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. 2019 Oct 18;8:e49291. doi: 10.7554/eLife.49291

Monosynaptic tracing maps brain-wide afferent oligodendrocyte precursor cell connectivity

Christopher W Mount 1,2,3,†, Belgin Yalçın 1,†, Kennedy Cunliffe-Koehler 1, Shree Sundaresh 1, Michelle Monje 1,4,5,6,✉
Editors: Klaus-Armin Nave7, Gary L Westbrook8
PMCID: PMC6800000  PMID: 31625910

Abstract

Neurons form bona fide synapses with oligodendrocyte precursor cells (OPCs), but the circuit context of these neuron to OPC synapses remains incompletely understood. Using monosynaptically-restricted rabies virus tracing of OPC afferents, we identified extensive afferent synaptic inputs to OPCs residing in secondary motor cortex, corpus callosum, and primary somatosensory cortex of adult mice. These inputs primarily arise from functionally-interconnecting cortical areas and thalamic nuclei, illustrating that OPCs have strikingly comprehensive synaptic access to brain-wide projection networks. Quantification of these inputs revealed excitatory and inhibitory components that are consistent in number across brain regions and stable in barrel cortex despite whisker trimming-induced sensory deprivation.

Research organism: Mouse

Introduction

Excitatory and inhibitory synapses between neurons and OPCs are well-established and the ultrastructural and electrophysiological features of these ‘axon->glial’ synapses have been investigated in slice preparations, generally by evoking potentials in local fiber bundles (Ziskin et al., 2007; De Biase et al., 2010; Kukley et al., 2007; Lundgaard et al., 2013). However, the afferent projections from neurons to OPCs providing this synaptic input have not been systematically mapped, and thus our understanding of the neuronal territories accessed by neuron->OPC synapses has been limited. Recent evidence has demonstrated that neuronal activity robustly regulates OPC proliferation, oligodendrogenesis, and myelination in both juvenile and adult rodents (Gibson et al., 2014; Mitew et al., 2018; Hughes et al., 2018) and also influences axon selection during developmental myelination in zebrafish (Mensch et al., 2015; Hines et al., 2015). These activity-regulated responses of oligodendroglial cells have been shown to confer adaptive changes in motor function (Gibson et al., 2014), are necessary for some forms of motor learning (McKenzie et al., 2014; Xiao et al., 2016) and contribute to cognitive behavioral functions such as attention and short-term memory (Geraghty et al., 2019). Appreciation for this plasticity of myelin has stoked interest in the axon->glial synapse as a means by which OPCs could detect and integrate activity-dependent neuronal signals. Here, we employ a modified rabies virus-based monosynaptically-restricted trans-synaptic retrograde tracing strategy to elucidate a map of neuronal synaptic inputs to OPCs in the corpus callosum (CC), secondary motor cortex (MOs), and primary somatosensory cortex (SSp) in vivo. We find brain-wide, functionally-interconnected inputs to OPCs and that the degree of this connectivity is stable across brain regions and is maintained despite whisker trimming-induced sensory deprivation in barrel cortex at the timepoint examined.

Results

Development and validation of retrograde monosynaptic OPC tracing strategy

Owing to the lack of viral tools to achieve specific transgene expression in OPCs, we employed a transgenic strategy by crossing Pdgfra::CreER mice (Kang et al., 2010), which permit OPC-specific Cre recombinase expression, with a Cre-inducible RABVgp4/TVA mouse (Takatoh et al., 2013). Offspring of this cross express rabies virus glycoprotein 4 (gp4) and the avian TVA receptor specifically in OPCs upon tamoxifen administration (Pdgfra::CreER-(gp4-TVA)fl). Subsequently, we performed stereotaxic injection of ASLV-A (EnvA)-pseudotyped gp4-deleted rabies virus encoding EGFP (SADΔG-EGFP(EnvA)). EnvA’s highly specific interaction with the TVA receptor ensures restriction of primarily-transduced cells – hereafter named starter cells – to the Pdgfra+ OPC cell population. Because these OPCs also express rabies gp4, virions can be assembled within these starter cells and spread retrogradely across single synaptic connections to presynaptic input neurons; however, because these input neurons do not express gp4, there is no additional spread of virus beyond these monosynaptic connections (Figure 1A) (Wickersham et al., 2007). A caveat to this approach is that OPCs that differentiate to oligodendrocytes (Ye et al., 2009) between tamoxifen administration and virus injection would still be susceptible to infection; likewise, infected OPCs that undergo differentiation could skew histological assessment of input to starter cell ratios. To mitigate these concerns, we followed a narrow injection time course (Figure 1A) beginning in adult (6 month old) mice, when rates of OPC differentiation are substantially lower than in juveniles (Young et al., 2013).

Figure 1. Monosynaptically-restricted rabies virus enables tracing of synaptic inputs to OPCs.

(a) Outline of experimental strategy used to label inputs to Pdgfra+ OPCs. (b) Injection of SADΔG-EGFP(EnvA) into sub-cingular corpus callosum results in widespread labeling of EGFP+ input neurons (representative injection site image from n = 10 animals. Green = EGFP, white = DAPI). (c) Injection of SADΔG-EGFP(EnvA) into animals lacking Pdgfra::CreER driver allele results in only minimal transduction, likely resulting from minimal quantities of EnvA- viral particles (representative image of n = 4 animals. Green = EGFP, white = DAPI). (d) Pdgfra+/Olig2+ OPC starter cells (left) are transduced with SADΔG-EGFP(EnvA) (right, same cell. Magenta = Pdgfra, white = Olig2, green = EGFP). (e) Immunostaining confirms Cre recombinase expression in Pdgfra+ OPCs (green) but not NeuN+ neurons (blue). Scale bars in (d,e) represent 10 microns.

Figure 1.

Figure 1—figure supplement 1. SADΔG-EGFP(EnvA) specificity and starter characterization.

Figure 1—figure supplement 1.

(a) Immunofluorescence staining for the white matter and reactive astrocyte marker Gfap (white; top panels) and the macrophage marker Iba1 (white; bottom panels) does not identify substantial evidence of EGFP+ (green) astroglial/microglial cells despite evidence of reactive gliosis in the injection site. (b) Quantification of colocalization studies represented in (a) demonstrates minimal overlap concentrated to the injection site that is not increased in Cre+ animals compared with minimal expected background labeling in Cre WT controls. Bars indicate the mean, n = 6 animals (Cre WT) or nine animals (Cre+) respectively; error bars represent SEM. (c) Example of EGFP+Pdgfra+Olig2+ starter cells with disrupted morphology. Blue = DAPI, green = EGFP; red = Pdgfra; white = Olig2. (d) Representative micrograph of SADΔG-mCherry compared against SADΔG-EnvA-GFP injections in Pdgfra::CreER and Cre WT animals after tamoxifen injection. In the absence of EnvA, widespread transduction occurs independently of Cre expression, confirming that viral EnvA confers starter cell specificity in Pdgfra::CreER animals. (e) Representative micrograph of CASPR (red) colocalization (indicated with white arrow) along myelinated GFP+ (green) input neurons, as well as unmyelinated axonal profiles lacking CASPR (green arrows). (f) Depth of Pdgfra+GFP+ OPC starter cells as measured from the pia. Histogram divided into 100 micron bins, with y-axis labels indicating center of bin; n = 68, 75, 93 cells respectively with injection site indicated by legend color. Scale bars in (a) are 50 microns, in (c) are 10 microns, in (d) are 500 microns, and in (e) is five microns.
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Viral injection occured three days following a single dose of tamoxifen. At 5 days following injection of SADΔG-EGFP(EnvA) into the genu of the corpus callosum inferior to the cingulum bundle, we observed substantial labeling of presynaptic neuronal inputs (Figure 1B). In control (gp4-TVA)fl mice lacking the Pdgfra::CreER transgene, injection of tamoxifen and modified rabies virus achieved only minor, local background labeling expected to result from small fractions of EnvA negative viral particles (Figure 1C). Pdgfra+/Olig2+/EGFP+ starter cells were present in the injection site (Figure 1D), while other glial subtypes including Gfap+ white matter astrocytes as well as Iba1+ macrophages and microglia were EGFP negative, confirming the specificity of glial infection to the targeted OPC population (Figure 1—figure supplement 1A,B). Immunostaining for Cre expression at this timepoint confirmed previously-reported driver specificity to OPCs (Kang et al., 2010), with no expression of Cre in NeuN+ neurons in this context (Figure 1E). Thus, the starter cell population is limited to the oligodendroglial lineage, with no evidence of non-synaptic ‘leak’ of virus into other cell populations. While viral infection did result in limited toxicity to infected OPCs as suggested by morphology, expression of the identifying markers, Pdgfra and Olig2, was retained (Figure 1—figure supplement 1C). To further confirm specificity of OPC transduction, we performed comparison injections with SADΔG-mCherry lacking EnvA – and therefore capable of unrestricted transduction – into Pdgfra::CreER and Cre WT control animals (Figure 1—figure supplement 1D). In stark contrast to (EnvA)-bearing virus, broad cortical transduction is observed in both animals. This difference in transduction patterns between EnvA-bearing virus and non-psuedotyped virus indicates that TVA expression in OPCs successfully restricts EnvA-bearing virus transduction.

