Here we used monosynaptic rabies virus based tracing, in situ molecular analysis of virallylabeled neurons, and optogenetic-assisted electrophysiological recordings to map and functionally characterize the premotor circuitry for vibrissal control in the neonatal mouse before and after a critical developmental landmark, namely the emergence of whisking behavior. We further assigned putative functions to vibrissal premotor inputs by using in situ hybridization to assess the neurotransmitter phenotypes of virally-labeled premotor neurons. The premotor wiring diagram with transmitter signs is summarized in . In parallel with the developmental onset of whisking, we detected the addition of a new premotor module that is well suited to bilaterally coordinate and potentially synchronize vibrissal movements, and facilitate cortical modulation of whisking, which are two characterictic features of this exploratory behavior.
A Wiring diagram of the premotor circuitry for vFMNs
This is the first study to selectively trace inputs to vFMNs during the first two weeks of postnatal life, when exploratory whisking first emerges. Although prior studies in adult rats have used conventional retrograde tracers (i.e., HRP or CTB) to trace inputs to the lateral FN (Hattox et al., 2002
; Isokawa-Akesson and Komisaruk, 1987
), the injection of these tracers directly into the FN is likely to have labeled not only inputs to vFMNs but also other neurons that either project axons through the injection site or that make synapses with facial motor neurons other than vFMNs. In contrast, the transsynaptic tracing method used here made it possible to infect specific groups of vFMNs with “monosynaptic” rabies viruses and subsequently label their inputs more precisely.
The different specificities of these approaches likely account for why conventional methods label a wider range of loci – including the PCRt, Sol, PAG, and peri-trigeminal motor nucleus – than observed with the monosynaptic rabies method. In fact, these additional loci were also labeled when we injected ΔG-RV directly into the lateral FN, an approach that is likely to label other inputs to the FN besides those that terminate on vFMNs. Additionally, previous conventional tracing of inputs to the FN also labeled numerous neurons in the midbrain red nucleus (Hattox et al., 2002
; Isokawa-Akesson and Komisaruk, 1987
), which were not labeled in our transsynaptic tracing. A possible explanation for this discrepancy is that diffusion of tracer from the lateral FN may have spread into the juxtaposed rubrospinal tract, resulting in false-positive labeling of neurons in the red nucleus.
The monosynaptic rabies tracing method employed here is not without limitations. Although prior studies have established that rabies virions produced from an infected neuron spread predominantly to its presynaptic partners (Kelly and Strick, 2000
; Ugolini, 1995
), at longer time points post infection, virus particles that are shed by dying neurons may infect nearby axon terminals and glial cells (e.g. Figures S2G–S2H
show labeled GFAP-positive glial cells in FN). Although we cannot entirely rule out the possibility of false-positive labeled neurons, we used relatively short survival times, and our transsynaptic tracing method labeled fewer loci than observed in other studies using conventional tracers, as described above. Furthermore, the LPGi premotor neurons are situated immediately outside the FN, and in fact direct injection of ΔG-RV into lateral FN in P6→P9 retrograde tracing experiments labeled these neurons (Figures S3 and Tables S1
) likely due to their proximity to FN. Had virus leaked from dying neurons in our trans-sysnaptic tracing experiment and infected nearby neurons, we would have also labeled LPGi neurons in our P1→P8 trans-synaptic tracing, yet this did not happen. Instead, LPGi neurons were only labeled in P8→P15 trans-synaptic tracing, arguing against non-synaptic spreading. A second potential limitation is that rabies virus jumps synapses in a stochastic fashion, and thus may fail to label neurons that make only very sparse inputs to the infected cell. This feature of the rabies tracing method could explain why we recovered only small numbers of labeled neurons in the cortex, and only when we injected larger volumes of the virus into multiple vibrissae (Figures S1E–S1H
). Notwithstanding these limitations, the monosynaptic rabies virus used here presents a useful strategy for selectively tracing inputs to motor neurons through intramuscular injections (Stepien et al., 2010
), an approach that greatly simplifies mapping of premotor circuitry at early developmental stages when precise targeting of injections to brainstem motor nuclei can be challenging. Here, this approach enabled us to detect developmental changes in the vFMN premotor circuitry.
