Tracing from Periphery to Cortex: Application of Herpes Simplex Virus to Somatosensory Neural Networks

Dear Editor,

Understanding the complex structure of the somatosensory neural network is crucial for deciphering how the brain processes sensory information. Recent advances in viral tracers, such as Herpes simplex virus (HSV) type 1 strain 129 (H129), have enabled researchers to effectively map somatosensory neural networks [1–3]. However, challenges such as low inoculation efficiency and difficulty in quantitative comparison of labeled neural networks arise due to the polysynaptic nature of H129 and the potential neuronal death induced by its toxicity.

Our previous research has revealed that bortezomib, an FDA-approved immunosuppressive drug, enhances H129 infection in dorsal root ganglia (DRG) neurons, addressing the challenge of low inoculation efficiency [3]. Bortezomib, a proteasome inhibitor, acts by inhibiting nuclear factor-κB activation [4] and inducing apoptosis in multiple myeloma cells [5]. Evaluating the efficiency and safety of bortezomib in enhancing H129 tracing in mice is imperative. The challenge of quantitatively comparing HSV-labeled neural networks stems from the polysynaptic properties of H129, where differences in labeling patterns can be attributed to varying propagation stages. Identifying the landmark of the nucleus representing the propagation stage is essential.

To address the main concern that lies in the potential toxicity of bortezomib, particularly in the context of H129 infection, we explored the impact of subcutaneous bortezomib injection on facilitating H129 infection in the lumbar DRG of adult mice. In a previous study, a dose of 0.8 mg/kg was administered to augment the anticancer efficacy of oncolytic HSV-1, with the primary objective of inducing cytotoxicity in the infected tumor cells [6, 7]. We evaluated the toxicity of bortezomib at the doses of 0.4 mg/kg, 0.8 mg/kg, and 1.6 mg/kg. The survival probability of mice in each group was determined within 14 days. Survival curves showed that half of the mice died within 7 days when given both 1.6 mg/kg and 0.8 mg/kg but not 0.4 mg/kg of bortezomib (Fig. 1A). Moreover, in the 1.6 mg/kg group, 36.3% of mice died 2–3 days post-injection and did not show any EGFP signals in the DRG, implying that bortezomib but not the virus induces toxicity and leads to death of the mice. Therefore, 1.6 mg/kg bortezomib was excluded from use for promoting H129 infection.

Fig. 1.

Optimizing the bortezomib dose and H129 titer for viral infection and propagation. A Kaplan–Meier curve of mice treated with different doses of bortezomib after H129-EGFP injection. B Comparison of infection efficiency by assessing the success rate of H129-EGFP inoculation in the DRG among groups with different doses of bortezomib. In A and B, the total numbers of mice used in the control, 0.4 mg/kg, and 0.8 mg/kg groups were 13, 14, and 10, respectively. C Representative images of DRGs infected by H129-EGFP with bortezomib at different doses. D Comparison of infection efficiency of H129-EGFP in the DRG among groups with different doses of bortezomib by assessing the percentage of EGFP labeled neurons in the DRG. E Analysis of the success rate of H129-EGFP propagating from the DRG to the RVM under different doses of bortezomib. The experiment was repeated 3 times. In D and E, the total numbers of mice used in the control, 0.4 mg/kg, and 0.8 mg/kg groups were 12, 13, and 9, respectively. F Kaplan–Meier curves of mice infected with different titers of H129-EGFP. G Comparison of the infection efficiency of H129-EGFP at different titers by assessing the success rate of inoculation. H Comparison of the infection efficiency of H129-EGFP at different titers by assessing the percentage of labeled neurons in the DRG. In F, G, and H, the total numbers of mice used in the 0.5 ×10⁹ PFU/mL, 1 ×10⁹ PFU/mL, and 2 ×10⁹ PFU/mL groups were 11, 8, and 10, respectively. I Representative images of H129-EGFP infection in the DRG and RVM. J Analysis of the success rate of H129-EGFP propagation from the DRG to the RVM under different titers. The experiment was repeated 3 times. The total numbers of mice used in the 0.5 ×10⁹ PFU/mL, 1 ×10⁹ PFU/mL, and 2 ×10⁹ PFU/mL groups were 11, 8, and 9, respectively. Data in D, E, H, and J are shown as the mean ± SEM and were analyzed by the unpaired Student’s t-test. P-values in A and F were determined by the log-rank test. n.s., no significant difference, *P <0.05, **P <0.01, ***P <0.001. Scale bars, 50 μm (DRG) and 100 μm (RVM).