OPCs in corpus callosum receive brain-wide synaptic input

Quantification of input neurons revealed extensive neuronal territories that synapse onto starter OPCs in the corpus callosum (Figure 2A,B). Summing all inputs identified to these white matter OPCs, viral input/starter ratios in this context are approximately 23 (slope of linear regression 23.46 ± 2.8 standard error, Figure 2C), with neuronal input cells clearly identifiable by morphology and EGFP expression (Figure 2D). To corroborate this ratio, we performed immunofluorescence staining of PSD95 puncta in the corpus callosum and quantified colocalized puncta with Pdgfra+ OPCs with the aid of 3D modeling software (Figure 2—figure supplement 1A). Assessing 95 cells across nine animals, we found a distribution of PSD95-colocalized puncta from 0 to 67, with a mean of 21.62 puncta ±2.8 standard error per OPC (Figure 2—figure supplement 1B,C). Both myelinated and non-myelinated input axons are present as indicated by immunofluorescence staining for paranodal CASPR (Figure 1—figure supplement 1E). Measuring starter OPC depth relative to the pia confirmed their subcortical location (Figure 1—figure supplement 1F). Neuronal inputs to OPCs in the genu of the corpus callosum inferior to the cingulum bundle are concentrated in dorsal and ventral mPFC (defined here to include anterior cingulate, pre- and infralimbic regions) and secondary motor cortex (MOs) (Figure 2E). Inputs from primary motor (MOp) and primary somatosensory (SSp) cortices are also present, along with substantial connectivity from the thalamus (TH, Figure 2E). These thalamic inputs are most consistently localized to ventroanterolateral (VAL), anteromedial (AM), and anterodorsal (AD) nuclei, consistent with thalamocortical projection neurons targeting motor and prefrontal cortical areas (Figure 5). The majority of inputs identified arise ipsilateral to the viral injection site; however, the relatively high contribution of inputs arising in the contralateral mPFC combined with high overall labeling densities in this region suggest that callosal OPCs are substantially innervated by contralateral intracortical projections (Figure 2F). By contrast, thalamic inputs are ipsilaterally restricted, further supporting the monosynaptic restriction of viral labeling (Figure 2F).

Figure 2. Neuronal inputs to callosal OPCs arise from functionally interconnected cortical and thalamic areas.

(a) Representative sections of neuronal input labeling to OPCs following stereotaxic injection of SADΔG-EGFP(EnvA) to corpus callosum underlying the secondary motor area. Green = EGFP, white = DAPI. (b) Schematic of injection site. (c) Linear regression fit of neuronal input/Pdgfra+ OPC starter cells. Each point represents one animal, n = 12 animals, R2 = 0.8732, slope = 23.46 ± 2.8 standard error. (d) Representative confocal micrograph of EGFP+ (green) input neurons in medial prefrontal (mPFC) cortex. (e) Inputs to callosal OPCs largely arise from frontal association cortices but also include primary motor and somatosensory areas and thalamic nuclei. Each bar represents mean input percentage, error bars indicate SEM, n = 10 total. mPFC = medial prefrontal cortex (anterior cingulate, prelimbic, infralimbic regions), MOs = secondary motor area, MOp = primary motor area, SSp = primary somatosensory area, TH = thalamus (including all thalamic nuclei). (f) Percent of input neurons ipsilateral or contralateral to the OPC starter cells. Bars indicate mean, error bars indicate SEM, n = 10 animals. (g) Representative image of parvalbumin+ (PV+, magenta) GFP+ (green) input neuron (arrowhead) and quantification of percentage of input neurons that co-label with immunofluorescence makers for PV, somatostatin (SOM), or vasoactive intestinal peptide (VIP). Bars represent mean, error bars indicate SEM, n = 6 animals total. Scale bars in (d,g) represent 20 microns.

Figure 2.

Figure 2—figure supplement 1. PSD95 puncta colocalization with OPCs.

Figure 2—figure supplement 1.

(a) Representative micrograph of PSD95 puncta (red) and Pdgfra+ (green) OPC in corpus callosum, with subsequent rendering of 3D filament model of OPC and colocalized puncta generated in Imaris. Scale bars 10 microns, (b) Frequency distribution of PSD95 colocalized puncta across all OPCs imaged in corpus callosum and rendered as in (a); bars indicate relative frequency, n = 95 cells across nine animals. (c) Comparison of mean PSD95 colocalized puncta per OPC calculated from the nine animals investigated with neuronal input:OPC starter ratio estimated by linear regression as in (Figure 2C). Error bars indicate standard error of each respective measurement.
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While GABAergic inputs to OPCs have been described (Kukley et al., 2008; Lin and Bergles, 2004), the majority of evidence for neuron-OPC synaptic connectivity in the corpus callosum arises from recordings of glutamatergic excitation either spontaneously or following callosal fiber stimulus (Ziskin et al., 2007; De Biase et al., 2010). Immunostaining for characteristic non-overlapping cortical inhibitory subpopulation markers accounting for the majority of total cortical inhibitory neurons (Pfeffer et al., 2013) – parvalbumin (PV), vasoactive intestinal peptide (VIP), and somatostatin (SOM) – revealed PV+/GFP+ co-labeled inputs encompassing approximately 3% of inputs to OPCs in CC (Figure 2G). The majority of these PV+GFP+ inputs were present ipsilaterally in the overlying MOs/mPFC. SOM or VIP co-labeled GFP+ input neurons comprised 1% or less of total inputs to OPCs in the CC. The excitatory to inhibitory neuron ratio of inputs to callosal OPCs is ~20:1, with inhibitory neurons defined by PV, VIP or SOM-expression.

OPCs in premotor cortex receive synaptic input from premotor cortical and thalamic neurons

Examining the afferents to cortical OPCs in the secondary motor (premotor, MOs, M2) cortex, injection of SADΔG-EGFP(EnvA) into MOs of Pdgfra::CreER-(gp4-TVA)fl mice (Figure 3B) again resulted in infection of Pdgfra+/EGFP+ starter OPCs. Labeled input neurons were strikingly predominant within cortical territory defining the boundaries of MOs (Figure 3A,D). Input to starter cell ratios for these gray matter OPCs were not substantially different from those in CC-injected animals (slope of best-fit linear regression = 18.76 ± 4.4 standard error, Figure 3C). Beyond MOs, a smaller fraction of inputs arise from primary motor cortex (MOp), nearby medial prefrontal cortex (mPFC), and to a lesser extent, projections from SSp, and thalamocortical projection neurons (Figure 3D,E), illustrating brain-wide premotor circuit inputs to premotor cortical OPCs. Immunostaining for markers of interneuron identity revealed PV+/GFP+ inputs averaging 6% of total input neurons to mPFC OPCs, while SOM+/GFP+ or VIP+/GFP+ costaining was present in approximately 1% or less of total inputs (Figure 3F). Input neurons to MOs OPCs are primarily ipsilateral, with a smaller proportion of afferent projections arising contralaterally than observed in OPCs within the CC (Figure 3G). Like CC OPCs, the excitatory to inhibitory ratio of inputs to premotor cortical OPCs is ~20:1, with inhibitory neurons defined by PV, VIP or SOM-expression.

