Dystonia-like behaviors and impaired sensory-motor integration following neurotoxic lesion of the pedunculopontine tegmental nucleus in mice

Abstract

Introduction: The pedunculopontine nucleus (PPTg) is a vital interface between the basal ganglia and cerebellum, participating in modulation of the locomotion and muscle tone. Pathological changes of the PPTg have been reported in patients and animal models of dystonia, while its effect and mechanism on the phenotyping of dystonia is still unknown.

Methods: In this study, a series of behavioral tests focusing on the specific deficits of dystonia were conducted for mice with bilateral and unilateral PPTg excitotoxic lesion, including the dystonia-like movements evaluation, different types of sensory-motor integrations, explorative behaviors and gait. In addition, neural dysfunctions including apoptosis, neuroinflammation, neurodegeneration and neural activation of PPTg-related motor areas in the basal ganglia, reticular formations and cerebellum were also explored.

Results: Both bilateral and unilateral lesion of the PPTg elicited dystonia-like behaviors featured by the hyperactivity of the hindlimb flexors. Moreover, proprioceptive and auditory sensory-motor integrations were impaired in bilaterally lesioned mice, while no overt alterations were found for the tactile sensory-motor integration, explorative behaviors and gait. Similar but milder behavioral deficits were found in the unilaterally lesioned mice, with an effective compensation was observed for the auditory sensory-motor integration. Histologically, no neural loss, apoptosis, neuroinflammation and neurodegeneration were found in the substantia nigra pars compacta and caudate putamen (CPu) following PPTg lesion, while reduced neural activity was found in the dorsolateral part of the CPu and striatal indirect pathway-related structures including subthalamic nucleus, globus pallidus internus and substantia nigra pars reticular. Moreover, the neural activity was decreased for the reticular formations such as pontine reticular nucleus, parvicellular reticular nucleus and gigantocellular reticular nucleus, while deep cerebellar nuclei were spared.

Conclusion: In conclusion, lesion of the PPTg could elicit dystonia-like behaviors through its effect on the balance of the striatal pathways and the reticular formations.

Keywords: dystonia; motor performance; pedunculopontine nucleus; reticular formation; sensory-motor integration; striatal pathways.

Figure 1

The flowchart of experimental design. TS, tail suspension test; OF, open field test; ART, adhesive removal test; FP, footprint test; PPI, prepulse inhibition test; CBT, challenge beam test; CT, cylinder test.

Figure 2

Lesioned areas in different layers of the PPTg for bilateral and unilateral PPTg IBO lesioned mice. (A) The overlay of lesioned areas in the bilateral PPTg IBO lesioned group is drawn according to the neuronal nuclei (NeuN) staining. The PPTg is outlined in black and every lesioned area is drawn in gray. (B) Representative photographs of NeuN staining in different layers of the PPTg for bilateral IBO lesioned mice. The PPTg is outlined in the dashed line. (C) The overlay of lesioned areas in the unilateral PPTg IBO lesioned group is drawn according to the NeuN staining. (D) Representative photographs of NeuN staining in different layers of the PPTg for unilateral IBO lesioned mice. Scale bar = 1000 μm.

Figure 3

Quantification of cholinergic neurons and lesion analysis in different layers of the PPTg for mice that underwent bilateral and unilateral PPTg IBO lesion. (A) The comparison of the absolute number of cholinergic neurons in the anterior (~AP −4.4 mm), central (~AP −4.8 mm), and posterior (~-5.1 mm) PPTg between the bilateral IBO lesioned and sham lesioned mice. (B) Representative photographs of ChAT staining of the anterior, central, and posterior PPTg for the bilateral IBO and sham lesioned mice. (C) The comparison of the absolute number of cholinergic neurons in the anterior (~AP −4.4 mm), central (~AP −4.8 mm), and posterior (~-5.1 mm) PPTg between unilateral IBO lesioned and sham lesioned mice. (D) Representative photographs of ChAT staining of anterior, central, and posterior PPTg for unilateral IBO and sham lesioned mice. The PPTg is outlined with the dotted line. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar = 1000 μm.

