Differential expression and function of nicotinic acetylcholine receptors in subdivisions of medial habenula

Abstract

Neuronal nAChRs in the medial habenula (MHb) to the interpeduncular nucleus (IPN) pathway are key mediators of nicotine's aversive properties. In this paper, we report new details regarding nAChR anatomical localization and function in MHb and IPN. A new group of knock-in mice were created that each expresses a single nAChR subunit fused to GFP, allowing high-resolution mapping. We find that α3 and β4 nAChR subunit levels are strong throughout the ventral MHb (MHbV). In contrast, α6, β2, β3, and α4 subunits are selectively found in some, but not all, areas of MHbV. All subunits were found in both ChAT-positive and ChAT-negative cells in MHbV. Next, we examined functional properties of neurons in the lateral and central part of MHbV (MHbVL and MHbVC) using brain slice patch-clamp recordings. MHbVL neurons were more excitable than MHbVC neurons, and they also responded more strongly to puffs of nicotine. In addition, we studied firing responses of MHbVL and MHbVC neurons in response to bath-applied nicotine. Cells in MHbVL, but not those in MHbVC, increased their firing substantially in response to 1 μm nicotine. Additionally, MHbVL neurons from mice that underwent withdrawal from chronic nicotine were less responsive to nicotine application compared with mice withdrawn from chronic saline. Last, we characterized rostral and dorsomedial IPN neurons that receive input from MHbVL axons. Together, our data provide new details regarding neurophysiology and nAChR localization and function in cells within the MHbV.

Keywords: habenula; interpeduncular; nicotine; nicotinic; tobacco; withdrawal.

Figure 1.

Production of GFP-labeled nAChR subunits. A, Functional characterization of α3-GFP and β4-GFP subunits. mRNA encoding WT or GFP-fused mouse α3 and β4 nAChR subunits was transcribed and microinjected (ratio of 2 α subunits:3 β subunits) into X. laevis oocytes. Using two-electrode voltage clamp (holding potential = −60 mV), inward current responses were recorded during application of ACh at the indicated concentrations. Concentration-response curves were constructed, and data were fitted to the Hill equation. The number of oocytes analyzed was 11 (WT α3β4), 11 (α3GFP β4), and 7 (α3 β4GFP). B, The nAChR genes modified and/or reported on in this study are the following: α3, α4, α6, β2, β3, and β4. For each of these genes, the M3–M4 intracellular loop is the site for insertion of monomeric GFP (abbreviated simply as “GFP”) and is encoded by exon 5. Except for the α6-GFP strain, for each of these nAChR subunit genes, a targeting vector was built that contained a modified (GFP inserted in-frame) exon 5, a neomycin selection cassette flanked by loxP sites, several kilobases of homologous 5′ and 3′ sequence (homology arms), and a diphtheria toxin-negative selection cassette. Targeting vectors were linearized and electroporated into mouse embryonic stem cells. GFP insertion into exon 5 was verified as described in Materials and Methods, targeted ES cell clones were injected into blastocysts, and blastocysts were implanted into pseudopregnant surrogates. Chimeric mice were crossed to Cre-deleter mice to remove the neomycin selection cassette in vivo, and correct gene targeting in neo-excised, F1 progeny was analyzed and verified.

Figure 2.

Localization of GFP-fused nAChR subunits in mouse brain. A, Brain atlas diagram for five bregma coordinates (bregma 2 mm, 1 mm, −1 mm, −3 mm, −4 mm) corresponding to approximate bregma coordinates for data presented in B–E. B–E, Brains from six mouse strains expressing GFP-fused nAChR subunits were fixed, sectioned at 35 μm, and stained with anti-GFP antibodies. B, C57BL/6 WT brains were processed as a staining negative control. A representative (of n = 3) image of a coronal section is shown for each nAChR-GFP mouse strain at the indicated anterior/posterior coordinate. α4 and β2 (C), α3 and β4 (D), and α6 and β3 (E) were grouped together based on their coexpression in several brain areas.

Figure 3.

Localization of GFP-fused nAChR subunits in MHb. A, Brain atlas diagram for three bregma coordinates (bregma −1 mm, −1.5 mm, and −2 mm) corresponding to anterior, medial, and posterior MHb (orange). B–E, Brains from six mouse strains expressing GFP-fused nAChR subunits were fixed, sectioned at 35 μm, and immunostained with anti-GFP antibodies. B, C57BL/6 WT brains were processed as a staining negative control. A representative (of n = 3) image of an MHb coronal section is shown for each nAChR-GFP mouse strain at the indicated anterior/posterior coordinate. α4 and β2 (C), α3 and β4 (D), and α6 and β3 (E) were grouped together based on coexpression in MHb and/or other brain areas.