Figure 3. Circuit-specific cortical and thalamic neuronal inputs to OPCs in secondary motor area (MOs).

Figure 3.

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(a) Representative sections of neuronal input labeling to OPCs following stereotaxic injection of SADΔG-EGFP(EnvA) to MOs. Green = EGFP, white = DAPI. (b) Schematic of injection site. (c) Linear regression fit of neuronal input/Pdgfra+ starter cells. Each point represents one animal, R2 = 0.7486, slope = 18.76 ± 4.4 standard error). (d) Representative confocal micrographs of EGFP+ (green) input neurons in secondary motor cortex (MOs) and thalamus. (e) Inputs to gray matter OPCs found in MOs are chiefly located within MOs, n = 8 animals total. mPFC = medial prefrontal cortex (anterior cingulate, prelimbic, infralimbic regions), MOs = secondary motor area, MOp = primary motor area, SSp = primary somatosensory area, TH = thalamus (including all thalamic nuclei). (f) Percentage of input neurons that co-label with immunofluorescence makers for parvalbumin (PV), somatostatin (SOM), or vasoactive intestinal peptide (VIP), n = 5 animals total. (g) Percent of input neurons ipsilateral or contralateral to OPC starter cells. Bars indicate mean, error bars indicate SEM, each point represents an individual animal (n = 8). Scale bars in (d) represent 20 microns.

OPCs in primary somatosensory cortex receive synaptic input from ipsilateral sensory cortex and thalamic neurons

To assess whether the pattern of cortical OPC inputs arising from local cortical neurons and functionally-associated thalamic nuclei was specific to MOs, we injected SADΔG-EGFP(EnvA) into primary somatosensory cortex (SSp) of Pdgfra::CreER-(gp4-TVA)fl mice (Figure 4B). As in MOs and CC, this resulted in primary infection of Pdgfra+/EGFP+ starter OPCs, and as in MOs, inputs were confined primarily to sensory cortical territory (SSp) (Figure 4A). Input to starter cell ratios at this site did not differ significantly from injections in CC or MOs (slope of best-fit linear regression = 22.57 ± 3.8 standard error, Figure 4C). Examination of GFP+ input neurons revealed inputs arising primarily from SSp across multiple cortical layers and from the thalamus (Figure 4D,E). In contrast to OPCs residing in CC, and to a lesser extent MOs, input neurons to OPCs in SSp are almost entirely ipsilaterally-restricted, and there is a small (<5%) contribution of input neurons from mPFC, MOs, or MOp. As in MOs, immunostaining for markers of interneuron identity revealed approximately 4% of GFP+ input neurons colabeled with PV, while SOM+ or VIP+ inputs comprised 1% or less of total GFP+ inputs (Figure 4F). Like CC and promotor cortex OPCs, the excitatory to inhibitory ratio of inputs to somatosensory cortical OPCs is ~20:1, with inhibitory neurons defined by PV, VIP or SOM-expression.

Figure 4. Circuit-specific cortical and thalamic neuronal neuronal inputs to SSp.

Figure 4.

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(a) Representative sections of neuronal input labeling to OPCs following stereotaxic injection of SADΔG-EGFP(EnvA) to SSp. Green = EGFP, white = DAPI. (b) Schematic of injection site. (c) Linear regression fit of neuronal input/Pdgfra+ starter cells. Each point represents one animal, R2 = 0.8145, slope = 22.57 ± 3.8 standard error). (d) Representative confocal micrographs of EGFP+ (green) input neurons in primary somatosensory cortex (SSp) and thalamus. (e) Inputs to gray matter OPCs found in SSp are chiefly located within SSp. n = 9 animals total. mPFC = medial prefrontal cortex (anterior cingulate, prelimbic, infralimbic regions), MOs = secondary motor area, MOp = primary motor area, SSp = primary somatosensory area, TH = thalamus (including all thalamic nuclei). (f) Percentage of input neurons that co-label with immunofluorescence makers for parvalbumin (PV), somatostatin (SOM), or vasoactive intestinal peptide (VIP), n = 5 animals. Bars indicate mean, error bars indicate SEM. Scale bars in (d) represent 20 microns.

Thalamic input neurons to OPCs arise from functionally-related thalamic nuclei

For OPCs in all brain regions studied, a substantial fraction of synaptic inputs arise from thalamic neurons. To assess whether these thalamic inputs arise from functionally-related nuclei, we registered acquired image tiles to the Allen Institute reference adult mouse brain atlas (Lein et al., 2007) and localized identified GFP+ inputs (Figure 5). Thalamic projections providing synaptic input to OPCs located in the corpus callosum underlying primary and secondary motor cortex arise primarily from ventral anterior-lateral (VAL) and anteromedial (AM) nuclei, consistent with known projections to motor planning territories (Figure 5A), along with projections from the anterodorsal (AD) nucleus. Strikingly, thalamic inputs to MOs OPCs also arose primarily from VAL and AM nuclei (Figure 5B). This is largely distinct from thalamic projections to SSp OPCs, which arise primarily within ventral posterolateral (VPL) and ventral posteromedial (VPM) regions, consistent with known projections to somatosensory targets (Figure 5C). Together, this suggests that particularly in the case of cortical OPCs, these thalamocortical synaptic inputs arise from functionally-related thalamic nuclei, consistent with expected thalamocortical projection patterns.

Figure 5. Thalamic inputs to OPCs arise from functionally-related nuclei.

Figure 5.

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Tiled immunofluorescence images of GFP+ input neurons were registered to the Allen Brain Atlas to determine the thalamic nuclei from which the inputs arise. Nuclei are color-coded here according to the primary function of their cortical projection targets – motor (yellow), sensory (blue), or mixed (green). (a) Thalamic inputs to OPCs in the CC underlying primary and secondary motor cortices arise primarily from ventral anterior-lateral (VAL) and anteriomedial (AM) nuclei. (b) Thalamic inputs to OPCs in MOs arise primarily from VAL and AM nuclei. (c) Thalamic inputs to SSp arise primarily from ventral posterolateral (VPL) and ventral posteromedial (VPM) nuclei. All bars indicate mean, error bars indicate SEM. Scale bars represent 100 microns. N = 10 mice (CC), eight mice (MOs), and six mice (SSp) respectively. Thalamic nuclei defined as presented in the Allen Brain Atlas and abbreviated as follows: VAL = ventral anterior-lateral, AMd = anteromedial dorsal part, AMv = anteromedial ventral part, VPL = ventral posterolateral, VPM = ventral posteromedial, PO = posterior complex, MD = mediodorsal, AD = anterodorsal.

Total synaptic connectivity to OPCs is consistent across brain regions despite reduced input neuron activity

To assess whether the degree of synaptic connections to OPCs varied across the injection sites assessed, we compared the average neuronal input ratios, assessed as the slope of the best-fit linear regression to total GFP+ inputs versus starter Pdgfra+/GFP+ OPCs. We found no significant difference in the synaptic input ratios between OPCs in the CC, MOs, or SSp (Figure 6A). To assess whether perturbations of synaptic input activity might modify the degree of synaptic connectivity, we performed daily whisker trimming of Pdgfra::CreER-(gp4-TVA)fl mice for 11 days prior to tamoxifen injection (Figure 6B). Anticipating that input activity to the cortical barrel field would be reduced in whisker-trimmed animals, we then injected SADΔG-EGFP(EnvA) into barrel field of trimmed and matched untrimmed control animals. Five days after viral injection, the animals were euthanized and total GFP+ input neurons and Pdgfra+/GFP+ starter OPCs were quantified. We found no significant difference in neuronal input to starter ratio between whisker-trimmed and untrimmed control animals as assessed by the slope of best-fit linear regression (Figure 6C,D). Moreover, we found no significant difference in the distribution of input neurons between trimmed and untrimmed animals, with primarily somatosensory cortical inputs and approximately 10% of inputs arising from thalamus (Figure 6E). Quantification of immunostaining for interneuron markers revealed PV+GFP+ inputs in equal proportion (3–4%) in trimmed and untrimmed groups (Figure 6F), indicating an unchanged excitatory to inhibitory (PV+ neuron) ratio of OPC inputs regardless of whisker trimming at this time point. Taken together, neither OPC location across white and gray matter territories, nor modification of input activity in barrel field by whisker trimming modified the quantity or pattern of neurons providing synaptic input to OPCs at this time point.