Figure 4

Dystonia-like behaviors following bilateral and unilateral PPTg IBO lesion. (A) Photographs of representative dystonia-like behaviors in mice following a bilateral and unilateral PPTg IBO lesion. Arrows indicated abnormal behaviors. (B) Dystonia score of the tail suspension test in mice that underwent a PPTg IBO lesion. Both the bilaterally and unilaterally PPTg IBO lesioned mice had higher dystonia scores after the lesion on day 8, and a significant difference persisted in the bilaterally lesioned group on day 15. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Impaired skilled motor functions and sensory–motor integration following PPTg IBO lesioned. (A) Both the bilaterally and unilaterally PPTg IBO lesioned mice showed elevated error per step in the challenge beam test compared with sham mice. (B) Significantly reduced latency to fall was seen in bilateral PPTg IBO lesioned mice at day 8 and day 15, while no difference was found between unilateral lesioned and sham mice in accelerating rotarod test. (C) No difference was found for time to contact and removal of the adhesive sticker on the snot between PPTg lesioned and sham mice at day 9 and day 16. (D) Decreased inhibition of prepulse stimulation was found in the bilateral PPT IBO lesioned mice at 73 db and 85 db, while no difference was seen between unilateral PPTg lesioned and sham group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

PPTg IBO lesion did not induce dopaminergic neuron loss and apoptosis in the SNpc. There was no change in the number of dopaminergic neurons (A, B) and apoptosis (C) in the SNpc following bilateral PPTg IBO lesion. In addition, there was no change in the number of dopaminergic neurons (D, E) and apoptosis (F) in the SNpc following unilateral PPTg IBO lesion. Scale bar = 1000 μm.

Figure 7

No neurodegeneration and microglial response were observed in the SNpc after the PPTg IBO lesion. Representative photographs of dopaminergic neurons, no fluorescence signal of alpha-synuclein (phosphor S129), and absent microglial response in the SNpc at AP −2.79 mm. Scale bar = 1000 μm.

Figure 8

PPTg IBO lesion did not induce cholinergic neuron loss and apoptosis in the CPu. There was no change in the number of cholinergic neurons (A, B) and apoptosis (C) in the CPu following the bilateral PPTg IBO lesion. In addition, there was no change in the number of cholinergic neurons (D, E) and apoptosis (F) in the CPu following unilateral PPTg IBO lesion. Scale bar = 1000 μm. DL, dorsolateral; DM, dorsomedial; VL, ventrolateral; VM, ventromedial.

Figure 9

Alterations of the neural activity in the BG following the PPTg IBO lesion. (A) Significantly reduced neural activity was found in the dorsolateral part of the CPu, GPi, STN, and SNpr, while no change was observed for the GPe and SNpc after the bilateral lesion. (B) representative photographs of c-Fos+ cells in the dorsolateral part of the right CPu, GPi, STN, SNpr, and SNpc following the bilateral lesion. (C) Significantly reduced neural activity was found in the dorsolateral part of the CPu, GPi, STN, and SNpr, while no change was observed for the GPe and SNpc after the unilateral lesion. (D) Representative photographs of c-Fos+ cells in the dorsolateral part of the right CPu, GPe, GPi, STN, SNpr, and SNpc following the unilateral lesion. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar = 1000 μm.

Figure 10

Alterations of the neural activity in the reticular formations and cerebellar structures following the PPTg IBO lesion. (A) Significantly reduced neural activity was found in the bilateral PnO, PnC, PCRt, and Gi, while no change was observed for the bilateral cerebellar nuclei of IntA and Lat after the bilateral lesion. (B) Representative photographs of c-Fos+ cells in the right PnO, PnC, PCRt, Gi, and IntA/Lat following the bilateral lesion. (C) Significantly reduced neural activity was found in the PnO (right), PnC (right), PCRt (bilateral), and Gi (bilateral), while no change was observed for the PnO (left), PnC (left), and bilateral cerebellar nuclei of IntA and Lat after the unilateral lesion. (D) representative photographs of c-Fos+ cells in the right PnO, PnC, PCRt, Gi, and IntA/Lat following the unilateral lesion. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar = 1000 μm.