Figure 4.

Localization of GFP-fused nAChR subunits in IPN. A, Brain atlas diagram for 3 bregma coordinates (bregma −3.2 mm, −3.5 mm, and −3.9 mm) corresponding to anterior, medial, and posterior interpeduncular nucleus. B–E, Brains from six mouse strains expressing GFP-fused nAChR subunits were fixed, sectioned at 35 μm, and immunostained with anti-GFP antibodies. B, C57BL/6 WT brains were processed as a staining negative control. A representative (of n = 3) image of an IPN coronal section is shown for each nAChR-GFP mouse strain at the indicated anterior/posterior coordinate. α4 and β2 (C), α3 and β4 (D), and α6 and β3 (E) were grouped together based on coexpression in MHb and/or other brain areas.

Figure 5.

nAChR MHb localization and function in ChAT(+) and ChAT(−) cells. A, For α3-GFP, α4-GFP, and α6-GFP mouse strains, coronal MHb sections were prepared, double-stained with anti-GFP and anti-ChAT antibodies, and imaged using confocal microscopy. Merged (green represents anti-GFP; red represents anti-ChAT; yellow represents colocalized pixels) micrographs of the MHb (top panels), and a high-resolution zoom of several cells in MHb (bottom panels) are shown. Single, diagonal arrowheads indicate cells expressing both ChAT and the nAChR-GFP subunit. Double, horizontal arrowheads indicate cells expressing nAChR-GFP subunits but not ChAT. Scale bars: top, 50 μm; bottom, 10 μm. B, For β2-GFP, β3-GFP, and β4-GFP mouse strains, coronal MHb sections were prepared, double-stained with anti-GFP and anti-ChAT antibodies, and imaged using confocal microscopy. Merged (green represents anti-GFP; red represents anti-ChAT; yellow represents colocalized pixels) micrographs of the MHb (top panels), and a high-resolution zoom of several cells in MHb (bottom panels) are shown. Single, diagonal arrowheads indicate cells expressing both ChAT and the nAChR-GFP subunit. Double, horizontal arrowheads indicate cells expressing nAChR-GFP subunits but not ChAT. Scale bars: top, 50 μm; bottom, 10 μm. C, Action potential firing patterns in MHb versus LHb neurons. Coronal habenular brain slices were prepared from C57BL/6 WT mice, and cell-attached recordings were made from MHb and LHb neurons. A representative trace is shown from a typical cell-attached recording from a MHb and LHb neuron. D, In whole-cell configuration, action potential firing in response to hyperpolarizing current (−50 pA) injections was recorded in MHb and LHb neurons. Representative traces show the latency to recovery of action potential firing, as well as the action potential firing frequency, after cessation of current injection. E, Responses to nicotine puffs in ChAT(+) and ChAT(−) MHb neurons. MHb neurons were voltage-clamped at −60 mV, and nicotine (30 μm, 250 ms) was puff-applied to the cell using a Picopump. Representative inward current deflections are shown for applications of nicotine at the indicated concentration. Responses from ChAT(+) and ChAT(−) neurons are shown. ChAT expression was later determined and assigned to each cell with a nicotine puff response as described in F. F, ChAT expression in MHb cells. For cells studied in E, neurobiotin was included in the patch pipette internal solution to mark the recorded cell. After successful nicotine puff experiments, neurobiotin-filled cells in slices were recovered and double-stained with streptavidin-Alexa-488 conjugates and anti-ChAT antibodies. A representative ChAT(+) and ChAT(−) MHb neuron that was studied with nicotine puffs is shown. The wide micrograph shows the location of the cell in the MHb. Inset shows whether the cell was ChAT (+) (yellow color) or ChAT (−) (green color). Scale bars: 50 μm; inset, 10 μm.

Figure 6.