Figure 6. Neuronal input to OPC starter ratios are consistent across brain region and despite whisker trimming-induced afferent activity deprivation.

Figure 6.

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(a) Neuronal input to OPC starter ratios, as measured by the slope of the best-fit linear regression of GFP+ input neurons against Pdgfra+/GFP+ OPC starter cells. Bars indicate mean, error bars indicate standard error of linear regression. (b) Outline of whisker trimming deprivation expriment and subsequent viral injection into barrel field of somatosensory cortex (SSp,BF). (c) Representative images of GFP+ input neurons in SSp, BF of non-trimmed and trimmed animals, white = DAPI, green = GFP. (d) Scatter plot of GFP+ input neurons against Pdgfra+/GFP+ starter OPCs and best-fit linear regression to assess average input to starter cell ratio in whisker-trimmed (Trimmed) and control (No Trim) groups. Each point represents an independent animal, n = 13 (Trimmed), n = 11 (No Trim). (e) Distribution of GFP+ neuronal inputs to OPCs in Trimmed and No Trim groups as a percentage of total inputs. n = 13 (Trimmed), n = 11 (No Trim). (f) Proportion of total GFP+ input neurons immunostaining for parvalbumin (PV) in Trimmed and No Trim groups, n = 5 animals each. Bars indicate mean, error bars indicate SEM. Scale bars in (c) represent 100 microns. Statistical testing performed by Tukey’s multiple comparisons test, n.s. indicates p>0.05.

Discussion

Substantial progress in characterizing the electrophysiological properties of neuron->OPC synapses has yet to clarify their potential role in modulating oligodendrocyte lineage dynamics and ultimately animal behavior. In particular, prior to this work little was known regarding the extent of neuronal input territories to OPCs beyond local neurons and fiber bundles accessible in a slice preparation. Using a monosynaptically restricted trans-synaptic retrograde tracing system, we have now elucidated a map of neuronal input territories to OPCs in three distinct regions of the mouse brain. OPCs in these territories – selected due to previously reported changes in local oligodendrocyte lineage dynamics in response to neuronal activity – all receive brain-wide synaptic input. Strikingly, the ratio of input neurons to starter OPCs is consistent across MOs, SSp, and CC, despite the greater local axon density in CC and despite higher OPC turnover rates in CC than either cortical territory. This suggests that regulation of the number of neuron->OPC synapses may be intrinsic to the OPC rather than specified by local neurons or other microenvironmental factors in these brain regions.

While the estimated number of synaptic inputs appears consistent across mapped regions, the localization of these strikingly extensive inputs is distinct by location and supports a pattern of brain-wide afferent connectivity to OPCs from brain regions known to be functionally-associated within the premotor or somatosensory circuits. For OPCs present in the CC genu inferior to the cingulum bundle, there is a relative bias of inputs from cortical regions involved in planning and execution of motor skills, and ~25% of these inputs arise contralateral to the targeted OPCs. While previous studies have demonstrated evoked synaptic inputs to these white matter OPCs by stimulation of callosal fibers, we now provide an unbiased assessment of the cortical projection neurons and interneurons responsible for these synapses, confirm the presence of thalamocortical inputs to OPCs in somatosensory cortex (Mangin et al., 2012), and further identify thalamocortical input to OPCs in MOs. Notably, behavioral paradigms shown to alter oligodendrocyte lineage dynamics in mice, including motor learning tasks and social isolation, are thought to drive dynamic changes in neuronal activity in MOs and mPFC. We now demonstrate that the majority of synaptic inputs to callosal white matter OPCs arise from these very brain regions. These synaptic inputs are also largely excitatory, with immunostaining revealing a relatively small fraction of OPC inputs arising from local PV+ interneurons. Strikingly, this fraction is relatively consistent across cortical and white matter territories investigated here, which may result either from higher regional density of excitatory projection axons or may indicate that OPCs actively regulate the number of interneuron inputs as a mechanism to maintain excitatory:inhibitory balance. Relative to transsynaptic tracing studies in neurons, both the relative and absolute degree of inhibitory connectivity is lower in OPCs. Recent studies with trans-synaptic tracing from principal neuron starter cells suggest GABAergic inputs range from 18–27% of all inputs in mouse mPFC and 9.5–15% in mouse barrel cortex (DeNardo et al., 2015).

Our assessment of synaptic inputs to gray matter OPCs maps neuronal connectivity to this regionally and perhaps functionally distinct cell population. In contrast to callosal white matter OPCs, neuronal inputs to OPCs present in SSp or MOs primarily arise within ipsilateral local cortex, although a smaller fraction (mean approximately 7.5%) of inputs to OPCs in MOs arise contralaterally. Additionally, we show thalamocortical projections from the expected, functionally-associated thalamic nuclei providing synaptic input to these OPCs. Taken together, the demonstrated map of input neurons to cortical OPCs suggests a mechanism by which OPCs could sense synchronized patterns of activity between thalamus and cortex. In turn, integration of this synaptic activity by individual OPCs might coordinate or regulate adaptive myelination of circuitry linking cortical and thalamic territories – a model that merits evaluation in future studies.

While the localization and laterality of neuronal input to OPCs varies depending on brain region, the total numerical extent of input connections – as measured by input:starter ratio – is remarkably consistent across territories. Given that the rate of OPC turnover in these regions has been shown to vary (Rivers et al., 2008), it follows that the extent of synaptic input must be regulated by a newly-generated OPC to result in equivalent connectivity. OPCs in afferent activity-deprived somatosensory cortex following unilateral whisker trimming are less likely to survive in a critical temporal window following division, which subsequently results in diminished generation of mature oligodendrocytes (Hill et al., 2014). Moreover, genetic ablation of AMPA receptors in OPCs reduces the survival of oligodendrocytes generated during development (Kougioumtzidou et al., 2017), and Ca2+-permeable AMPARs may have an important role in balancing OPC response to proliferation/differentiation cues (Chen et al., 2018). We now demonstrate that deprivation of input activity to barrel field OPCs by whisker trimming does not alter the synaptic input ratios of surviving cells at 15 days after trimming, nor does it impact the distribution of neuronal inputs at the time point evaluated. We selected this timepoint based on 2-photon microscopy studies of spine plasticity in barrel cortex following whisker trimming which have identified new-persistent spine formation in the 12–20 day window after trimming begins (Knott et al., 2006; Holtmaat et al., 2006). We therefore suspected that a similar window might represent a period of ‘new persistent’ changes in OPC synaptic connectivity, yet input:starter ratios and input distribution remained similar at the timepoint examined. This may suggest that the pool of OPCs giving rise to early oligodendrocytes in the above studies begin from the same level of synaptic connectivity. From this baseline, activity deprivation-related deficits resulting in decreased survival may accumulate at later stages of cell differentiation. Alternatively, OPCs that fail to attain sufficient synaptic input in the critical window after division may fail to survive, resulting in deficient oligodendrogenesis despite apparently normal starter to input ratios. Future studies at these early and late timepoints will be required to definitively evaluate potential synaptic remodeling in OPCs following sensory deprivation.

