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. Author manuscript; available in PMC: 2023 Jun 7.
Published in final edited form as: Brain Res. 2022 Aug 4;1793:148040. doi: 10.1016/j.brainres.2022.148040

Eyeblink tract tracing with two strains of herpes simplex virus 1

Deidre E O’Dell a,b,*,3, Carrie A Smith-Bell a,b,2, Lynn W Enquist c,d,e,1, Esteban A Engel d,e,1, Bernard G Schreurs a,b,*,4
PMCID: PMC10245079  NIHMSID: NIHMS1901699  PMID: 35932812

Abstract

Background:

Neuroinvasive herpes simplex-1 (HSV-1) isolates including H129 and McIntyre cross at or near synapses labeling higher-order neurons directly connected to infected cells. H129 spreads predominately in the anterograde direction while McIntyre strains spread only in the retrograde direction. However, it is unknown if neurons are functional once infected with derivatives of H129 or McIntyre.

New method:

We describe a previously unpublished HSV-1 recombinant derived from H129 (HSV-373) expressing mCherry fluorescent reporters and one new McIntyre recombinant (HSV-780) expressing the mCherry fluorophore and demonstrate how infections affect neuron viability.

Results and comparison with existing methods:

Each recombinant virus behaved similarly and spread to the target 4 days post-infection. We tested H129 recombinant infected neurons for neurodegeneration using Fluoro-jade C and found them to be necrotic as a result of viral infection. We performed dual inoculations with both HSV-772 and HSV-780 to identify cells comprising both the anterograde pathway and the retrograde pathway, respectively, of our circuit of study. We examined the presence of postsynaptic marker PSD-95, which plays a role in synaptic plasticity, in HSV-772 infected and in dual-infected rats (HSV-772 and HSV-780). PSD-95 reactivity decreased in HSV-772-infected neurons and dual-infected tissue had no PSD-95 reactivity.

Conclusions:

Infection by these new recombinant viruses traced the circuit of interest but functional studies of the cells comprising the pathway were not possible because viral-infected neurons died as a result of necrosis or were stripped of PSD-95 by the time the viral labels reached the target.

Keywords: H129, McIntyre, Transsynaptic Tracers, Neuroinvasive Viruses, Eyeblink Conditioning Circuit

1. Introduction

Neuroinvasive alphaherpes viruses, including strains of herpes simplex virus-1 (HSV-1) and pseudorabies virus (PRV), can not only invade neurons at the site of infection in the peripheral nervous system, but can also enter the central nervous system by traversing multiple synapses to spread to higher-order neurons directly connected to an initially infected cell (Dix et al., 1983; Card et al., 1990). Researchers have taken advantage of the ability of transsynaptic viruses to travel through the nervous system by using them to trace neuronal tracts in vivo including the projections to and from the “arm area” in the primary motor cortex (Zemanick et al., 1991), trigeminal pathways from tooth pulp to cortex (Barnett et al., 1995), subcortical projections to the inferior parietal lobule (Clower et al., 2001), and the contribution of brainstem nuclei in sensory airway inputs (McGovern et al., 2015). This methodology allows not only a determination of which neurons form circuits, but also allows identification of specific neurons in a circuit for further study (Barnett et al., 1993; Ding et al., 1993; Sík et al., 2006; Zeng et al., 2017).

We have used PRV as a retrograde viral tracer injected into the orbicularis oculi muscle and have been able to successfully study the membrane changes of virally-labeled cells in the rat deep cerebellar nuclei (DCN) involved in eyeblink conditioning (EBC) (Wang et al., 2018). However, the PRV recombinants we used spread only in the retrograde direction, and it is unclear if there are membrane changes in cells that are along the sensory circuitry involved in EBC using periorbital stimulation. To trace these sensory inputs, we used derivatives of H129, a strain of HSV-1, which was first isolated from the brain of a patient with encephalitic herpes (Dix et al., 1983). This virulent strain travels through synaptically connected neurons in a predominantly anterograde direction (Wojaczynski et al., 2015). H129 is taken up by the neuron at the dendrites innervating infected epithelial tissue. H129 virions bind to and fuse with the plasma membrane secreting only the viral capsid and tegument proteins into the cytoplasm. DNA replication occurs after the capsid delivers the genome to the nucleus and assembly of new virus particles occurs in the cytoplasm. It is postulated that the newly assembled virions associate with kinesin motors that move virions along the microtubule network to the axon terminal (anterograde transport) (Fields et al., 2013). Once virions arrive at axon terminals, they are released at or near synapses and infect the dendrites of an uninfected neuron. Infection then continues as more virions are produced and spread to synaptically connected cells along the pathway. The HSV-1 strain McIntyre, unlike H129 derivatives, spreads only in the retrograde direction beginning with an infected cell, from infected presynaptic to a post-synaptically connected cell (LaVail et al., 1997; Szpara et al., 2014).