The in situ-GFP co-localization analysis performed here represents an initial step in characterizing the functional properties of the various synaptic inputs onto vFMNs ( and ). For example, we found that GFP-labeled LPGi neurons express glutamatergic markers, and whole cell recordings from vFMNs combined with optogenetic stimulation of LPGi axons show that LPGi axon terminals can evoke robust fast inward synaptic currents in vFMNs, consistent with fast excitatory synaptic transmission involving ionotropic glutamate receptors. In contrast, a previous study showed that direct electrical stimulation in the LPGi region elicited vibrissal movements, and ascribed these behavioral effects to the actions of serotonin (5-HT) because of the presence of 5-HT fibers in LPGi and the observation that 5-HT application can elicit periodic firing in vFMNs (Hattox et al., 2003
). The present findings suggest that the glutamatergic synapses that LPGi premotor neurons form on vFMNs also contribute significantly to vibrissal movements evoked by electrical stimulation of the LPGi. Although a full characterization of the functional nature of various components of the vFMN premotor circuitry is beyond the scope of the current study, the transmitter phenotype analysis we performed here highlights the remarkable diversity of the synaptic inputs onto vFMNs.
The developmental addition of bilateral-projecting neurons in the LPGi to the vFMN premotor circuitry provides a plausible anatomical substrate for the emergence of whisking, which is characterized by cortically modulated, bilaterally coordinated movements of the vibrissae. When rodents whisk freely in air, vibrissae on both sides of the head move synchronously at similar amplitudes and frequencies (Gao et al., 2001
; Sellien et al., 2005
). Even when asymmetric bilateral vibrissal movements occur as a result of contact with objects on one side of the head or head turning, both sides usually continue to whisk at the same frequency and/or in phase, and hence are still bilaterally coordinated (except in object-localization tasks in head-fixed mice) (Mitchinson et al., 2007
; O'Connor et al., 2010
; Sachdev et al., 2003
; Towal and Hartmann, 2006
). Electrophysiological recordings show that LPGi axons provide potent excitatory input bilaterally to vFMNs, suggesting that LPGi premotor neurons are well suited to synchronize and coordinate bilateral vFMN firing. Furthermore, cortical axons form numerous synapses on LPGi neurons, suggesting that LPGi premotor neurons are especially well positioned to relay cortical command to vFMNs to facilitate cortical modulation of whisking patterns, kinematics, and behaviors (Berg et al., 2005
; Berg and Kleinfeld, 2003
; Brecht et al., 2004
; Carvell et al., 1996
; Donoghue and Parham, 1983
; Hill et al., 2011
; Huber et al., 2012
; Li and Waters, 1991
; Matyas et al., 2010
The developmental addition of new premotor modules, as seen here for murine vibrissal circuitry, may be a general theme underlying the postnatal acquisition and refinement of behaviors in vertebrates. For example, juvenile zebra finches initially produce babbling-like subsongs, but abruptly begin to produce “plastic” songs with identifiable syllables and phrases near the end of the seventh week after hatching (Aronov et al., 2008
; Immelman, 1969
). This behavioral transition follows the formation of synaptic connections between the song premotor nucleus HVC and the song motor nucleus RA, suggesting that the emergence of plastic songs depends on the developmental addition of the HVC module to the premotor pathway (Aronov et al., 2008
; Mooney, 1992
; Mooney and Rao, 1994
). Human infants also transition from infant stepping to toddler walking, from babbling to talking, and from grasping-like hand movements to fine finger movements (Gerber et al., 2010
; Kuhl and Meltzoff, 1996
; Wallace and Whishaw, 2003
), and these behavioral changes likely require the addition of new control modules to the relevant motor pathways (Dominici et al., 2011
). Therefore, vertebrate nervous system development may first involve the formation of neural circuitry for primitive movements, followed later by the addition of premotor modules that enable the generation of behaviors necessary for environmental and social exploration.