An important limitation of our current study is the inability to delineate the connectivity of single cells, only the population total. This raises the possibility that a mixture of high and low-connectivity OPCs could exist – a possibility supported by the distribution of OPC-colocalized PSD95 puncta we observe in these animals – and newly-generated cells could tend to sort into one pool or the other under the control of local factors. In our hands, we were unable to titrate viral injections to achieve single starter cell transductions. Recently, several groups have achieved transduction of single neurons under 2-photon microscopy guidance using targeted electroporation and/or guided microinjection of rabies virus (Marshel et al., 2010; Wertz et al., 2015; Rompani et al., 2017), suggesting that if technical barriers can be overcome, input networks to single OPCs might be elucidated in the future. This discovery of widespread and remarkably stable neuronal afferents to OPCs thus indicates a need to probe context-specific roles of neuron->OPC synaptic connectivity and ultimately to determine the function of these enigmatic structures.

Materials and methods

Key resources table.

Reagent type
(species) or
resource		 Designation Source or reference Identifiers Additional
information
Antibody Anti-Pdgfra (goat polyclonal) R and D systems AF1062
RRID:AB_2236897 		1:500
Antibody Anti-Olig2 (rabbit monoclonal) Abcam EPR2673
RRID:AB_10861310 		1:500
Antibody Anti-GFP (chicken polyclonal) Abcam Ab13970
RRID:AB_300798 		1:1000
Antibody Anti-parvalbumin (rabbit polyclonal) Abcam Ab11427
RRID:AB_298032 		1:250
Antibody Anti-somatostatin (rat monoclonal) Millipore MAB354
RRID:AB_2255365 		1:200
Antibody Anti-VIP (rabbit polyclonal) Immunostar 20077
RRID:AB_572270 		1:500
Antibody Anti-Iba1 (rabbit polyclonal) Wako 019–19741
RRID:AB_839504 		1:500
Antibody Anti-Cre recombinase (mouse monoclonal) Millipore MAB3120
RRID:AB_2085748 		1:1000
Antibody Anti-PSD95 (rabbit polyclonal) Invitrogen 51–6900
RRID:AB_2533914 		1:100
Antibody Anti-CASPR (rabbit monoclonal) Cell Signaling Technologies 97736
RRID:AB_2800288 		1:250
Software Imaris Bitplane/Oxford Instruments v8.1.2
RRID:SCR_007370
Software Matlab Mathworks R2017b
RRID:SCR_001622
Strain (Mus musculus) B6N.Cg-Tg(Pdgfra-cre/ERT)467Dbe/J The Jackson Laboratory 018280
RRID:IMSR_JAX:018280
Strain (Mus musculus) B6;129P2-Gt(ROSA)26Sortm1(CAG-RABVgp4,-TVA)Arenk/J The Jackson Laboratory 024708
RRID:IMSR_JAX:024708
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Animal breeding

All animal studies were approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC). Animals were housed on a 12 hr light cycle according to institutional guidelines. Mice expressing CreER under the control of Pdgfra promoter/enhancer regions (Pdgfra::Cre/ERT) were purchased from The Jackson Laboratory (stock number 018280) and have been previously described (Kang et al., 2010). Mice expressing a recombinant rabies G glycoprotein gene (RABVgp4) along with the gene encoding avian leucosis and sarcoma virus subgroup A receptor (TVA) preceded by a loxP-flanked STOP fragment and inserted into the GT(ROSA)26Sor locus (R26(gp4-TVA)fl/fl) have been previously described (Takatoh et al., 2013) and were purchased from The Jackson Laboratory (stock number 024708). Hemizygous Pdgfra::Cre/ERT mice were then crossed with homozygous R26(gp4-TVA)fl/fl mice to generate animals used in subsequent experiments. Genotyping was performed by PCR according to supplier protocols.

Viral tracing

EGFP-expressing G-deleted rabies virus pseudotyped with EnvA (SADΔG-EGFP(EnvA)) (Wickersham et al., 2007) was prepared at and obtained from the Salk Institute Gene Transfer, Targeting, and Therapeutics Facility vector core (GT3). Virus used in these studies originated in two lots with reported titers of 7.92 × 107 and 1.94 × 109 TU/mL. 3 days prior to stereotaxic injections, Cre/ERT-mediated recombination was induced by a single IP injection of 100 mg/kg of tamoxifen (Sigma) solubilized in corn oil. Stereotaxic delivery of virus occurred under isofluorane anesthesia in BSL2+ conditions. 300 nL of SADΔG-EGFP(EnvA) was delivered to the corpus callosum (coordinates AP +1 mm, ML – 1 mm, DV −1.2 mm) or the overlying secondary motor area (coordinates AP + 1 mm, ML – 0.8 mm, DV −0.5 mm) or primary somatosensory cortex (coordinates AP −1 mm, ML −3 mm, DV −0.7 mm) over 5 min (Stoelting stereotaxic injector). Animals were monitored for general health, and no adverse symptoms of viral administration were observed. 5 days following viral injection, animals were deeply anesthetized with tribromoethanol and transcardially perfused with PBS followed by 4% PFA, then brains were removed and post-fixed overnight in 4% PFA. Brains were then transferred to 30% sucrose, and after sinking serial 40 micrometer floating coronal sections were prepared on a freezing-stage microtome for subsequent immunolabeling and imaging.

Whisker trimming

Pdgfra::CreERT; R26(gp4-TVA)fl mice generated as described above were trimmed of whiskers bilaterally to the level of the skin using electric clippers daily beginning at P25. At P37, tamoxifen was injected as described above, and whisker trimming continued daily until P40, when SADΔG-EGFP(EnvA) was injected as described above. Animals were then sac’d and perfused at P45 as described above.

Immunofluorescence and confocal microscopy

Antibodies and dilutions used for immunofluorescence staining were as follows: polyclonal goat anti-mouse Pdgfra (R and D Systems, AF1062, 1:500), monoclonal rabbit anti-mouse Olig2 (Abcam EPR2673, 1:500), polyclonal chicken anti-GFP (Abcam, ab13970, 1:1000), polyclonal rabbit anti-parvalbumin (Abcam, ab11427, 1:250), monoclonal rat anti-somatostatin (Millipore, MAB354, 1:200), polyclonal rabbit anti-VIP (Immunostar 20077, 1:500), polyclonal rabbit anti-Iba1 (Wako, 1:500), mouse anti-Cre recombinase (Millipore, MAB3120, clone 2D8, 1:1000), rabbit anti-PSD95 (Invitrogen, 51–6900, 1:100), and rabbit anti-CASPR (Cell Signaling Technologies, clone D813V). Tissues collected at serial intervals of 1 in every six sections were blocked and permeabilized with 3% normal donkey serum and 0.3% Triton X-100 in Tris-Buffered Saline (3%NDS/TBST) for 30 min at room temperature, followed by incubation with antibodies at the indicated dilution factors in 1%NDS/TBST for 18 hr at four degrees C. For mouse anti-Cre recombinase staining, NDS block was followed by treatment with mouse-on-mouse staining reagent (Vector Laboratories, BMK-2202) prior to incubation with primary antibody. Following a series of washes, secondary AlexaFluor-tagged antibodies raised in donkey (Jackson Immunoresearch) in 1%NDS/TBST were incubated for 4 hr at room temperature, and following a series of washes, sections were counterstained with DAPI (1 ug/mL) and mounted on slides with ProlongGold media (ThermoFisher Scientific). Tile scanning images were acquired at 10X magnification on a Zeiss AxioObserver upright fluorescence microscope with automated stage and tile-scanning capability (Microbrightfield). For identification of atlas regions for labeling quantification, acquired images were manually registered to the closest available section from the Allen Brain Mouse Reference Atlas (Lein et al., 2007) (ImageJ) using DAPI fluorescence of the section outline and major neuroanatomical structures to guide fitting. Analysis participants were blinded to injection conditions, and independent adjustment of atlas registration maps did not substantially impact counting results. Cell counting was performed by two independent reviewers on every 6th 40 micrometer tissue section throughout the brain, and total cell count estimates were derived by multiplying the number of counted cells by 6. Multichannel immunofluorescence microscopy to identify starter cell populations, neuronal identity, and other high-resolution imaging was conducted by acquiring Z-stacks through the target region with a Zeiss LSM710 confocal microscope.

