Pitx2 cholinergic interneurons are the source of C bouton synapses on brainstem motor neurons

Abstract

Cholinergic neuromodulation has been described throughout the brain and has been implicated in various functions including attention, food intake and response to stress. Cholinergic modulation is also thought to be important for regulating motor systems, as revealed by studies of large cholinergic synapses on spinal motor neurons, called C boutons, which seem to control motor neuron excitability in a task-dependent manner. C boutons on spinal motor neurons stem from spinal interneurons that express the transcription factor Pitx2. C boutons have also been identified on the motor neurons of specific cranial nuclei. However, the source and roles of cranial C boutons are less clear. Previous studies suggest that they originate from Pitx2+ and Pitx2- neurons, in contrast to spinal cord C boutons that originate solely from Pitx2 neurons. Here, we address this controversy using mouse genetics, and demonstrate that brainstem C boutons are Pitx2+ derived. We also identify new Pitx2 populations and map the cholinergic Pitx2 neurons of the mouse brain. Taken together, our data present important new information about the anatomical organization of cholinergic systems which impact motor systems of the brainstem. These findings will enable further analyses of the specific roles of cholinergic modulation in motor control.

Figure 1

C boutons on brainstem motor neurons are Pitx2 derived. In green anti-ChAT antibody labels motor neuron somata and cholinergic C boutons on them, in red anti-dsRed antibody labels tdTomato+ Pitx2 derived synapses. (A–D) The first column contains images of the cranial motor nuclei and the next three columns contain close up images of motor neurons (merge, red only, green only). (Ai–iv) Pitx2 derived (red) C boutons (green) on the accessory nucleus (green) appear yellow. (Bi–iv) Dorsal nucleus of vagus does not receive C bouton inputs. (Ci–iv) Pitx2 derived (red) C boutons (green) on the hypoglossal nucleus (green) appear yellow. (Di–iv) Pitx2 derived (red) C boutons (green) on the nucleus ambiguus (green) appear yellow. Scale bar (A–D) 10 μm. (Ei) Half section of medulla showcasing the position of accessory nucleus, (Eii) dorsal nucleus of vagus, hypoglossal nucleus, nucleus ambiguus, in white boxes. Scale bar (E) 100 μm.

Figure 2

C boutons on brainstem motor neurons are Pitx2 derived. In green anti-ChAT antibody labels motor neuron somata and cholinergic C boutons on them, in red anti-dsRed antibody labels tdTomato+ Pitx2 derived synapses. (A–D) The first column contains images of the cranial motor nuclei and the next three columns contain close up images of motor neurons (merge, red only, green only). (Ai–iv) Pitx2 derived (red) C boutons (green) on the facial nucleus (green) appear yellow. (Bi–iv) Nucleus abducens does not receive C bouton inputs. (Ci–iv) Pitx2 derived (red) C boutons (green) on the trigeminal nucleus (green) appear yellow. (Di–iv) Trochlear and oculomotor nuclei do not receive C bouton inputs. Scale bar (A–D) 10 μm. (Ei) Half section of medulla showcasing the position of facial nucleus, (Eii) nucleus abducens, (Eiii) trigeminal nucleus, (Eiv) trochlear/oculomotor nucleus, in white boxes. Scale bar (E) 100 μm.

Figure 3

Verification of C bouton identity via postsynaptic m2 immunoreactivity. In white anti-m2 antibody, in green anti-ChAT antibody labels motor neuron somata and cholinergic C boutons on them, in red endogenous tdTomato+ fluorescence from Pitx2 derived synapses. (A) Pitx2 derived (red) C boutons (green) on the accessory nucleus (green) in alignment with m2 (white). (B) Dorsal nucleus of vagus lacks m2 immunoreactivity and C boutons. (C) Pitx2 derived (red) C boutons (green) on the hypoglossal nucleus (green) in alignment with m2 (white). (D) Pitx2 derived (red) C boutons (green) on the nucleus ambiguus (green) in alignment with m2 (white). (E) Pitx2 derived (red) C boutons (green) on the facial nucleus (green) in alignment with m2 (white). (F) Nuclus abducens lacks m2 immunoreactivity and C boutons. (G) Pitx2 derived (red) C boutons (green) on the trigeminal nucleus (green) in alignment with m2 (white). (H) Trochlear/oculomotor nucleus lacks m2 immunoreactivity and C boutons. Scale bar: 2 μm.

