics we observed were similar to α7 currents previously recorded by others (Hurst et al., 2005) and by our group (Banala et al., 2018) in stratum radiatum hippocampal interneurons. Our demonstration that IPL neurons also express functional α3β4 nAChRs suggests that some ACh-evoked currents we recorded are summation currents resulting from simultaneous activation of α7 and α3β4 nAChRs. Our pharmacology studies do not exclude the possibility that IPL neurons express heteromeric α7β2 nAChRs (George et al., 2021; Nielsen et al., 2018; Wu et al., 2016; Zwart et al., 2014) and/or metabotropic α7 nAChRs (Grau et al., 2018; Horenstein and Papke, 2017; Kabbani and Nichols, 2018; Pismataro et al., 2020), but future studies will be required to probe these questions.

Limitations –

We were unable to study the electrical characteristics (using current clamp recordings) of α7(+) IPL neurons due to the inclusion of QX314 in the internal pipette solution, which interferes with voltage-gated sodium channel function. Our study included predominantly male mice, so we are unable to draw any conclusions about sex differences. We did not exhaustively document the number of IPL neurons that were not responsive to ACh, so we do not know what fraction of IPL neurons do or do not express functional nAChRs.

Conclusions –

These results indicate that α7 nAChRs likely contribute to cholinergic modulation of neurotransmission in the habenulo-interpeduncular pathway. IPL α7 nAChRs are also likely to respond to nicotine during chronic exposure such as passive delivery via osmotic minipump or volitional intake via self-administration. Given the importance of IPN GABA neurons in nicotine dependence, α7 nAChRs on IPL Gad2⁺ cells may participate in the expression of nicotine withdrawal behavioral phenotypes.

Highlights.

• α7 nAChR subunit mRNA expression in interpeduncular nucleus (IPN) is selective for GABAergic neurons in the lateral subnucleus (IPL).

• IPL neurons expressing functional α7 nAChRs also exhibit nAChR currents that are sensitive to α3β4 nAChR antagonists.

• IPL α7 nAChR currents are potentiated by a Type I and a Type II allosteric modulators.

• α7⁺ IPL neurons have modest to low dendritic complexity.