PSD95 colocalization

Floating sections immunostained for Pdgfra and PSD95 as above were imaged as Z stacks on a Zeiss LSM710 confocal microscope with a 63X objective. Acquired Z-stacks were loaded in Imaris software (Bitplane) with MATLAB integration. Filament models were constructed using the Imaris Filament plugin. PSD95 puncta were selected with the Imaris Spots plugin. Colocalized puncta were identified with the MATLAB plugin for Imaris ‘Find spots close to filament.’ Modeling parameters were held consistent across individual cohorts.

Statistics and reproducibility

Stereotaxic injections were repeated in three independent cohorts (litters) of animals for each injection location, and both male and female mice were used. Sample sizes were established based upon similar studies in the literature and were not pre-determined. Cell counters were blinded to injection location, and counts were performed independently by two reviewers. All statistical tests were performed using Graphpad Prism software and details of individual tests are described in figure legends.

Acknowledgements

We thank Brady Weissbourd for helpful discussion and Pamelyn Woo for assistance with animal colony maintenance. The authors gratefully acknowledge support from the California Institute for Regenerative Medicine (CIRM RN3-06510), National Institute of Neurological Disorders and Stroke (NINDS R01NS092597 and F31NS098554), NIH Director’s Pioneer Award (DP1NS111132), SFARI Foundation, Maternal and Child Health Research Institute at Stanford.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Michelle Monje, Email: mmonje@stanford.edu.

Klaus-Armin Nave, Max Planck Institute of Experimental Medicine, Germany.

Gary L Westbrook, Oregon Health and Science University, United States.

Funding Information

This paper was supported by the following grants:

  • NIH Office of the Director DP1NS111132 to Michelle Monje.

  • California Institute for Regenerative Medicine CIRM RN3-06510 to Michelle Monje.

  • National Institute of Neurological Disorders and Stroke R01NS092597 to Michelle Monje.

  • National Institute of Neurological Disorders and Stroke F31NS098554 to Christopher W Mount.

  • Simons Foundation The Simons Foundation Autism Research Initiative to Belgin Yalçın, Michelle Monje.

  • Stanford University Maternal and Child Health Research Institute to Michelle Monje, Belgin Yalçın.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Formal analysis, Investigation, Writing—original draft.

Formal analysis, Investigation, Writing—review and editing.

Formal analysis, Investigation, Writing—review and editing.

Software, Investigation, Methodology.

Conceptualization, Supervision, Funding acquisition, Writing—original draft.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal studies were performed in accordance with a protocol approved by the Stanford Administrative Panel on Laboratory Animal Care (APLAC, protocol number 27215).

Additional files

Source data 1. Raw data presented in the paper.
elife-49291-data1.xlsx (24.2KB, xlsx)
DOI: 10.7554/eLife.49291.010
Transparent reporting form
elife-49291-transrepform.docx (244.8KB, docx)
DOI: 10.7554/eLife.49291.011

Data availability

The data that support the findings of this study are presented in Source data 1.

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Decision letter

Editor: Klaus-Armin Nave1
Reviewed by: David A Lyons2

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Monosynaptic tracing maps brain-wide afferent oligodendrocyte precursor cell connectivity" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gary Westbrook as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: David A Lyons (Reviewer #1). The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Using a modified rabies virus to specifically label "presynaptic" neurons by an unbiased retrograde monosynaptic tracing approach, Mount et al. map out the neuronal inputs that oligodendrocyte precursor/ progenitor cells (OPCs) receive in three different anatomical locations in the forebrain of mice (corpus callosum, motor cortex and somatosensory cortex). Although it has been known for quite some time that OPCs receive synaptic input from both excitatory and inhibitory neurons, the techniques employed in this manuscript provide the opportunity to further explore this circuitry. The authors provide a robust dataset confirming the connectivity between OPCs in white matter and grey matter regions with various neurons and neuronal subtypes across the brain.

The greatest strength of this study is the strategy employed for monosynaptic tracing of connectivity. On the other hand, the study still misses the individual variance and in-depth information on whether certain types of OPCs receive mainly glutamatergic or GABAergic inputs, or whether GABAergic inputs to OPCs are more frequent in certain layers of the cortex. This information would be helpful as some oligodendrocytes myelinate only GABAergic, or glutamatergic neurons, whereas others myelinate both. Given the large number of labelled OPCs it is also surprising that no neuromodulatory input, such as cholinergic synapses, were detected. Overall, the findings complement previously published works. The novelty is in the methodology, not the insight of the input that OPCs receive.

Overall, the reviewers thought that the conclusion (including the negative data), see below) were a little bit overstated and should be toned down. Terms, such as "functionally related" and "functionally associated" suggest a specificity of OPC targeting that remains unproven, as it may merely reflect the specificity of underlying neuronal circuitry, with synaptic targeting of the omnipresent OPC being a secondary effect (see also question 2 below).

Essential revisions:

1) What proportion of OPCs have synapses? How does the overall number of neuron-OPC connections relate to the structure of individual OPCs? Antibody-based staining of postsynaptic components and imaging of OPCs at highest resolution should reveal what proportion of OPCs make connections with axon-like profiles? Similarly, and perhaps a bit easier, it should be possible to count post-synaptic clusters per OPC and relate that to the overall ratio predicted from the connectivity maps. The data in general related to the ratio of inputs to OPCs look very convincing, but the left-most value in Figure 2C is unclear. It looks like very few OPCs contact almost 5000 neurons. Can the authors clarify that point?

2) Are the neurons that make synapses with OPCs strictly unmyelinated or do they include myelinated or partially myelinated cells? That issue could also be addressed by immunostaining of labelled neurons.

3) How easy is it to control the mosaicism, i.e. the number/density of the starting population? It would be useful if the authors provide more details on the method throughout, which could make this manuscript potentially suitable for eLife's Tools and Resources format. A more detailed schematic of the molecular mechanism behind the tracing strategy would also be helpful).

4) One experimental concern was the possibility of off-target recombination of neurons using the PDGFRa-CreERT2 line. Although the authors note that Cre protein expression was only detectable in OPCs, sporadic recombination in neurons has been reported in the PDGFRa-CreERT2 lines from the Richardson lab line as well as the Bergles lab (used in this study). It seems unlikely that off-target neuronal recombination and subsequent rabies virus infection would occur in a widespread manner, but it is important to categorically rule out this possibility, given that the entire paper hinges on the neuronal populations expressing GFP being infected trans-synaptically rather than directly. Staining for Cre protein is an imperfect approach as sub-detectable amounts of protein may still be sufficient to drive recombination. Can staining or ISH for the gp4 protein be used to confirm that GFP-expressing neurons were not recombined and infected directly? Alternatively, a very early time-point could be assessed post-infection to confirm GFP expression in the OPC starter cells but not (yet) in any presynaptic neuron.

5) One point that the authors were unable to address is the connectivity patterns of individual OPCs. The authors' comment that it "cannot be tested with existing methods" – but is that really the case? Can the authors elaborate in the Discussion about how one might go about tackling this issue, e.g. generate animals with very few starting OPCs, applying methods to automate tracing across what are very large datasets (not suggesting that the authors need to trace these inputs as part of the current paper).

6) How does the excitatory-inhibitory ratio of input onto OPCs compare with actual excitatory-inhibitory ratios of neurons? Can the authors comment on whether their observed ratios represent a skew towards specific neuronal subtypes? It is also interesting that there appears to be input from neurons that are not myelinated. Perhaps the authors could speculate on possible functions associated with such connectivity.

7) The manuscript would benefit from further information on where exactly the transfected OPCs are located. For example, was the highest proportion of infected OPCs located in layer 4? This information is essential to interpret the data and understand the anatomy of the neuronal inputs to OPCs.