We further investigated the effect of bortezomib on viral infection at doses of 0.4 mg/kg and 0.8 mg/kg. The majority of mice were sacrificed on day 7 following virus and bortezomib injection, with a minority becoming moribund on post-injection days 5 and 6. The moribund animals were promptly sacrificed for sample collection. The success rate of viral inoculation was determined as the percentage of mice with detectable EGFP signals in the DRG among the control, 0.4 mg/kg, and 0.8 mg/kg groups (92.4%, 92.9%, and 90.0%, respectively) (Fig. 1B). However, the infection efficiency of H129-EGFP was much higher in the 0.8 mg/kg bortezomib group (31.1% ± 4.7%) than in the 0.4 mg/kg bortezomib group (10.7% ± 1.0%) (Fig. 1C, D). Administration of 0.4 mg/kg bortezomib had no significant effect on viral infection compared to the control group (11.7% ± 2.0%; Fig. 1C, D).

Meanwhile, we evaluated the effect of the bortezomib dose on viral propagation. Our previous study has reported that the rostral ventromedial medulla (RVM), known for its spinomedullary projection [8], emerged as the initial region with detectable viral signals [3]. Consequently, cases displaying H129-EGFP signals in the RVM were considered indicative of successful propagation to the brain. The success rate of HSV propagation to the RVM was assessed by determining the percentage of mice with viral signals in the RVM. We found that 0.8 mg/kg of bortezomib significantly enhanced viral propagation efficiency (Fig. 1E).

In addition, to assess the impact of bortezomib on neuronal function, we made whole-cell patch clamp recordings from cortical pyramidal neurons in brain slices of mice that had been given a subcutaneous injection of 0.8 mg/kg bortezomib 7 days prior. Neurons were selected based on their morphological features (Fig. S1A) and resting membrane potential (Fig. S1B), ensuring that only those with similar types and normal electrophysiological activity were analyzed. The initial action potential, triggered by step current injection, was chosen for analysis (Fig. S1C, D). Assessing the threshold, peak amplitude, and slope of rise showed that bortezomib does not alter the excitability of cortical neurons (Fig. S1E–G). Thus, considering the infection efficiency of H129 in DRG neurons, the survival probability of mice, and the success rate of viral propagation to the brain, 0.8 mg/kg of bortezomib emerged as the preferred dose for subcutaneous injection to promote H129 infection.

The titer of H129 also influences viral infection and propagation. We assessed the effects of commercially available titers at 0.5 ×10⁹ PFU/mL, 1×10⁹ PFU/mL, 2×10⁹ PFU/mL, and 3×10⁹ PFU/mL on the HSV infection and propagation. Survival curves showed that the median survival time (MST) of mice infected with H129-EGFP at 1×10⁹ PFU/mL and 2×10⁹ PFU/mL in the lumbar DRG was 7 days (Fig. 1F). Based on this result, day 7 was selected as the endpoint for detecting viral infection. However, the MST of the 3×10⁹ PFU/mL group was 6 days (Fig. S2A), and this titer led to significant DRG neuron apoptosis in 6 out of 8 cases (Fig. S2B), suggesting increased cytotoxicity. Both the success rate of viral inoculation (100%, 100%, and 90%, respectively) (Fig. 1G) and the percentage of labeled neurons (11.5% ± 1.9%, 16.0% ± 3.3%, and 14.8% ± 2.9%, respectively) in the injected DRG were similar at the titers of 0.5×10⁹ PFU/mL, 1 × 10⁹ PFU/mL, or 2 × 10⁹ PFU/mL H129-EGFP (Fig. 1H). Thus, the titer of H129 with acceptable toxicity does not affect infection efficiency in DRG neurons. Further investigation revealed that increasing the H129 titer from 0.5 × 10⁹ PFU/mL (8.0% ± 8.3%) to 1 × 10⁹ PFU/mL (67.0% ± 19.2%) significantly improved the success rate of viral propagation to the RVM (Fig. 1I, J). However, no additional enhancement of viral propagation to the brain was found with a titer increase from 1 × 10⁹ PFU/mL to 2 × 10⁹ PFU/mL (Fig. 1I, J). Further comparison of labeled neurons in the RVM between 1 × 10⁹ PFU/mL and 2 × 10⁹ PFU/mL groups revealed no apparent differences (Fig. S3). Consequently, we recommend the use of H129 at a titer of 1 × 10⁹ PFU/mL for optimal tracing from the DRG to the brain.