Using viral strains that spread in opposite directions allows for dual inoculations to identify cells receiving both anterograde and retrograde projections. We have experience using transynaptic viruses to label cells in the eyeblink conditioning circuit (EBCC). Fig. 1 shows the EBCC. As illustrated, one of the deep cerebellar nuclei, the interpositus nuclei is a key site involved in integrating excitatory and inhibitory information. Previously, we had tracked the motor pathway of the EBCC in the rabbit using PRV strains that spread only in the retrograde direction, allowing us to study how EBC altered synaptic markers (Gonzalez-Joekes and Schreurs, 2012). More recently, we were able to track the same motor pathway underlying EBC in juvenile rats (Wang et al. 2018). Since the EBCC can be modified as a result of the animal learning EBC (Beylin and Shors, 1998; Person and Raman, 2010; Brown and Woodruff-Pak, 2011; Maiz et al., 2012; Li et al., 2017), by tagging infected cells in the EBCC, we could analyze changes to the intrinsic properties of these cells. This EBCC is well-characterized and has been used to determine the location of learning- and memory-related changes in the brain (Nicholson and Freeman, 2002; Christian and Thompson, 2003; Freeman et al., 2005; Thompson and Steinmetz, 2009). However, the neurons comprising the EBCC do not have a single lineage, suggesting that transgenic reporters would be unable to identify all of the cells in the EBCC. By infecting rats with H129 and McIntyre recombinants expressing different fluorophores, it may be possible to identify cells that, respectively, comprise the anterograde sensory pathway, starting at the periorbital region innervated by the ophthalmic division of the trigeminal nerve to the DCN, particularly in the anterior interpositus nucleus (AIN), and the retrograde, motor pathway, from the periorbital region innervated by the zygomatic branch of the facial nerve to the AIN. An experimental outline can be seen in Fig. 2, which shows the overall timeline of the experiments, as well as the novel HSV-1 strains used in the study.

Fig. 1.

Fig. 1.

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A schematic showing the rat eyeblink conditioning circuit. The anterior interpositus nucleus is shown receiving several excitatory and inhibitory inputs, suggesting this region is involved in cerebellar learning and memory.

Fig. 2.

Fig. 2.

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Experimental Timeline. The figure shows the overall timeline used to carry out our experiments.

It is unknown if the very aspects of neuroinvasive viruses that make them useful as polysynaptic tracers, would compromise them for functional characterization of the neurons in a circuit. We chose an approach that examined neuron viability following viral infection. The goal of this paper was to use H129 and McIntyre recombinants to locate the neurons comprising the anterograde and retrograde pathway of the EBCC in rats and determine if infected neurons in either pathway were suitable for further downstream assays that rely upon neuron viability. We determined if neurons were functional by using Fluoro-jade C, a marker of neuronal death, and immunofluorescent synaptic markers labeling post-synaptic machinery.

2. Results

2.1. Viral time course

2.1.1. Infections with H129 recombinants:

As the HSV-772 traveled along the sensory pathway it also spread to direct connections in the brainstem. Table 1 includes a list of brain regions infected with HSV-772 at the maximum survival time point, 4.5 days post viral inoculation. In addition to the structures comprising the EBCC, structures with direct connections to these regions were also positive for HSV-772.

Table 1.

Brain regions with HSV-772 reactivity 4.5 days post infection. The table lists the structures with at least one cell infected with HSV-772.