Electrophysiological properties of MHbVL and MHbVC neurons. A, Diagram of approximate location of MHbVL and MHbVC. B, Hyperpolarization-activated cation currents (I_h) in MHbVC and MHbVL. MHb neurons were voltage-clamped at −60 mV, and the command voltage was pulsed to −120 mV for 1 s. The amplitude of the voltage-dependent inward current response was measured beginning with the current value at the end of the capacitive transient (indicated by the broken line), ending with the current value at the end of the voltage step. Representative I_h current traces are shown for MHbVL and MHbVC cells. Calibration: 100 pA, 200 ms. C, Quantification of I_h currents. A bar graph is shown for all recorded MHbVC (n = 13) and MHbVL (n = 18) cells. **p < 0.01 (unpaired t test). D, Measurement of rebound (anode break) delay in MHbVC and MHbVL neurons. MHb neurons recorded in whole-cell configuration were current-clamped (I = 0), followed by injection of hyperpolarizing (−75 pA) current. The latency to firing an action potential after the end of the pulse (from the broken line to the arrowhead) was measured for MHbVC and MHbVL neurons. Representative current traces are shown for MHbVL and MHbVC cells. Calibration: 50 mV, 200 ms. E, Quantification of rebound delay responses. A bar graph is shown for all responses from MHbVL (n = 18) and MHbVC (n = 13) neurons. **p < 0.01 (unpaired t test). F, Action potential amplitude adaptation in MHb neurons. MHb neurons were current-clamped (I = 0), and depolarizing current (50 pA) was injected. The amplitude of the first and last spike in the train was measured (between the two broken lines), and the adaptation ratio (last spike amplitude/first spike amplitude) was calculated for each cell. Calibration: 50 mV, 200 ms. G, Quantification of adaptation ratio measurements from F. A bar graph is shown for all adaptation ratio responses from MHbVL (n = 18) and MHbVC (n = 13) cells. **p < 0.01 (unpaired t test). H, J, For each recorded MHbVL (H; black open circles) and MHbVC (J; red open circles) neuron, I_h current amplitude was plotted on the x-axis, and rebound delay was plotted on the y-axis. Linear regression analysis was performed on the plotted data points, and r² and p values are shown. I, K, For each recorded MHbVL (I; black open circles) and MHbVC (K; red open circles) neuron, I_h current amplitude was plotted on the x-axis, and adaptation ratio was plotted on the y-axis. Linear regression analysis was performed on the plotted data points, and r and p values are shown.

Figure 7.

Nicotine-elicited inward currents in MHbV neurons. A, MHbVL or MHbVC neurons were voltage-clamped at −60 mV, and nicotine at the indicated concentration was puff-applied. Representative traces are shown for each nicotine concentration and cell type. Calibration: 1 μm nicotine, 25 pA, 5 s; 30 μm nicotine, 0.5 nA, 5 s. B, Mean nicotine (1 μm) responses from MHbVL (n = 7) and MHbVC (n = 8) neurons are shown. *p < 0.05 (unpaired t test). C, Mean nicotine (30 μm) responses from MHbVL (n = 7) and MHbVC (n = 7) neurons are shown. *p < 0.05 (unpaired t test). D, Data from B and C are plotted together on a logarithmic scale. *p < 0.05 (unpaired t test).

Figure 8.

α3β4* and/or α4* nAChRs drive nicotine-elicited firing in MHbVL neurons. A, Cell-attached recordings from MHbVL (n = 8) and MHbVC (n = 8) neurons were established, and baseline firing was recorded for several minutes, followed by superfusion of nicotine (1 μm). Normalized firing rate is plotted, with baseline firing before nicotine application set to 1.0. B, Cell-attached recordings from α4 KO MHbVL neurons (n = 9) were conducted. As in A, baseline firing was recorded followed by firing in response to superfusion of 1 μm nicotine. Data from MHbVL WT neurons shown in A are replotted for reference using dashed lines and no symbols. C, Cell-attached recordings from WT MHbVL neurons (n = 6) were conducted in response to SR16584, a putative α3β4* nAChR antagonist. Baseline firing was recorded for several minutes, followed by superfusion of SR16584 (20 μm), then superfusion of nicotine (1 μm) plus SR16584. Data from MHbVL WT neurons shown in A (response to 1 μm nicotine alone) are replotted for reference using dashed lines and no symbols. D, Representative traces from cell-attached firing experiments described in A–C. A sample current trace from t = 2.5 min (baseline) and t = 10 min (in 1 μm nicotine) is shown for the indicated cell type, genotype, and/or pharmacological treatment. Calibration: 20 pA, 200 ms. E, Quantification of data shown in A–C. For each cell from each of 4 conditions described in A–C, the fold change in action potential firing was derived compared with predrug firing. A bar graph is shown for these 4 conditions. *p < 0.05 (paired t test comparing predrug firing and peak firing during drug application for each cell).

