45. Adenoviral tetanus toxin light chain gene expression in the brainstem induces specific motor inhibition

Introduction: Focused neural inhibition is an important potential therapy for neural disorders that stem from an imbalance in neural activity. Gene based delivery of neuromodulators such as the tetanus toxin light chain fragment (TeTxLC), could help rectify these imbalances with inherent advantages over currently available methods.Tetanus toxin has a ∼100 kDa heavy chain mediating neuronal membrane binding and penetration, and a ∼50 kDa light chain (LC) which is an intracelluar protease. TeTxLC specifically cleaves synaptobrevin, a protein involved in synaptic membrane vesicle fusion, thereby disrupting neurotransmitter release and "turning off" the affected neuron. We previously reported the creation of an adenoviral vector (AdTeTxLC) to induce transient neural expression of TeTxLC. This vector triggered synaptobrevin digestion in vitro. TeTxLC expression caused neither in vitro nor in vivo neuronal death. Injections of AdTeTxLC into rat spinal cord induced transient and reproducible hindlimb dysfunction indicative of synaptic inhibiton.In the current study, we used a the rat startle system to test our hypothesis that AdTeTxLC could achieve specific inhibition of a subset of neurons within the brainstem. The rat acoustic startle reflex (ASR) is a trisynaptic reflex consisting of cochlear spiral ganglion cells, cochlear root neurons, neurons within the nucleus reticularis pontis caudalis and spinal motor neurons. The ASR is modulated by the amygdala via a multisynaptic pathway featuring the dSC nucleus.Methods: We cloned the 1496 bp synthetic gene encoding TeTxLC (AdTeTxLC), as well as the LacZ (AdLacZ) and GFP (AdGFP) genes into E1- and E3-deleted adenovirus vectors. To track gene expression, we cloned the IRES.GFP sequence into the AdTeTxLC vector. The CMV promoter drove expression of TeTxLC and LacZ. The Rous sarcoma virus (RSV) promoter drove GFP expression. We utilized published protocols to produce and purify recombinant adenovirus. Anti-LC western blots verified the presence of TeTxLC in AdTeTxLC infected HEK 293 cells.ASR was evoked in rats by a 50 ms white noise burst and was measured by an accelerometer affixed to the bottom of each test cage. Bilateral dSC of 36 rats were injected stereotactically (AP -6.8, ML =/- 1.5, DV-5.0) with 2 μl of ADTeTxLC, AdGFP (8 × 10 7 PFU), or PBS. Post operative fear potentiated startle of each rat was measured as previously described. Rats were then sacrificed and brainstems were sectioned, mounted and stained.Re