Structure Name
ambiguus nucleus, loose part nucleus of Roller
ascending fibers of the facial nerve nucleus of the solitary tract, intermediate part
caudoventrolateral reticular nucleus nucleus X
dorsal paragigantocellular nucleus olivocerebellar tract
dorsal spinocerebellar fibers and olivocerebellar fibers paramedian reticular nucleus
dorsal spinocerebellar tract parapyramidal nucleus
dorsomedial spinal trigeminal nucleus paratrigeminal nucleus
external cuneate nucleus parvicellular reticular nucleus, alpha part
facial nucleus perifacial zone
facial nucleus, dorsolateral subnucleus prepositus nucleus
facial nucleus, lateral subnucleus principal sensory trigeminal nucleus, ventrolateral part
gigantocellular reticular nucleus raphe magnus nucleus
gigantocellular reticular nucleus, alpha part rubrospinal tract
inferior cerebellar peduncle solitary tract
inferior olive, subnucleus A of medial nucleus spinal trigeminal nucleus, caudal part
inferior olive, subnucleus B of medial nucleus spinal trigeminal nucleus, interpolar part
inferior olive, subnucleus C of medial nucleus spinal trigeminal nucleus, oral part
intermediate reticular nucleus, alpha part spinal trigeminal tract
lateral (dentate) cerebellar nucleus spinal vestibular nucleus
lateral cerebellar nucleus, parvicellular part superior cerebellar peduncle
lateral paragigantocellular nucleus, external part superior vestibular nucleus
lateral reticular nucleus trapezoid body
lateral reticular nucleus, parvicellular part trigeminal-solitary transition zone
lateral reticular nucleus, subtrigeminal part vagus nerve
lateral superior olive vestibulospinal tract
lateral vestibular nucleus
matrix region of the medulla
medial lemniscus
medial longitudinal fasciculus
medial superior olive
medial vestibular nucleus, magnocellular part
medial vestibular nucleus, parvocellular part
medullary reticular nucleus, dorsal part
medullary reticular nucleus, ventral part
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Fig. 3 shows HSV-772 reactivity at 3 (A), 3.5 (B), 4 (C), and 4.5 (D) days after viral exposure at 4x magnification. We did not detect HSV-772 in any area of the DCN at 3 (A) and 3.5 (B) days post-inoculation. The first signs of HSV-772 reactivity in the AIN (black circle) were observed at 4 days (C) and also at 4.5 days (D) post infection (shown at a slightly higher magnification (10x) in C’ and D’).

Fig. 3.

Fig. 3.

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HSV-772 Reactivity in the AIN appears at 4 days post infection. A.) HSV-772 at 3 days post infection shows little to no HSV-772 reactivity. Coordinates: Bregma:10.80 mm, Interaural: −1.80 mm. B.) HSV-772 has some reactivity in the brainstem but none in any of the DCN at 3.5 days post infection. Coordinates: Bregma: 11.28 mm, Interaural: −2.28 mm. C.) Shows minor reactivity of HSV-772 at 4 days post infection at the earliest. Coordinates: Bregma: 11.16 mm, Interaural: −2.16 mm. C’.) Shows a zoomed in image of the AIN. There are at least 3 HSV-772 positive cells in this region at 4 days post infection. Same coordinates as in C. D.) Shows HSV-772 is present not only at the AIN but also in the cerebellar cortex. The circle outlines the location of the AIN. Coordinates: Bregma: 10.68 mm, Interaural: −1.68 mm. D’.) Shows a zoomed in image of the AIN. A large group of HSV-772–positive cells is visible. Same coordinates as in D. AIN: Anterior Interpositus Nucleus, DCN: Deep Cerebellar Nuclei. Coordinates are taken from Paxinos and Watson Rat Atlas. Magnification 4x.

2.1.2. Infections with McIntyre Recombinants:

Although HSV-780 traveled retrogradely, HSV-780 also arrived at the AIN at 4 days post infection or later. Fig. 4 shows positive HSV-780 reactivity in the AIN of one rat at 4 days. Similar to H129 recombinant HSV-772, McIntyre recombinant HSV-780 also had spread extensively in other brain regions with direct synaptic connections to the motor component of the EBCC. These results indicate that the earliest HSV-772 or HSV-780 arrives in the AIN is at 4 days post-infection.

Fig. 4.

Fig. 4.

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HSV-780 reactivity appears in the AIN 4 days post infection. Discrete labeling of HSV-780 is found in the AIN 4 days following infection. The circle identifies where the AIN is located. There is also extensive infection throughout the brainstem in areas associated with the facial nerve and the red nucleus, which are identified by the black rectangle. Coordinates: Bregma: 11.04 mm, Interaural: −2.04 mm. AIN: Anterior Interpositus Nucleus. Coordinates are taken from Paxinos and Watson Rat Atlas. Magnification 4x.