Understanding the propagation stage of H129 is crucial when comparing the labeled networks among different types or subtypes of DRG neurons, as variations in H129 propagation may lead to different labeling patterns. Previous studies have documented that spinal projection neurons target specific nuclei in the hindbrain, midbrain, and interbrain (Fig. 2A) [8]. Theoretically, the speed of viral propagation is correlated with the viral life cycle. However, individual variabilities in the time of trans-synaptic spread from the DRG neurons to the spinal cord neurons have been reported [3]. Therefore, relying solely on the post-injection time to define the viral propagation could be difficult. To address this, we analyzed H129-EGFP propagation in 14 mice, harvesting brains 3 to 7 days post-injection into the DRG. These post-injection periods were chosen because the viral signals in the spinal cord could be detected as early as 2 days after injection, allowing the brain analysis on post-injection day 3. The RVM was initially infected by H129-EGFP administered at the titer of 1 × 10⁹ PFU/mL with 1 mg/kg of bortezomib in the L5 DRG. Subsequently, the paraventricular hypothalamic nucleus (PVH) and the parabrachial nucleus (PBN) in the pons were labeled, followed by the red nucleus (RN) in the midbrain and the primary sensory/motor cortex (SSp/MOp) in the cerebrum. Further propagation led to the labeling of other cortical areas receiving projections from the SSp/MOp (Fig. 2B). Therefore, H129 propagates gradually along ascending spinal pathways.

Fig. 2.

Propagation process of H129-EGFP along ascending somatosensory pathways. A Schematic of ascending pathways from DRG to the cortex. B Viral signals in nuclei as landmarks of propagation. Mice are infected by H129-EGFP at the titer of 1×10⁹ PFU/mL in the L5 DRG with subcutaneous injection of 1 mg/kg of bortezomib. Brains were harvested within 3 to 7 days post-DRG injection. Each column presents nuclei from one mouse. The triangular arrows point to neurons labeled by H129-EGFP. Scale bar, 100 μm.

We categorized the propagation process into three stages. Stage I involved infection limited to the RVM, the initial site of H129 infection in the brain. Stage II was a protracted phase of viral infection from the PVH to the SSp, and could be further divided into early, medium, and late phases. Early stage II showed the viral signal in the PVH and PBN, which received direct spinal cord projections. Medium stage II included labeled neurons in the SSp/Mop, which were likely targeted by the longest spinal cord projections [9]. Late stage II featured the viral labeling of nuclei that did not directly receive spinal cord inputs, such as the ipsilateral RN receiving inputs from the contralateral RN. The indistinct boundaries of the stage II phases suggest that the complexity of viral propagation is influenced by several factors, such as the distance between the downstream neurons and the injection site, the strength of neural activity and the connectivity of the neurons in the circuits. Stage III involved the trans-synaptic propagation within the somatosensory networks. Thus, tracking the sequential H129 labeling provides the possibility of determining the propagation stages of neural networks from various DRG neuron types or subtypes, facilitating quantitative comparisons among different networks.

