The Role of C3-C4 Propriospinal Interneurons on Reaching and Grasping Behaviors Pre- and Post-Cervical Spinal Cord Injury

Abstract

Greater than 50% of all spinal cord injuries (SCIs) in humans occur at the cervical level and the biggest desire of quadriplegic patients is recovery of hand and digit function. Several weeks after spinal cord injury, re-organization and re-modeling of spared endogenous pathways occurs and plasticity of both supraspinal and interneuronal networks are believed to mediate functional recovery. Propriospinal interneurons (PNs) are neurons found entirely in the spinal cord with axons projecting to different spinal segments. PNs function by modulating locomotion, integrating supraspinal motor pathways and peripheral sensory afferents. Recent studies have postulated that if PNs are spared following SCI, these neurons can contribute to functional recovery by establishing synaptic connections onto motor neurons. However, to what extent cervical PNs are involved in recovery of reaching behavior is not known. In our first study, we generated a lentiviral vector that permits highly efficient retrograde transport (HiRet) upon uptake at synaptic terminals in order to map supraspinal and interneuronal populations terminating near forelimb motoneurons (MNs) innervating the limb. With this vector, we found neurons labeled within the C3-C4 spinal cord and in the red nucleus, two major populations which are known to modulate forelimb reaching behavior. We also proceeded to use a novel two-viral vector method to specifically label ipsilateral C3-C4 PNs with tetracycline-inducible GFP. Histological analysis showed detailed labeling of somas, dendrites along with axon terminals. Based on this data, we proceeded to determine the contribution of C3-C4 PNs and rubrospinal neurons on forelimb reaching and grasping before and after cervical SCI. In our second study, we have examined a double-infection technique for shutdown of PNs and rubrospinal neurons (RSNs) in adult rats. Adult rats were microinjected with a lentiviral vector expressing tetracycline-inducible inhibitory DREADDs into C6-T1 spinal levels. Adeno-associated viral vectors (AAV2) expressing TetON mixed with GIRK2 were injected into the red nucleus and C3-C4 spinal levels respectively. Rats were tested for deficits in reaching behaviors upon application of doxycycline and clozapine-n-oxide (CNO) administration. No behavioral deficits were observed pre-injury. Rats then received a C5 spinal cord lesion to sever cortical input to forelimb motoneurons and were allowed four weeks to spontaneously recover. Upon re-administration of CNO to activate inhibitory DREADDs, deficits were observed in forelimb reaching. Histological analysis of the C3-C4 spinal cord and red nucleus showed DREADD+ neurons co-expressing GIRK2 in somas and dendrites of PNs and RSNs. PN terminals expressing DREADD were observed near C6-T1 motoneurons and in the brainstem. Control animals did not show substantial deficits with CNO administration. These results indicate both rubro- and propriospinal pathways are necessary for recovery of forelimb reaching. In a separate study, we sought to determine if promoting severed CST sprouting rostral to a C5 lesion near C3-C4 PNs could improve behavioral recovery post SCI. Past studies have examined sprouting and regeneration of corticospinal tract (CST) fibers post-cervical SCI through viral upregulation of key components of the PI3K/Akt/mTOR cascade. We examined the regenerative growth potential of CST fibers that are transduced with AAV2 expressing constituively active Akt3 or STAT3 both separately and in combination (Akt3 + STAT3). We have observed significant increases in CST axonal sprouting and regeneration in Akt3 and Akt3 + STAT3 transduced samples. However, no recovery was observed as animals transduced with viral constitutively active Akt3 displayed an epileptic phenotype. Further, epileptic animals with constitutively active Akt3 were found to have significant cortical neuron cell hypertrophy, activatived astrogliosis, increased dendritic arbors and hemimegencephalitis (HME). These results indicate a new model for examining mechanisms of HME and mTOR hyperactivity-induced epilepsy in adult rodents.