The next set of experiments were designed to determine if the cells positive for these strains of HSV-1 were viable by the time viral spread reached the AIN. Neurons were tested with Fluoro-jade C to detect neurodegeneration following HSV-1 infection. Fluoro-jade C is an anionic fluorescein derivative that stains neurons undergoing cell death (Schmued and Hopkins, 2000; Schmued et al., 2005; Gu et al., 2012). Virally-labeled cells were also tested to determine if they retained synaptic receptors associated with learning and memory changes, such as PSD-95 (Migaud et al., 1998; Almeida et al., 2005; Nithianantharajah et al., 2008; Broadhead et al., 2016; Horner et al., 2018). PSD-95 is an excitatory post-synaptic scaffolding marker. It is found on the large excitatory neurons within the deep cerebellar nuclei and is used to transmit information from the excitatory connections of the EBCC including mossy and climbing fiber collaterals.

2.2. HSV recombinant infected cell viability

Fluoro-jade C was used to locate neurons undergoing neurodegeneration in order to determine if neurons infected with HSV-373 were necrotic. Fig. 5 shows cells of the AIN from a rat euthanized 3.5 days post-inoculation with HSV-373 at 63x magnification. This virus has an mCherry reporter instead of EGFP and was used in order to differentiate between the virally-labeled cells and necrotic cells identified by the Fluoro-jade staining. The figure shows that the majority of virally-labeled cells were already necrotic. Fig. 6 shows the AIN of a rat inoculated with HSV-373 that was perfused 4 days post-infection. Not only were the infected neurons necrotic, but they also displayed abnormal morphology with cells appearing amorphous and with swollen somas. The nuclei of these cells also appeared to be displaced and invaginated at this time point. These observations were made previously and are considered a marker of advanced H129 infection (Wojaczynski et al., 2015).

Fig. 5.

Fig. 5.

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HSV-373 infected cells of the AIN. 3.5 days post infection appear necrotic. The figure shows the Fluoro-jade C (upper right panel) is present in cells that are also infected with HSV-373 mCherry (upper left panel). Magnification 63x.

Fig. 6.

Fig. 6.

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HSV-373 infected cells in the AIN 4 days post infection. Neurons infected with HSV-373 have amorphous shape and abnormal cell bodies with displaced nuclei. Neurons with H129 recombinant infection are also green with fluoro-jade C reactivity (upper right panel). Magnification 63x.

Meanwhile, rats that received saline did not show neurodegeneration and Fig. 7 shows the AIN of a control rat. The control animal was euthanized 4 days following saline inoculation had no cells reactive for Fluoro-jade C at 63x magnification. These results indicate that these recombinant strains of H129-positive neurons underwent cytotoxic effects of infection. Neurons positive for HSV-373 at this stage would not be viable for any downstream applications that rely on living cells.

Fig. 7.

Fig. 7.

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The AIN of uninfected rats lacks Flour-Jade C positive cells. There are no cells positive for Fluoro-Jade C staining in the AIN of a control uninfected rat. Magnification 63x.

We next investigated if neurons dual labeled with H129 and with McIntyre recombinants or those labeled with H129 recombinant virus alone possessed the post-synaptic marker PSD-95. As HSV-1 spreads through the circuit, the host immune system activates astrocytes, which work to prevent additional transsynaptic viral transmission by stripping synaptic contacts from infected cells (Fields et al., 2013). In the AIN of rats exposed to HSV-772 and HSV-780 there was no visible PSD-95 reactivity presumably due to an attempt by the immune system to prevent further viral spread by isolating infected cells. Fig. 8 shows neurons labeled with both HSV-1 strains. The upper right panel, which shows the channel used to detect PSD-95, was completely blank. This indicated that dual-infected neurons had been stripped of synapses in an attempt to sequester HSV-772 and HSV-780. In addition, the neurons positive for both strains of HSV-1 had an abnormal morphology. Cells lost their pyramidal shape and demonstrated a bulging soma.

Fig. 8.

Fig. 8.

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Neurons with dual infection in the AIN lack PSD-95. 4 days post infection with HSV-780 and HSV-772 leads to synaptic stripping of infected neurons. There is no visible PSD-95 in the AIN of dual inoculated rats. AIN: Anterior Interpositus Nucleus. Magnification 63x.

Although neurons exposed to both strains of HSV-1 did not retain PSD-95, this was not the case in neurons from animals only inoculated with H129. Fig. 9 shows a neuron positive for HSV-772 in the rat AIN. PSD-95 staining was minimal but still present in the AIN of rats only inoculated with one virus. The HSV-772 infected cell had a swollen cell body but had a normal shape compared to the virally-labeled cells in Figs. 6 and 8. These results suggest that when glial cells, most likely astrocytes, detect the presence of two strains of recombinant HSV-1 in the central nervous system they may cause an exaggerated host response in order to contain the spread of infection. Also, structures comprising both the sensory pathway of the EBCC and the motor pathway of the EBCC would be exposed to virus as well in the dual inoculated animals. In contrast to Figs. 8 and 79, Fig. 10 shows the AIN of an animal given a saline inoculation at the same magnification. Unlike the tissue in Figures 8 and 9 there was extensive PSD-95 reactivity. Without any viral products to activate the immune system, cells in the rat AIN retained their post-synaptic machinery.