Figure 9.

nAChR function and nicotine-evoked action potential firing in MHbVL neurons after nicotine withdrawal. A, Nicotine withdrawal procedure. C57BL/6 WT mice were implanted with saline (SAL)- or nicotine (NIC)-containing osmotic minipumps. After 14 d, minipumps were removed to initiate withdrawal, and mice were killed 1 d later for slice preparation and electrophysiology. B, Representative traces from nicotine puff-application experiments. The indicated nicotine concentration was puff-applied to MHbVL cells in slices from animals that underwent withdrawal from chronic saline or chronic nicotine. Calibration: 1 μm nicotine, 10 pA, 5 s; 30 μm nicotine, 0.5 nA, 5 s. C, Mean nicotine (1 μm) responses from saline-withdrawn (n = 6) and nicotine-withdrawn (n = 9) MHbVL neurons are shown. D, Mean nicotine (30 μm) responses from saline-withdrawn (n = 6) and nicotine-withdrawn (n = 8) MHbVL neurons are shown. **p < 0.01 (unpaired t test). E, Time course for cell-attached firing in response to nicotine (1 μm) for saline-withdrawn (n = 4) and nicotine-withdrawn (n = 6) MHbVL neurons. MHbVL neurons were held in the cell-attached configuration and action potential firing was recorded before and after bath application of 1 μm nicotine. F, Raw firing frequencies from experiment described in E. Baseline and nicotine-elicited peak action potential firing frequency in MHbVL neurons from saline-withdrawn (n = 6) and nicotine-withdrawn (n = 6) mice is shown. G, Fold change in action potential firing in response to nicotine. Using raw firing frequency data from F, we calculated the fold change in action potential firing in response to nicotine application in MHbVL neurons from saline-withdrawn (n = 6) and nicotine-withdrawn (n = 6) mice. **p < 0.01 (unpaired t test). H, c-Fos induction in IPN neurons after mecamylamine-precipitated withdrawal. C57BL/6 WT mice were implanted with saline- or nicotine-containing osmotic minipumps as in A. On day 14, mice were given an injection of mecamylamine (1 mg/kg, i.p.) and were killed and perfused 90 min later. Coronal IPN sections were stained with anti c-Fos antibodies, and a representative image is shown. Scale bars, 30 μm.

Figure 10.

IPR/IPDM neuron populations receiving input from MHbVL. A, IPR/IPDM neurons recorded in whole-cell configuration were held in current-clamp mode, and action potential firing was recording while the indicated amount of positive or negative current was injected. Based on the pattern of action potential firing, cells were classified as Type I or Type II. B, Type I or Type II IPR/IPDM neurons were voltage-clamped at −60 mV, and AMPA (100 μm) was puff-applied. Representative traces are shown for each cell type. Calibration: 300 pA, 2 s. C, Type I or Type II IPR/IPDM neurons were voltage-clamped at −60 mV, and nicotine (10 μm) was puff-applied. Representative traces are shown for each cell type. Calibration: 100 pA, 2 s. D, Quantification of action potential firing from A. Action potential firing after injection of the indicated amount of positive current was recorded, and the maximum instantaneous firing rate was derived and plotted for each current injection. The number of neurons analyzed was as follows: n = 16 (Type I), n = 9 (Type II). E, AMPA concentration response relation for Type I and Type II IPR/IPDM neurons. AMPA-evoked inward currents were recorded for a range of AMPA concentrations (in μm: 1, 3, 10, 30, 100, and 300). Data from Type I neurons were fitted to a single-component function, whereas data from Type II neurons were fitted to a biphasic function. The number of Type I neurons analyzed for each AMPA concentration was as follows: n = 3 (1 μm), n = 3 (3 μm), n = 7 (10 μm), n = 4 (30 μm), n = 16 (100 μm), n = 6 (300 μm). The number of Type II neurons analyzed for each AMPA concentration was as follows: n = 2 (1 μm), n = 2 (3 μm), n = 4 (10 μm), n = 3 (30 μm), n = 9 (100 μm), n = 3 (300 μm). F, Quantification of nicotine-evoked currents in Type I and Type II IPR/IPDM neurons. Cells were voltage-clamped at −60 mV, nicotine (1 and 10 μm) was puff-applied, and mean values are plotted. The number of Type I neurons analyzed for each nicotine concentration was as follows: n = 7 (1 μm), n = 7 (10 μm). The number of Type II neurons analyzed for each nicotine concentration was as follows: n = 7 (1 μm), n = 7 (10 μm). *p < 0.05 (unpaired t test).

Figure 11.

nAChRs and the MHbV to IPN pathway: summary schematic. A, nAChR-expressing cells of the MHbVI project preferentially to ventral IPN, and the indicated (red text) nAChR subunits are expressed in MHb somata and in presynaptic terminals in IPN. B, MHbVC projects preferentially to central regions of IPN. C, MHbVL projects preferentially to IPR/IPDM. At least two cell types exist in IPR/IPDM. Type I neurons exhibit relatively small inward currents in response to puffed nicotine (NIC) and AMPA and fire tonic action potentials in response to positive current injection. Type II neurons exhibit relatively (compared with Type I cells) large inward currents in response to puffed NIC or AMPA and fire bursts of action potentials following positive current injection.