The use of viruses as transneuronal tracers has become an increasingly powerful technique for defining the synaptic organization of neural networks. Although a number of recombinant alpha herpesviruses are known to spread selectively in the retrograde direction through neural circuits only one strain, the H129 strain of herpes simplex virus type 1, is reported to selectively spread in the anterograde direction. However, it is unclear from the literature whether there is an absolute block or an attenuation of retrograde spread of H129. Here we demonstrate efficient anterograde spread, and temporally delayed retrograde spread, of H129 and three novel recombinants. In vitro studies revealed no differences in anterograde and retrograde spread of parental H129 and its recombinants through superior cervical ganglion neurons. In vivo injections of rat striatum revealed a clear bias of anterograde spread, although evidence of deficient retrograde transport was also present. Evidence of temporally delayed retrograde transneuronal spread of H129 in the retina was observed following injection of the lateral geniculate nucleus. The data also demonstrated that three novel recombinants efficiently express unique fluorescent reporters and have the capacity to infect the same neurons in dual infection paradigms. From these experiments we conclude that H129 and its recombinants efficiently infect neurons through anterograde transneuronal passage, but also are capable of temporally delayed retrograde transneuronal spread. In addition, the capacity to produce dual infection of projection targets following anterograde transneuronal passage provides an important addition to viral transneuronal tracing technology.

Rett syndrome is a devastating childhood neurological disorder caused by mutations in MECP2. Of the many symptoms, motor deterioration is a significant problem for patients. In mice, deleting Mecp2 from the cortex or basal ganglia causes motor dysfunction, hypoactivity, and tremor, which are abnormalities observed in patients. Little is known about the function of Mecp2 in the cerebellum, a brain region critical for motor function. Here we show that deleting Mecp2 from the cerebellum, but not from its neuronal subtypes, causes a delay in motor learning that is overcome by additional training. We observed irregular firing rates of Purkinje cells and altered heterochromatin architecture within the cerebellum of knockout mice. These findings demonstrate that the motor deficits present in Rett syndrome arise, in part, from cerebellar dysfunction. For Rett syndrome and other neurodevelopmental disorders, our results highlight the importance of understanding which brain regions contribute to disease phenotypes.

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Cerebellar inhibitory interneurons are important regulators of neural circuit activity for diverse motor and nonmotor functions. The molecular layer interneurons (MLIs), consisting of basket cells (BCs) and stellate cells (SCs), provide dendritic and somatic inhibitory synapses onto Purkinje cells, respectively. They are sequentially generated in an inside-out pattern from Pax2 1 immature interneurons, which migrate from the prospective white matter to the ML of the cortex. However, little is known about how MLI subtype identities and pool sizes are determined, nor are their contributions to motor learning well understood. Here, we show that GABAergic progenitors fated to generate both BCs and SCs respond to the Sonic hedgehog (Shh) signal. Conditional abrogation of Shh signaling of either sex inhibited proliferation of GABAergic progenitors and reduced the number of Pax2 1 cells, whereas persistent Shh pathway activation increased their numbers. These changes, however, did not affect early born BC numbers but selectively altered the SC pool size. Moreover, genetic depletion of GABAergic progenitors when BCs are actively generated also resulted in a specific reduction of SCs, suggesting that the specification of MLI subtypes is independent of Shh signaling and their birth order and likely occurs after Pax2 1 cells settle into their laminar positions in an inside-out sequence. Mutant mice with reduced SC numbers displayed decreased dendritic inhibitory synapses and neurotransmission onto Purkinje cells, resulting in an impaired acquisition of eyeblink conditioning. These findings also reveal an essential role of Shh signaling-dependent SCs in regulating inhibitory dendritic synapses and motor learning.

Rett syndrome is a devastating childhood neurological disorder caused by mutations in MECP2. Of the many symptoms, motor deterioration is a significant problem for patients. In mice, deleting Mecp2 from the cortex or basal ganglia causes motor dysfunction, hypoactivity, and tremor, which are abnormalities observed in patients. However, little is known about the consequences of deleting Mecp2 from the cerebellum, a brain region critical for motor function. Here we show that deleting Mecp2 from the entire cerebellum, but not from individual cerebellar cell types, causes a delay in motor learning that is overcome by additional training. We also observed irregular firing rates of Purkinje cells and transcriptional misregulation within the cerebellum of knockout mice. These findings demonstrate that the motor deficits present in Rett syndrome arise, in part, from cerebellar dysfunction. For Rett syndrome as well as other neurodevelopmental disorders, our results highlight the importance of understanding which brain regions contribute to disease phenotypes.