Fig. 9.

Fig. 9.

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Neurons with HSV-772 infection in the AIN have diminished PSD-95 reactivity. PSD-95 reactivity is minimal in the AIN of a rat euthanized 4 days after HSV-772 infection. AIN: Anterior Interpositus Nucleus. Magnification 63x.

Fig. 10.

Fig. 10.

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PSD-95 reactivity is extensive in an uninfected rat AIN. Control rats without H129 or McIntyre have PSD-95 in their AIN. AIN: Anterior Interpositus Nucleus. Magnification 63x.

Taken together, these results indicate that HSV-772 alone or in conjunction with McIntyre is a useful tool for tracing neuronal circuits. However, the functional study of cells labeled with the virus may be limited. Studies using electrophysiology or immunofluorescence that aim to compare changes in the membrane properties or synaptic changes in naïve or trained animals could be compromised by these strains of HSV-1.

3. Discussion

The major findings of this paper were that: (1) Both H129 and McIntyre recombinants traced the eyeblink sensory and motor pathway respectively to the AIN, (2) neurons infected with HSV-373 underwent neurodegeneration, as evidenced by Fluoro-jade C reactivity, a marker for cell death, by the time this HSV-1 strain reached the AIN, (3) HSV-772 cells were stripped of the majority of PSD-95 four days post inoculation, and (4) AIN neurons positive for both HSV-772 and HSV-780 did not have any reactivity for the excitatory post-synaptic marker PSD-95. These results suggest that although these recombinant strains of HSV-1 successfully label cells in the EBCC, they likely limit functional studies of these neurons, including electrophysiological slice recordings that would be compromised by the viral infection.

H129 and McIntyre traced the sensory and motor pathway respectively; sensory neurons comprising the EBCC from the periorbital region and motor neurons from the eyelid to the AIN were identified. These two viruses had similar rates of transmission through this circuit, allowing for animals to be inoculated with both HSV-1 strains on the same day. This is an important consideration since different viral strains can exclude each other in hosts or require specific glycoproteins to enter and infect the cell (Barnett et al., 1993; Kim et al., 1999; Banfield et al., 2003; Mettenleiter, 2003; Pomeranz et al., 2005; Sík et al., 2006). The first virus that enters and begins replicating inside a neuron could impede the second virus from effectively spreading to that same cell (Kim et al., 1999). The time of viral inoculation may also influence the ability of both HSV-1 strains to enter the same cell. If the delay between individual viral strain inoculations is too long, the probability of locating dual-labeled neurons is sharply decreased. Delaying inoculation with the second virus by merely-two hours decreases the percentage of dual labeled cells from 100 % to 27 % in the rat spinal cord most likely by superinfection-exclusion (Banfield et al., 2003). Despite these caveats, careful planning increases the likelihood of achieving dual infections in neurons. Combining viruses that travel in opposite directions through the central nervous system is a useful method for identifying cells that are not ideal for transgenic identification.

Neurons labeled with H129 recombinants alone had noticeably altered morphology at four days after viral exposure. Cell bodies were swollen, and nuclear displacement was also noted. These findings are consistent with previous work examining the invasive profiles of various strains of H129 that also classified the stages of H129 infection (Wojaczynski et al., 2015). Although this virus is first visible in the AIN four days post inoculation, the altered morphology of H129-positive cells in the AIN appeared to be at an advanced stage of infection by this time. In the future, perhaps attenuated strains with reduced neurovirulence may prevent cell death or synaptic stripping, allowing downstream assays of infected cells to be successfully performed.

Cells that were positive for both H129 and McIntyre recombinants also displayed abnormal neuronal morphology that appears to be symptomatic of progressed viral spread. In addition to their aberrant appearance, these dual-labeled cells lacked post-synaptic machinery when examined with PSD-95 reactivity. Further work could investigate the presence of other post-synaptic machinery on cells positive for both H129 and McIntyre recombinants, like those cells in the deep cerebellar nuclei receiving GABAergic or glycinergic inputs (Laurie et al., 1992; Morishita and Sastry, 1996; Chaudhry et al., 1998; Gaiarsa et al., 2002; Takayama and Inoue, 2004; Bagnall et al., 2009; Uusisaari and Knöpfel, 2011), since they may also be removed by viral transmission.

