Recombinant adeno-associated virus 2 (rAAV2) has been shown to deliver genes to neurons effectively in the brain, retina, and spinal cord. The characterization of new AAV serotypes has revealed that they have different patterns of transduction in diverse tissues. We have investigated the tropism and transduction frequency in the central nervous system (CNS) of three different rAAV vector serotypes. The vectors contained AAV2 terminal repeats flanking a green fluorescent protein expression cassette under the control of the synthetic CBA promoter, in AAV1, AAV2, or AAV5 capsids, producing the pseudotypes rAAV2/1, rAAV2/2, and rAAV2/5. Rats were injected with rAAV2/1, rAAV2/2, or rAAV2/5 into selected regions of the CNS, including the hippocampus (HPC), substantia nigra (SN), striatum, globus pallidus, and spinal cord. In all regions injected, the three vectors transduced neurons almost exclusively. All three vectors transduced the SN pars compacta with high efficiency, but rAAV2/1 and rAAV2/5 also transduced the pars reticulata. Moreover, rAAV2/1 showed widespread distribution throughout the entire midbrain. In the HPC, rAAV2/1 and rAAV2/5 targeted the pyramidal cell layers in the CA1-CA3 regions, whereas AAV2/2 primarily transduced the hilar region of the dentate gyrus. In general, rAAV2/1 and rAAV2/5 exhibited higher transduction frequencies than rAAV2/2 in all regions injected, although the differences were marginal in some regions. Retrograde transport of rAAV1 and rAAV5 was also observed in particular CNS areas. These results suggest that vectors based on distinct AAV serotypes can be chosen for specific applications in the nervous system.

Pompe disease is a severe form of muscular dystrophy due to glycogen accumulation in all tissues, especially striated muscle. Disease severity is directly related to the deficiency of acid ␣-glucosidase (GAA), which degrades glycogen in the lysosome. Respiratory dysfunction is a hallmark of the disease, muscle weakness has been viewed as the underlying cause, and the possibility of an associated neural contribution has not been evaluated previously. Therefore, we examined behavioral and neurophysiological aspects of breathing in 2 animal models of Pompe disease-the Gaa ؊/؊ mouse and a transgenic line (MTP) expressing GAA only in skeletal muscle, as well as a detailed analysis of the CNS in a Pompe disease patient. Glycogen content was elevated in the Gaa ؊/؊ mouse cervical spinal cord. Retrograde labeling of phrenic motoneurons showed significantly greater soma size in Gaa ؊/؊ mice vs. isogenic controls, and glycogen was observed in Gaa ؊/؊ phrenic motoneurons. Ventilation, assessed via plethysmography, was attenuated during quiet breathing and hypercapnic challenge in Gaa ؊/؊ mice (6 to >21 months of age) vs. controls. We confirmed that MTP mice had normal diaphragmatic contractile properties; however, MTP mice had ventilation similar to the Gaa ؊/؊ mice during quiet breathing. Neurophysiological recordings indicated that efferent phrenic nerve inspiratory burst amplitudes were substantially lower in Gaa ؊/؊ and MTP mice vs. controls. In human samples, we demonstrated similar pathology in the cervical spinal cord and greater accumulation of glycogen in spinal cord compared with brain. We conclude that neural output to the diaphragm is deficient in Gaa ؊/؊ mice, and therapies targeting muscle alone may be ineffective in Pompe disease.glycogenosis ͉ motor neuron ͉ muscular dystrophy ͉ myopathy

While monosynaptic bulbospinal projections to phrenic motoneurons have been extensively described, little is known about the organization of phrenic premotor neurons in the adult rat spinal cord. As interneurons may play an important role in normal breathing and recovery following spinal cord injury, the present study has used anterograde and transneuronal retrograde tracing to study their distribution and synaptic relations. Exclusive unilateral, first-order labeling of the phrenic motoneuron pool with pseudorabies virus demonstrated a substantial number of second-order, bilaterally-distributed cervical interneurons predominantly in the dorsal horn and around the central canal. Combined transneuronal and anterograde tracing revealed ventral respiratory column projections to pre-phrenic interneurons suggesting some propriospinal relays exist between medullary neurons and the phrenic nucleus. Dual-labeling studies with pseudorabies virus recombinants also showed pre-phrenic interneurons integrated with either contralateral phrenic or intercostal motoneuron pools. The stability of interneuronal pseudorabies virus labeling patterns following lateral cervical hemisection was then addressed. Except for fewer infected contralateral interneurons at the level of the central canal, the number and distribution of phrenicassociated interneurons was not significantly altered two weeks post-hemisection (i.e. when the earliest post-injury recovery of phrenic activity has been reported). These results demonstrate a heterogeneous population of phrenic-related interneurons. Their connectivity and relative stability after cervical hemisection raises speculation for potentially diverse roles in modulating phrenic function normally and post-injury.

Following C2 spinal hemisection (C2HS) in adult rats, ipsilateral phrenic motoneuron (PhMN) recovery occurs through a time-dependent activation of latent, crossed-spinal collaterals (i.e., spontaneous crossed phrenic phenomenon; sCPP) from contralateral bulbospinal axons. Ventilation is maintained during quiet breathing after C2HS, but the ability to increase ventilation during a respiratory stimulation (e.g. hypercapnia) is impaired. We hypothesized that long-term expression of the sCPP would correspond to a progressive normalization in ventilatory patterns during respiratory challenge. Breathing was assessed via plethsymography in unanesthetized animals and phrenic motor output was measured in urethane-anesthetized, paralyzed and vagotomized rats. At 2-week post-C2HS, minute ventilation (VE) was maintained during baseline (room air) conditions as expected but was substantially blunted during hypercapnic challenge (68±3% of VE in uninjured, weight-matched rats). However, by 12 weeks the spinal-lesioned rats achieved a hypercapnic VE response that was 85±7% of control (p = 0.017 vs. 2 wks). These rats also exhibited augmented breaths (AB's) or "sighs" more frequently (p<0.05) than controls; however, total AB volume was significantly less than control at 2-and 12-week post-injury (69±4% and 80±5%, p<0.05, respectively). We also noted that phrenic neurograms demonstrated a consistent delay in onset of the ipsilateral vs. contralateral inspiratory phrenic burst at 2-12-week post-injury. Finally, the ipsilateral phrenic response to respiratory challenge (hypoxia) was greater, though not normalized, at 4-12-vs. 2-week post-injury. We conclude that recovery of ventilation deficits occurs over 2-12-week post-C2HS; however, intrinsic neuroplasticity remains insufficient to concurrently restore a normal level of ipsilateral phrenic output.