Knockouts reveal overlapping functions of M(2) and M(4) muscarinic receptors and evidence for a local glutamatergic circuit within the laterodorsal tegmental nucleus

Abstract

Cholinergic neurons in the laterodorsal tegmental (LDT) and peduncolopontine tegmental (PPT) nuclei regulate reward, arousal, and sensory gating via major projections to midbrain dopamine regions, the thalamus, and pontine targets. Muscarinic acetylcholine receptors (mAChRs) on LDT neurons produce a membrane hyperpolarization and inhibit spike-evoked Ca(2+) transients. Pharmacological studies suggest M(2) mAChRs are involved, but the role of these and other localized mAChRs (M(1-)-M(4)) has not been definitively tested. To identify the underlying receptors and to circumvent the limited receptor selectivity of available mAChR ligands, we used light- and electron-immunomicroscopy and whole cell recording with Ca(2+) imaging in brain slices from knockout mice constitutively lacking either M(2), M(4), or both mAChRs. Immunomicroscopy findings support a role for M(2) mAChRs, since cholinergic and noncholinergic LDT and pedunculopontine tegmental neurons contain M(2)-specific immunoreactivity. However, whole cell recording revealed that the presence of either M(2) or M(4) mAChRs was sufficient, and that the presence of at least one of these receptors was required for these carbachol actions. Moreover, in the absence of M(2) and M(4) mAChRs, carbachol elicited both direct excitation and barrages of spontaneous excitatory postsynaptic potentials (sEPSPs) in cholinergic LDT neurons mediated by M(1) and/or M(3) mAChRs. Focal carbachol application to surgically reduced slices suggest that local glutamatergic neurons are a source of these sEPSPs. Finally, neither direct nor indirect excitation were knockout artifacts, since each was detected in wild-type slices, although sEPSP barrages were delayed, suggesting M(2) and M(4) receptors normally delay excitation of glutamatergic inputs. Collectively, our findings indicate that multiple mAChRs coordinate cholinergic outflow from the LDT in an unexpectedly complex manner. An intriguing possibility is that a local circuit transforms LDT muscarinic inputs from a negative feedback signal for transient inputs into positive feedback for persistent inputs to facilitate different firing patterns across behavioral states.

Fig. 1.

Neurons in the laterodorsal tegmental (LDT) express M₂ muscarinic acetylcholine receptors (mAChRs). A: a coronal section through the mouse brain stem of an M₂−/− mouse stained with an antibody against choline acetyltransferase (ChAT) reveals a grossly normal distribution of cholinergic neurons in the LDT region. The box indicates the approximate area stained with an antibody against the M₂ receptor in sections from wild-type (WT; B) and M₂−/− mice (C). In WT mice, the M₂ mAChR antibody outlines somata (examples indicated by arrows in B and by “S+” in D) and dendrites (D+). Scale in A = 250 μm, scale in C = 50 μm and also applies to B. Scale in D = 10 μm.

Fig. 2.

Neuronal nitric oxide synthase (nNOS) neurons in the LDT express M₂ mAChRs. Confocal images illustrate LDT cells labeled with antibodies against the M₂ mAChR (green) and nNOS (purple). A: a merged stack of optical sections. B and C: single 1-μm optical sections within the stack illustrated in A. M₂ mAChRs are expressed on the membranes of nNOS+ cells (arrows) and nNOS− cells (asterisk). Scale bar in A = 20 μm. Scale bar in B = 10 μm and also applies to C.

Fig. 3.

M₂ mAChRs are distributed in pre- and postsynaptic profiles in the LDT. Electron micrographs illustrate the distribution of M₂ mAChR staining in the LDT. A and B: M₂ mAChR staining is distributed near the outer membranes of somata (S+) and proximal dendrites (D+). C and D: M₂ mAChR staining is distributed throughout smaller dendrites (D+) that are contacted (arrows) by unlabeled synaptic terminals (T−). E and F: M₂ mAChR staining is distributed in synaptic terminals (T+) that contact (arrows) unlabeled dendrites (D−). G and H: M₂ mAChR staining is distributed in synaptic terminals (T+) that contact (arrows) labeled dendrites (D+). Scale in A = 5 μm (also applies to B). Scale in H = 0.5 μm (applies to C–G).

Fig. 4.

Neither M₂ nor M₄ mAChRs are necessary for the carbachol (CCh)-mediated membrane hyperpolarization or inhibition of spike-evoked Ca²⁺ transients (SpECTs) in LDT neurons. A: CCh (10 μM; 90 s) elicited a membrane hyperpolarization, as well as a decrease in SpECTs in cholinergic LDT neurons from WT mice. Similar inhibitory actions were elicited in slices from either M₂−/− (B) or M₄−/− (C) mice, indicating that neither M₂ nor M₄ mAChRs alone are necessary for these actions. Asterisks in A1, B1, and C1 mark the 8-Hz train of five spikes producing the SpECTs in A2, B2, and C2. D: histograms detailing CCh-induced hyperpolarization (D1) and decrease in SpECTs (D2) in normal C57, M₂−/−, and M₄−/− mice. *P < 0.05. E: a representative example of a LDT cell from which recordings in this report were obtained. Copresence of Alexa-594 (arrow, left) with Alexa-488 immunofluorescence for nNOS (arrow, right) indicate that this recorded cell is cholinergic.