Figure 4

Pitx2 neurons of the brainstem. In green anti-ChAT antibody labels motor neuron somata, cholinergic interneurons and cholinergic synapses from interneurons and motor neurons, in red anti-dsRed antibody labels tdTomato+ Pitx2 neurons and Pitx2 derived synapses. Sections (A–C) presented in caudal to rostral succession (A) Caudal medulla section showcasing the localization of Pitx2 cholinergic and non-cholinergic neurons, in white box. (B) Pons section at the level of trigeminal nucleus. In red Pitx2 non-cholinergic neurons ventromedial and ventral of the laterodorsal tegmental nucleus indicated in white box. (Ci,ii) Midbrain sections, i. in white boxes the populations of the superior colliculus, of the anterior tegmental nucleus and of the penduculopontine tegmental nucleus, ii. in white box the population in the supraoculomotor area. Scale bar (A–C) 100 μm. (D) Close up image of the caudal medulla Pitx2 neurons among the dorsal nucleus of vagus and the hypoglossal. (Ei–iii) Close up image of caudal medulla cholinergic Pitx2 neurons, indicated with white arrows (i. merge, ii. red only, iii. green only). (F) Occasional non-cholinergic Pitx2 neurons appear in a lateral position away from the central canal. (G) The most rostral Pitx2 neurons in the medulla appear approximately in the middle of the dorsal nucleus of vagus and hypoglossal nucleus in the rostrocaudal axis. (H) Close up image of pons Pitx2 neurons. (I) Close up image of the Pitx2 neurons in the superior colliculus among cholinergic axons and synapses. (J) Close up of Pitx2 neurons in the red nucleus. (K) Close up of the Pitx2 neurons around the anterior tegmental nucleus (ATg). (L) Close up of Pitx2 neurons in the oral part of the pontine reticular nucleus (PnO), lateral to the tectospinal tract (ts) ventral to the penduculopontine tegmental nucleus (PPTg). (M) Close up image of Pitx2 neurons in the supraoculomotor area Scale bar (D–K) 20 μm.

Figure 5

Pitx2 neurons of the anterior brain. In green anti-ChAT antibody labels motor neuron somata, cholinergic interneurons and cholinergic synapses from interneurons and motor neurons, in red anti-dsRed antibody labels tdTomato+ Pitx2 neurons and Pitx2 derived synapses. Sections (A–F) presented in caudal to rostral succession. (A) Pitx2 neurons in the mammillary area of the hypothalamus are present in the ventral part of the section (white box). The vast majority are non-cholinergic and very few at the boundary of supramammillary and medial mammillary nucleus are cholinergic. At the dorsal part of the section the midbrain Pitx2 neurons are still evident. (B) More rostral section with Pitx2 neurons in the mammillary area. (C) Pitx2 neurons evident in the posterior hypothalamus in the medial part of the section and the parasubthalamic nucleus at the lateral sides (white box). (D) At the lateral sides of the section, Pitx2 neurons appear in the subthalamic nucleus and in the medial area in the posterior hypothalamus (white boxes). (E) Subthalamic Pitx2 neurons and a line of Pitx2 neurons in the dorsal part of the zona incerta, dorsally to subthalamic nucleus (white box). (F) More rostral section that lacks Pitx2 somata, but few Pitx2 axons are still present. Scale bar (A–F) 100 μm. (Gi–iii) Close up image of cholinergic mammillary Pitx2 neurons indicated with arrows (i: merge, ii: green only, iii: red only). (Giv) Close up image of the mammillary area with non-cholinergic Pitx2. (Hi) Close up image of the non-cholinergic Pitx2 neurons in the posterior hypothalamus. (Hii) Close up image of the non-cholinergic Pitx2 neurons in the parasubthalamic nucleus. (Hiii) Close up image of the non-cholinergic Pitx2 neurons in the subthalamic nucleus. (Hiv) Close up image of the non-cholinergic Pitx2 neurons in the dorsal part of zona incerta, dorsally to the subthalamic nucleus. Scale bar (G,H) 20 μm.