8) The author states "These previously unidentified thalamic inputs are…" Then the author states "These previously unidentified thalamocortical synaptic inputs arise from functionally-related thalamic nuclei." And again the author states "…as well as previously unrecognized OPC inputs from thalamocortical projections." These statements are surprising given that the thalamocortical inputs in OPCs have been well described by Gallo's group. Particularly, in focusing on the barrel cortex, they have shown functional thalamic cortical inputs to OPCs, by stimulating the VB area of the thalamus and record the inputs in the OPCs at different locations within the cortex, and they found a difference in the number of thalamic inputs OPCs receive depending on their location within the cortex (Mangin et al., 2012). These statements need to be rephrased to adhere to the literature.

9) The whisker trimming experiment was carried out from one time-point with analysis at one other time. Is this a period over which other aspects of cellular and functional remodelling occur in the barrel field? Reviewers were unsure how much to take from the negative data shown. Moreover, the lack of an effect is difficult to interpret when observing a large population of cells in a large cortical area. Another concern is the duration of the repeated trimming, as in adult mice, some plasticity changes in the barrel cortex take more time to be detected than those in younger mice (in some papers testing for synaptic structural changes in adult mice, which are often found to layer specific, use repeated trimming protocol for up to 4 weeks). Thus, it needs to be discussed whether the method is sensitive enough to detect regional and small changes in synaptic inputs.

eLife. 2019 Oct 18;8:e49291. doi: 10.7554/eLife.49291.014

Author response

Summary:

Using a modified rabies virus to specifically label "presynaptic" neurons by an unbiased retrograde monosynaptic tracing approach, Mount et al. map out the neuronal inputs that oligodendrocyte precursor/ progenitor cells (OPCs) receive in three different anatomical locations in the forebrain of mice (corpus callosum, motor cortex and somatosensory cortex). […]

Overall, the reviewers thought that the conclusion (including the negative data), see below) were a little bit overstated and should be toned down. Terms, such as "functionally related" and "functionally associated" suggest a specificity of OPC targeting that remains unproven, as it may merely reflect the specificity of underlying neuronal circuitry, with synaptic targeting of the omnipresent OPC being a secondary effect (see also question 2 below).

We are grateful for the thoughtful comments. We have now made significant updates based on these helpful suggestions: We have further confirmed specificity of labeling by adding control non-EnvA-bearing tracer injections (Figure 1—figure supplement 1). We performed PSD95 puncta colocalization studies as a quantitative estimate of potential synaptic densities on OPCs, and find that this estimate correlates well with the input:starter cell ratios that we measured throughout our transsynaptic tracing experiments (Figure 2—figure supplement 1). We have also performed additional studies and made textual modifications as suggested, including clarifying that our intended meaning of “functionally-related/associated” was simply that we observed inputs to OPCs from the expected regions of brain known to be functionally related, not to suggest a specificity of OPC targeting apart from this. Specific responses to reviewer comments are detailed below:

Essential revisions:

1) What proportion of OPCs have synapses? How does the overall number of neuron-OPC connections relate to the structure of individual OPCs? Antibody-based staining of postsynaptic components and imaging of OPCs at highest resolution should reveal what proportion of OPCs make connections with axon-like profiles? Similarly, and perhaps a bit easier, it should be possible to count post-synaptic clusters per OPC and relate that to the overall ratio predicted from the connectivity maps.

With regard to the synapse density per OPC question, we sought to address this as suggested by immunostaining for PSD95 and assessing for co-localization with OPC processes as defined by PDGFRa expression (subsection “OPCs in corpus callosum receive brain-wide synaptic input”). On average, approximately 20 PSD95+ puncta co-localize with 3D models of OPC processes generated from PDGFRa+ immunostaining; this density is not significantly different from the average input:starter cell ratio calculated by linear regression from the transsynaptic tracing data (Figure 2—figure supplement 1). On the level of individual cells, the distribution of PSD95+ colocalized puncta ranges from 0-67 across the 95 individual cells imaged; ~95% of OPCs analyzed in the corpus callosum exhibit PSD95+ puncta. While PSD95 would not account for potential inhibitory inputs, overall the similarity between the two approaches further supports our earlier estimates. We thank the reviewer for this helpful question.

The data in general related to the ratio of inputs to OPCs look very convincing, but the left-most value in Figure 2C is unclear. It looks like very few OPCs contact almost 5000 neurons. Can the authors clarify that point?

Thank you for pointing out this important point to address. The above PSD95 puncta analysis also suggests that with regard to Figure 2C, there are not individual OPCs making 5000 connections. Rather, we suspect that regional effects in the corpus callosum (CC) – either increased relative OPC cell density, enhanced susceptibility of CC OPCs to viral-induced toxicity, or increased relative rate of OPC differentiation in the CC contributed to systematic underestimates of the true starter cell ratio, resulting in an overall left-shift of the input:starter ratio curve and hence a y-intercept > 0. To address this, we performed diluted rabies virus injections at a 1:5 ratio to better sample low starter cell ratios and attempt to reduce regional effects (Figure 2C). This approach was somewhat successful, and the y-intercept of the revised curve is now 1610 without a substantial impact on the calculated starter:input ratio. Therefore, it is likely that we are still missing starter cells, and further dilutions of virus did not lead to reliable labeling patterns useable for quantification. We have now discussed this limitation in the Discussion section.

2) Are the neurons that make synapses with OPCs strictly unmyelinated or do they include myelinated or partially myelinated cells? That issue could also be addressed by immunostaining of labelled neurons.

This is an excellent question. We observe transsynaptic labeling of both myelinated and unmyelinated input neurons (subsection “OPCs in corpus callosum receive brain-wide synaptic input”). To better illustrate this, we performed immunofluorescence labeling with CASPR to identify paranode/juxtaparanode of myelinated axons (Figure 1—figure supplement 1E). Many labeled axons are devoid of any CASPR labeling through their visualized path, consistent with previous reports of synapses with unmyelinated callosal axons.

3) How easy is it to control the mosaicism, i.e. the number/density of the starting population? It would be useful if the authors provide more details on the method throughout, which could make this manuscript potentially suitable for eLife's Tools and Resources format. A more detailed schematic of the molecular mechanism behind the tracing strategy would also be helpful).

Mosaicism of the starting population can be controlled to some extent by adjusting the amount of total viral particles delivered, as we describe above. Optimizing for specific starter cell densities also depends on the native cell density at the target site, as a sufficient volume must be infused to propagate virus through the target site. We have updated the text to offer more description of the methodology as suggested (subsection “Development and validation of retrograde monosynaptic OPC tracing strategy”). We have also updated the diagram in Figure 1A to provide additional clarification as suggested.

4) One experimental concern was the possibility of off-target recombination of neurons using the PDGFRa-CreERT2 line. Although the authors note that Cre protein expression was only detectable in OPCs, sporadic recombination in neurons has been reported in the PDGFRa-CreERT2 lines from the Richardson lab line as well as the Bergles lab (used in this study). It seems unlikely that off-target neuronal recombination and subsequent rabies virus infection would occur in a widespread manner, but it is important to categorically rule out this possibility, given that the entire paper hinges on the neuronal populations expressing GFP being infected trans-synaptically rather than directly. Staining for Cre protein is an imperfect approach as sub-detectable amounts of protein may still be sufficient to drive recombination. Can staining or ISH for the gp4 protein be used to confirm that GFP-expressing neurons were not recombined and infected directly? Alternatively, a very early time-point could be assessed post-infection to confirm GFP expression in the OPC starter cells but not (yet) in any presynaptic neuron.

Due to the limited ability to directly stain for TVA or gp4 (we inquired after aliquots of previously-described anti-TVA antibodies, which are not available commercially and seem to have fallen into disuse in host laboratories, and available gp4 antibodies are non-reactive in our tissue) we sought to address this by comparison injection of non-EnvA SADΔG19. In the absence of EnvA, viral uptake is no longer restricted to TVA-expressing OPCs and can be taken up more broadly. As now illustrated in Figure 1—figure supplement 1D, with injection of SADΔG19 into primary somatosensory cortex we observe broad uptake throughout the entirety of the injection site in both Cre+ and Cre negative animals with little discernable difference between the two (subsection “Development and validation of retrograde monosynaptic OPC tracing strategy”). By contrast, SADΔG19-EnvA exhibits minimal labeling in Cre WT animals (Figure 1B) – which we would now expect if there were substantial leak of TVA expression in neurons.