Fig. 5.

Only M₂ and M₄ mAChRs mediate the CCh-induced membrane hyperpolarization and inhibition of SpECTs in LDT neurons. In slices from M₂M₄−/− mice, CCh elicited a depolarization rather than a hyperpolarization (A1) and did not alter SpECTs (A2). Asterisk in A1 marks the 8-Hz train of five spikes producing the SpECTs in A2. B: presence of Alexa 594+ (top) and nNOS+ (bottom) in the cell from which electrophysiological recordings were obtained, as shown in A, indicate that these CCh-induced effects are elicited in cholinergic neurons. Scale: 20 μm. C: bar graphs summarizing the depolarizing action of CCh and the failure of CCh to inhibit SpECTs in neurons recorded in slices from M₂M₄−/− mice. ΔVm, change in membrane potential. Nevertheless, serotonin (5-HT) elicited a hyperpolarizing current (D1) in the same cells in which CCh was found to subsequently induce a depolarizing current (D2). These data indicate failure of CCh to induce hyperpolarization is not due to alterations in the ability of G protein-coupled inwardly rectifying potassium (GIRK) channels to be activated.

Fig. 6.

CCh induces excitatory synaptic activity [spontaneous excitatory postsynaptic currents (sEPSCs)] and does not induce inhibitory [spontaneous inhibitory postsynaptic currents (sIPSCs)] synaptic activity in slices from M₂M₄−/− mice. A1: application of strychnine and bicuculline did not block CCh-induced inward currents or postsynaptic currecnts (PSCs). A2: however, adding (2R)-amino-5-phosphonovaleric acid (APV) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) in the artificial cerebrospinal fluid (ACSF) blocked CCh-induced PSC activity. A3: partial recovery following washout of APV and DNQX. B: summary graph of effects of CCh on frequency and amplitude of excitatory postsynaptic currents (EPSCs). *P < 0.05.

Fig. 7.

The CCh-mediated inward current (I_CCh) results from a direct postsynaptic action, while the increase in sEPSCs results from action potentials in presynaptic glutamatergic neurons. A: in slices from M₂M₄−/− mice, the I_CCh was not attenuated by application of tetrodotoxin (TTX) (A2 and A3), while the increase in EPSC frequency was abolished. B: application of low-calcium ACSF did not attenuate the I_CCh, but prevented the increase in EPSC frequency (B1 and B3). B2: reapplication of normal calcium ACSF reinstated the increase in EPSC frequency produced by CCh.

Fig. 8.

M₁ and/or M₃ mAChRs mediate pre- and postsynaptic actions of CCh in slices from M₂M₄−/− mice. A: pirenzepine greatly attenuated the CCh-mediated inward current and excitatory synaptic activity. In this neuron, the direct and indirect excitatory actions were reinstated following the wash-out of pirenzepine. B: bar graph summarizing the pirenzepine attenuation of the CCh-induced inward currents (B1; n = 5) and increase in sEPSC frequency (B2; n = 5). C, D, and E: confocal images of a section through the LDT illustrates immunocytochemical staining for M₁ mAChRs (C), nNOS (D), and both M₁ receptors and nNOS (E). M₁ receptors are expressed by nNOS+ cells (asterisks) and nNOS− cells (arrows). Scale bar in C = 20 μm and applies to D. Scale bar in E = 5 μm.

Fig. 9.

CCh activates local glutamatergic neurons. A: low power fluorescence image of the LDT which has been surgically isolated from more ventral portions of the slice. Midline is indicated by the dotted line, and the dorsal-to-ventral flow of ACSF is indicated by the dotted arrow (d to v flow). Both the recording pipette (right) and puff pipette (Puff #1) contain Alexa 594 and are fluorescent, as are two recorded and filled neurons which are visible. Inset shows the leftmost neuron at higher magnification during the recording (pipette seen on right side). Responses shown in B were obtained from this neuron, with the puff pipette located at the corresponding positions. B: the increase in sEPSC frequency and postsynaptic inward current decreased as the puff location was moved out of the LDT. C: immunocytochemistry following fixation revealed the recorded cell was nNOS+ and hence cholinergic.

Fig. 10.

Recordings from LDT neurons obtained from WT slices reveal that CCh can induce an inward current and barrage of EPSPs in cholinergic neurons. A: current clamp recording from a cell in the LDT that responded to bath-applied CCh with an initial hyperpolarization and a decrease in the SpECT (right). This hyperpolarization was followed by depolarization and a barrage of EPSP activity. Asterisks (left) mark the 8-Hz train of five spikes producing the SpECTs shown at higher temporal resolution to the right. B: cumulative distribution of excitatory postsynaptic potential (EPSP) barrage latency recorded in slices from M₂M₄−/− and WT mice. The barrage latency was shifted to longer times in WT mice. C: voltage clamp recordings from an identified LDT cholinergic neuron from a WT slice following puffer application of CCh before (C1) and after the application of atropine (C2). CCh evoked an increase in EPSC frequency and an outward current that were blocked following application of atropine. Note the early atropine-resistant inward current which is attributable to activation of nicotinic acetylcholine receptors by the rapid application method.