Compared to other strains of alphaherpes viruses like PRV, H129′s and McIntyre’s virulence may increase the likelihood of neuron degeneration. Attenuated strains of PRV are capable of transsynaptic tracing without compromising cellular viability. Rabbits exposed to PRV possess infected neurons that retain multiple synaptic markers and PRV-positive neurons in the DCN and cerebellar cortex still possess immunoreactivity for glutamate, GABA, and glycine (Gonzalez-Joekes and Schreurs, 2012). We previously used PRV to identify deep cerebellar neurons in young rats trained with EBC; after performing whole cell recordings on labeled neurons, we observed membrane changes associated with motor learning including a reduction of the after-hyperpolarization amplitude and shortened latency following evoked deep cerebellar nuclei evoked potentials and rebound spikes (Wang et al., 2018). In addition to our work in young rats, other studies using cell culture have successfully performed electrophysiological recordings on superior cervical ganglion neurons infected with silenced PRV-Bartha that were not significantly different from unlabeled cells while using wild type PRV-Bartha or -Kaplan viruses led to continuous and spontaneous firing (McCarthy et al., 2007). Although we have experience using PRV, combining PRV and H129 in one animal is a safety risk that could result in the formation of a hazardous mutant strain of PRV that is transmissible to humans, which is why we ultimately decided to use recombinants of H129 and McIntyre. Others have found that cell cultures of virally-exposed neurons could be treated with antiviral drugs to retain normal neuronal physiological properties. Cell cultures of primary murine trigeminal neurons were treated with the antiviral drug foscarnet. Foscarnet did not halt viral spread between cells but protected virally-infiltrated cells from exhibiting abnormal membrane properties (Damann et al., 2006; Rothermel et al., 2009).

Although H129 and McIntyre recombinants may not be suitable for studies requiring neuron viability, these HSV-1 strains could be used to study circuit reformation following neuronal injury. Previous work with PRV examined circuit reorganization of the phrenic nerve following cervical spinal cord injury (Lane et al., 2012; Zholudeva et al., 2017). By performing dual inoculation following injury to an area of the central nervous system, H129 and McIntyre recombinants could be used to examine the reorganization of the circuit as it recovers from injury. Dual inoculations of genetically engineered viral strains that travel in opposite directions through the nervous system could identify restructuring along the anterograde and retrograde pathway (McGovern et al., 2012).

4. Conclusion

These findings suggest that although viral strains are useful tools to label neurons in transsynaptic circuits, infected cells must be closely analyzed for overall health and for synaptic markers. It is known that transsynaptic viruses are useful tracers of discrete neuronal circuits, however, it is generally unknown how these viral tracers may negatively impact the infected neurons in the circuit. Determining how neurons are altered following exposure to transsynaptic neuroinvasive viruses is necessary to ensure these viruses do not compromise experimental integrity. Combinations of viruses allow for great precision of labeling and identification of populations of neurons. However, these viruses could also affect the ability to detect changes in synaptic markers or neuron activity and researchers employing them should investigate cell viability in addition to effects on synaptic markers.

5. Methods and materials

5.1. Animals

Twenty-nine juvenile male and female Long Evans rats (Rattus norvegicus) between post-natal day 22 (P22) and P28 were supplied by Charles River (Wilmington, MA). At least 3 rats were assigned to six different time points for H129 anatomical tracing (3 d, 3.25 d, 3.5 d, 3.75 d, 4 d, 4.5 d). Three additional rats were euthanized at 3.5 d or 4 d post H129 recombinant infection for Fluoro-jade C experiments. Three rats were given dual inoculations of H129 and McIntyre recombinants. Two rats were used for McIntyre recombinant-only inoculation. Finally, three rats were used as controls for comparison against infected animals. Instead of viral application, the control animals were exposed to saline alone. All rats were housed with littermates of the same sex after weaning but were housed individually once inoculated with either HSV-1 #772-EGFP (HSV-772), HSV-772 and HSV-1 #780-mCherry (HSV-780), HSV-1 #373-mCherry (HSV-373), or saline control. Rats were given ad libitum access to food and water and maintained on a 12 h light/dark cycle, along with crinkle paper for enrichment all in accordance with the National Institute of Health guidelines. All procedures were approved by the West Virginia University Animal Care and Use Committee and the WVU Biosafety Committee.