It is known that HSV replication in neurons induces cytotoxicity and apoptosis [10, 11], and may trigger immune responses leading to animal death and release virions that could reduce the viral tracing accuracy. Therefore, avoiding neuronal apoptosis is crucial for reliable HSV tracing. Evidently, not only the virulence but also the viral titer and duration of infection with HSV are correlated with the level of cytotoxicity, which in turn affects the tracing accuracy. To assess the onset of apoptosis, we assessed caspase-3 expression, a key apoptosis mediator [12], in the injected DRG on post-injection days 2, 4, and 6. Caspase-3 expression was found in a limited number of neurons on days 2 (108.5 ± 15.0 a.u.) and 4 (102.5 ± 18.70 a.u.) (Fig. S4A). The density of caspase-3, calculated as the total fluorescent intensity of caspase-3 divided by the area of DRG sections, increased more than threefold on day 6 (360.6 ± 111.5 a.u.) compared to that on day 2 (Fig. S4B), and apoptotic cell debris was also noted. This indicates that DRG neuron apoptosis begins around day 2 and intensifies on post-injection day 6. However, not all mice showed apoptosis on post-injection day 6 or 7. Among 7 mice at late stage II of propagation characterized by labeled SSp and ipsilateral RN but not SSs, caspase-3 signals were detected in 3 cases (435.8 ± 48.4 a.u.) (Fig. S4C), while the remaining 4 cases showed no caspase-3 signals (Fig. S4D). This result suggests that approximately 42.9% of mice at late stage II experienced contamination at the injection site. Thus, evaluating neuronal apoptosis at the injection site is recommended to exclude contaminated cases.

Using the HSV strain H129 for tracing somatosensory neural networks presents both advantages and challenges. One major challenge is achieving effective viral infection while managing toxicity. Bortezomib, when combined with HSV-1, induces the expression of heat shock protein 90, which may facilitate HSV-1 amplification and improve H129 infection in DRG neurons [13]. Bortezomib also suppresses the immune system, leading to severe HSV infection, as found in inflammatory arthropathy [14] and organ transplant patients [15]. To balance efficiency and toxicity, we tested bortezomib doses of 0.4, 0.8, and 1.6 mg/kg. Our results showed that subcutaneous injection of bortezomib at 0.8 mg/kg enhanced the H129 infection in DRG neurons with manageable toxicity, overcoming previous inefficiencies. Meanwhile, determining the optimal H129 titer for effective trans-synaptic propagation from the DRG to the brain is also crucial. Few studies have examined the impact of viral titer on H129 propagation. A previous report has suggested that the initial virus titer affects the viral propagation rate from initially infected cells to surrounding cells [16]. We found that a titer of 1×10⁹ enhanced H129 propagation to the brain compared to 0.5×10⁹, underscoring the need for sufficient initial viral particles for effective propagation.

For a quantitative comparison of somatosensory neural networks, we defined three stages of H129 propagation. These stages aid in evaluating the extent of neural network labeling. Somatosensory signals from DRG neurons are transmitted through various spinal cord pathways, including spinomedullary projections, the spinoparabrachial tract, the spinothalamic tract, and the spinohypothalamic tract [8]. The first region with viral signals was the RVM, followed by the PVH and PBN. Sequential differences in H129 labeling among the targeted nuclei are related to the distances of the spinal projections. Notably, the VPL, a terminal of the spinothalamic tract, showed delayed labeling compared to SSp, suggesting an alternative ascending pathway to the cortex that bypasses the thalamus [9]. The temporal dynamics of H129 propagation highlight the influence of nucleus grade in somatosensory ascending pathways and propagation stage on labeling, which is critical for accurate network comparison.

Despite the benefits of H129 for tracing neural networks in the brain, managing its toxicity and verifying the results are essential. Bortezomib combined with HSV-1 may induce intense endoplasmic reticulum stress, and therefore activate the apoptosis pathways [17]. Apoptotic neurons may release H129 which can mislabel upstream neurons. Our assessment of caspase-3 expression revealed apoptosis in nearly half of the infected DRGs, suggesting the need for the exclusion of contaminated cases. Evaluating caspase-3 expression is crucial for neuron-type-specific tracing. In addition, to further elucidate the fine structure of neural networks traced by HSV, it is essential to apply other viral tracing tools, such as adeno-associated viruses for anterograde or retrograde tracing and rabies virus for retrograde monosynaptic tracing, to cross-validate the results obtained with HSV.

Supplementary Information

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Conflict of interest

The authors declare that there are no conflict of interest.