5) One point that the authors were unable to address is the connectivity patterns of individual OPCs. The authors' comment that it "cannot be tested with existing methods" – but is that really the case? Can the authors elaborate in the Discussion about how one might go about tackling this issue, e.g. generate animals with very few starting OPCs, applying methods to automate tracing across what are very large datasets (not suggesting that the authors need to trace these inputs as part of the current paper).

While this remains beyond our current capabilities in the specific context of OPCs, single cell network tracing has been achieved by several groups in neurons and some discussion reveals the challenges that must be overcome before this could be approached in OPCs. Both (Marshel et al., 2010) and (Wertz et al., 2015) achieve single-cell access by targeted electroporation of plasmids into single neurons by “shadow patching” under two-photon (2P) guidance in mice (Kitamura et al., 2008, Nat Meth). While to our knowledge this has not been attempted in OPCs, several obstacles are apparent. First, OPCs in our hands at least have been challenging cells to electroporate after isolation forin vitro studies, and substantial optimization may be necessary to achieve efficiencies compatible with the multi-plasmid electroporation necessary to both label the starter cell and provide the requisite packaging and payload elements for transsynaptic tracing. Additionally, the ability of OPCs to divide and dilute any epigenomic plasmids may limit one’s ability to subsequently track the starter cell. More recently, (Rompani et al., 2017) achieved single-cell-initiated tracing in the LGN by a combination of targeted electroporation and guided microinjection of rabies virus under 2P guidance targeting individual principal cells. In summary, 2P guidance might be utilized in the future to guide single-OPC targeted transduction, potentially with the assistance of automated tracing methods to assess distant inputs. The reviewer’s point is well-taken and we have updated the Discussion section accordingly to reflect this (Discussion, last paragraph).

6) How does the excitatory-inhibitory ratio of input onto OPCs compare with actual excitatory-inhibitory ratios of neurons? Can the authors comment on whether their observed ratios represent a skew towards specific neuronal subtypes? It is also interesting that there appears to be input from neurons that are not myelinated. Perhaps the authors could speculate on possible functions associated with such connectivity.

On a structural level, excitatory/inhibitory input ratios to neurons have been measured in several recent trans-synaptic tracing studies. In their study of mouse mPFC and barrel cortex, DeNardo et al. report ratios ranging from 18-27% GABAergic inputs to principal neurons across cortical layers of mPFC, and between 9.5-15% in mouse barrel cortex (DeNardo et al., 2015). In a more recent study by Yetman et al., in SS cortex supragranular principal neurons, a reported average of 7.4 +/- 1.2 parvalbumin inputs to individual principal neurons are present (Yetman et al., Nat Neuro, 2019). Compared with these studies, both the absolute number and fractional percentage of inhibitory inputs to OPCs are lower than seen in neurons in these territories, and we have updated the text to reflect this (Discussion).

7) The manuscript would benefit from further information on where exactly the transfected OPCs are located. For example, was the highest proportion of infected OPCs located in layer 4? This information is essential to interpret the data and understand the anatomy of the neuronal inputs to OPCs.

We thank the reviewers for this suggestion, and have consequently included measurements of cortical location for starter cells in CC, MOs, and SSp (see subsection “OPCs in corpus callosum receive brain-wide synaptic input”; Figure 1—figure supplement 1F). These data demonstrate that cortical distribution of targeted starter cells in both MOs and SSp are broad without evidence of layer-specific targeting. With our injection strategy, this was a necessary trade-off to balance adequate starter cell transduction against injection site specificity. OPCs within layer 4 are indeed sampled, but do not comprise a dominant fraction and we feel there is insufficient resolution at this time to comment more thoroughly on OPC inputs at the level of cortical microcircuitry.

8) The author states "These previously unidentified thalamic inputs are…" Then the author states "These previously unidentified thalamocortical synaptic inputs arise from functionally-related thalamic nuclei." And again the author states "…as well as previously unrecognized OPC inputs from thalamocortical projections." These statements are surprising given that the thalamocortical inputs in OPCs have been well described by Gallo's group. Particularly, in focusing on the barrel cortex, they have shown functional thalamic cortical inputs to OPCs, by stimulating the VB area of the thalamus and record the inputs in the OPCs at different locations within the cortex, and they found a difference in the number of thalamic inputs OPCs receive depending on their location within the cortex (Mangin et al., 2012). These statements need to be rephrased to adhere to the literature.

We thank the reviewers for their attention to this unintended implication, as it was our desire to highlight these findings in secondary motor area and the language was inadvertently carried forward in subsequent discussion of thalamocortical projections to barrel cortex. We have rephrased these statements appropriately and discuss the Mangin et al., 2012 paper prominently (Discussion, second paragraph).

9) The whisker trimming experiment was carried out from one time-point with analysis at one other time. Is this a period over which other aspects of cellular and functional remodelling occur in the barrel field? Reviewers were unsure how much to take from the negative data shown. Moreover, the lack of an effect is difficult to interpret when observing a large population of cells in a large cortical area. Another concern is the duration of the repeated trimming, as in adult mice, some plasticity changes in the barrel cortex take more time to be detected than those in younger mice (in some papers testing for synaptic structural changes in adult mice, which are often found to layer specific, use repeated trimming protocol for up to 4 weeks). Thus, it needs to be discussed whether the method is sensitive enough to detect regional and small changes in synaptic inputs.

We agree with the reviewers that definitive evaluation of potential synaptic remodeling will require systematic analysis at multiple additional timepoints. For the purposes of our study, we sought to select a timepoint that was both amenable to input:starter ratio quantification and consistent with prior studies of persistent synaptic alterations – at least on a structural level – from prior studies in mouse barrel cortex. Regarding animal age, elegant work (Hill et al., 2014) has shown that whisker trimming in the early postnatal (P6-8) window over a short timecourse of several days reduced survival of differentiating OPCs, suggesting that studies of input:starter cell ratios would be complicated in this window – an already challenging one given that there is substantial oligodendrocyte lineage expansion and differentiation underway. Therefore, we decided to perform our study in young adult animals. As far as duration of whisker trimming, (Hughes et al., 2018) demonstrated that sensory enrichment for 21 days in adult mice enhanced oligodendrocyte generation that was abrogated with whisker trimming, but found no effect with whisker trimming alone. Thus, without definite guidance from the OPC literature, we turned to the synaptic plasticity literature with special attention to structural studies of spine generation and persistence in barrel cortex following whisker trimming. The seminal work of (Holtmaat et al., 2006) assessed this question with whisker trimming in mice with a median age of 2.3 months and evaluated spine formation in barrel cortex pyramidal neurons by 2P microscopy daily after the initiation of trimming. They binned newly-formed spines as those appearing within 0-12 days of trimming initiation, and new-persistent spines as those continuing to be present through 12-20 days of trimming. Additional studies, including (Knott et al., 2006) further suggest that persistent spines – in particular those lasting 4 days or longer, are more likely to acquire synapses. On this basis, we felt that a total trimming period of 14 days was reasonable to assess a possible period of “new persistent” changes in OPC synaptic connectivity. However, we are of course unable to rule out alterations to connectivity in other temporal domains or brain regions, which could be a valuable area of investigation. We have updated the text to reflect this discussion (Discussion, fourth paragraph). We have also clarified throughout that our data represent only one timepoint.

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    Supplementary Materials

    Source data 1. Raw data presented in the paper.
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    DOI: 10.7554/eLife.49291.010
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    DOI: 10.7554/eLife.49291.011

    Data Availability Statement

    The data that support the findings of this study are presented in Source data 1.

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