5.2. Viral inoculation

5.2.1. Inoculation with H129 recombinants

Either HSV-772 or HSV-373, H129 recombinants expressing enhanced green fluorescent protein or mCherry respectively, was used as the anterograde transsynaptic tracer. HSV-373 expressed diffusible mCherry from the CMV promoter. The reporter was inserted in the UL26/UL26.5-UL27 intergenic region. HSV-373 and HSV-772 are isogenic recombinants with the fluorescent reporter being the only difference. H129 recombinants were administered at a concentration of 7 × 108 plaque-forming units per milliliter (PFU/mL). H129 recombinants were aliquoted and stored at −80 °C. A 100 μl aliquot was thawed on wet ice and sonicated for 1 s on and 1 s off ten times. Before inoculation, rats were given 5 % isoflurane to induce anesthesia and then reduced to 2 % isoflurane to maintain anesthesia while supplemented with 1 % O2. The left periorbital region was shaved, and a #10 scalpel blade was scraped at the periorbital region until a mild abrasion, but no blood, was present. A 10 μl Hamilton syringe was primed with 3 draws and expressions of saline. A 31-gauge, beveled needle was then attached and up to 10 μl of H129 was drawn up and expressed 3 times. The needle was then loaded with the intended inoculation volume and the virus inoculum was applied to the abraded periorbital region and further scraped to achieve viral absorption. The same procedure was used in control animals except that saline was applied to the abraded periorbital region. This process has been used to induce bacterial infections in lab animals and was a precursor to injected vaccinations in humans (Cantey, 2011; Imperato and Imperato, 2014; Helfert, 2015).

5.2.2. Inoculation with McIntyre recombinants

An HSV-1 McIntyre recombinant that expressed mCherry (HSV-1 #780-mCherry), referred to as HSV-780 from here on, was used as the retrograde transsynaptic tracer. HSV-780 was the McIntyre strain that expressed diffusible mCherry from the CMV promoter. The reporter was inserted in the UL26/UL26.5-UL27 region. HSV-780 was administered at a concentration in a range from 2.0 × 108 to 7.8 × 108 PFU/mL. HSV-780 was aliquoted and stored at −80 °C. A 100 μl aliquot was thawed on wet ice and sonicated for 1 s on and 1 s off ten times. Before inoculation, rats were given 5 % isoflurane to induce anesthesia, and then reduced to 2 % isoflurane to maintain anesthesia while supplemented with 1 % O2. The left eyelid was shaved. A 10 μl Hamilton syringe was primed with 3 draws and expressions of saline. A 31-gauge, beveled needle was attached and up to 10 μl of HSV-780 was drawn up and expressed 3 times. The needle was the slightly overfilled beyond the intended inoculation volume. HSV-780 was injected into the left orbicularis oculi muscle with 1 μl at each of 5 injection sites and the needle was left at each site for 2 min to ensure complete viral absorption.

5.3. Tissue processing

Following induction of anesthesia with 5 % isoflurane supplemented with 1 % O2, the rats were given an intramuscular injection of 0.2–0.3 cc of ketamine hydrochloride (80 mg/kg) mixed with xylazine (8.0 mg/kg) at 3 d, 3.25 d, 3.5 d, 3.75 d, 4 d, or 4.5 d after viral inoculation. Animals were perfused transcardially with 150–200 mL of 0.9 % saline (pH 7.4 at room temperature) followed by 150–200 mL of 4 % paraformaldehyde. For anti-H129 immunohistochemistry, Fluoro-jade C, and synaptic marker immunofluorescence, brains were collected and placed in fixative for 24 h and transferred to 30 % sucrose for cryoprotection until they sank, then 50-μm sections were cut on a freezing microtome.

5.4. Immunohistochemistry and immunofluorescence

5.4.1. anti-HSV-1 immunohistochemistry

Free-floating sections were washed in 0.5 M Tris and placed in 3 % H2O2 for 30 min to quench endogenous peroxidases. After washing, the sections were blocked for 1 h in 5 % normal goat serum and incubated in a polyclonal, rabbit anti-HSV-1 primary antibody (1:5000, Dako, B011402–2) overnight at 4 °C in 0.5 M Tris-1 % Triton 100. After washing, the sections were placed in secondary antibody (1:400, biotinylated goat anti-rabbit) for 1 h. The sections were then washed and placed in an avidin/ biotin complex (ABC) based peroxidase system (Vector Laboratories, PK-6100) for 1 h. Finally, tissue was reacted with 3, 3 –diaminobenzidine or DAB horseradish peroxidase substrate (Vector Laboratories, SK-4100) for 2–5 min until color developed. Sections were mounted onto slides using cresyl gelatin and were counterstained with hematoxylin. Slides were coverslipped using Permount media (Fisher Scientific) and #1 coverslips (Fisher Scientific).

The location of each HSV-1-labeled neuron was plotted on sequential coronal sections of the rat brain, but a specific structure was only considered to be labeled and reported here if more than one neuron was labeled in that structure for each of the 3 rats used for a given time point. Figures were compared to the Paxinos and Watson Rat Stereotaxic Atlas. Labeled cells were investigated from the spinal cord until almost into the midbrain. We looked from Bregma: −8.04 mm, Interaural: 0.96 mm to Bregma: −15.96 mm, Interaural: −6.96 mm, roughly figures 100 – 161 within the atlas. Only the most relevant images were included.

5.4.2. Synaptic marker immunofluorescence

Brain sections were mounted onto 3 % gelatin coated slides. Slides were first washed in 0.1 M phosphate buffered saline (PBS)- 1 % Tween then blocked for 2 h with 5 % normal donkey serum. Mounted sections were then incubated in primary antibody, a mouse monoclonal anti-PSD-95 (1:2000, ThermoFisher, MA1–046) overnight at 4 °C. Following washing in 0.1 M PBS, slides were incubated in secondary antibody, AlexaFluor 546 donkey anti-mouse (1:250, Invitrogen, A10036), and 1 % normal donkey serum for 4 h. After completing the secondary antibody reaction, slides were washed again in 0.1 M PBS. Following the final wash, slides were coverslipped with DAPI Fluoromount-G mounting media (SouthernBiotech) and #1.5 coverslips (Fisher Scientific), to label nuclei.

5.4.3. Fluoro-jade C staining

Brain sections were mounted onto 3 % gelatin coated slides and then processed with the Fluoro-jade C Staining Kit with DAPI counterstain, to label nuclei (Histo-Chem Inc., Product # FJC-SK-DAPI). Slides were immersed in 9 mL 70 % ethanol to 1 mL Solution A (sodium hydroxide) for 5 min. Slides were then placed into 10 mL of 70 % ethanol for 2 min before being transferred to 10 mL of distilled water for an additional 2 min. 9 mL of distilled water were added to 1 mL of Solution B (potassium permanganate) and the slides were left in this solution for approximately 6 min. The slides were removed from this solution and rinsed in 10 mL of distilled water for 2 min. 9 mL of distilled water were added to 1 mL Solution C (Fluoro-jade C) and 1 mL Solution D (DAPI) and slides were placed into this solution for 10 min. Immediately after, slides were transferred and underwent 3 rinses in 10 mL of distilled water for 1 min each. Following rinsing, slides were allowed to dry on a slide warmer after which they were placed in xylene for 1–5 min for clearing. Slides were coverslipped with DPX mounting media (Sigma-Aldrich, 06522–100ML) and #1.5 coverslips (Fisher Scientific).

5.5. Image acquisition

5.5.1. Light microscopy

Images were acquired using an upright Olympus BX51 light microscope with a motorized stage and with 1.25X (NA 0.04), 4X (NA 0.15), or 10X (NA 0.4) objectives. Images were exported to ImageJ (NIH) for cell counts.

5.5.2. Confocal microscopy

Synaptic marker immunofluorescent reactivity in the AIN of rats previously inoculated with HSV-1 H129 recombinants alone or H129 and McIntyre recombinants together were visualized using a confocal laser-scanning microscope (Zeiss LSM 710; Carl Zeiss International). Images were acquired using 405 (Diode 405–30), 488 (Argon), 546 (DPSS 561–10), and 633 (HeNe633) nm lasers, sequential multichannel line scan, averaged twice, pinhole aperture of 54 AU, 1024 X 1024 resolution, and the filters were set manually to detect the spectral peak of each fluorophore. Cells were imaged at 10X (NA 0.4) and 63X oil-immersion (NA 1.4) objectives. Images were exported to Adobe Photoshop CC 2017 and minor adjustments to the brightness and contrast were made.

Acknowledgments

This research was supported by NIH Grant NS094009 awarded to B. G. Schreurs and by NIH P40 OD010996 awarded to L.W. Enquist.

Footnotes

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Deidre E. O’Dell: Investigation, Writing – original draft, Visualization. Carrie A. Smith-Bell: Investigation, Writing – review & editing. Lynn W. Enquist: Methodology, Resources. Esteban A. Engel: Methodology, Resources. Bernard G. Schreurs: Supervision, Writing – review & editing.

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