# Pseudorabies Virus (PRV) for DRG retrograde labeling? **URL:** \ **Category:** Reagents and Equipment\ **Tags:** viral\_vectors\ **Created:** [March 13, 2018, 6:43pm UTC](https://forum.painresearcher.net/t/pseudorabies-virus-prv-for-drg-retrograde-labeling/414 "2018-03-13T18:43:42Z")\ **Posts on this page:** 3\ **Page:** 1
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [March 13, 2018, 6:43pm UTC](https://forum.painresearcher.net/t/pseudorabies-virus-prv-for-drg-retrograde-labeling/414/1 "2018-03-13T18:43:42Z")
Anyone have thoughts or experiences with this? ---
**Author:** ![liz](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/liz/32/267_2.png) [@liz](https://forum.painresearcher.net/u/liz)\ **Post date:** [April 9, 2018, 2:19pm UTC](https://forum.painresearcher.net/t/pseudorabies-virus-prv-for-drg-retrograde-labeling/414/2 "2018-04-09T14:19:13Z")
I’m not sure it would adequately label DRG and afferents. In our experience, we injected PRV-152 (Bartha strain expressing PRV) into the hindlimb muscle. We saw robust infection of spinal cord motor neurons but not substantial DRG infection. Moreover we definitely did not see GFP-labeled afferents in the spinal cord. > > > Pacemaker neurons with an intrinsic ability to generate rhythmic burst-firing have been characterized in lamina I of the neonatal spinal cord, where they are innervated by high-threshold sensory afferents. However, little is known about the output of these pacemakers, as the neuronal populations that are targeted by pacemaker axons have yet to be identified. The present study combines patch-clamp recordings in the intact neonatal rat spinal cord with tract-tracing to demonstrate that lamina I pacemaker neurons contact multiple spinal motor pathways during early life. Retrograde labeling of premotor interneurons with the trans-synaptic pseudorabies virus PRV-152 revealed the presence of burst-firing in PRV-infected lamina I neurons, thereby confirming that pacemakers are synaptically coupled to motor networks in the spinal ventral horn. Notably, two classes of pacemakers could be distinguished in lamina I based on cell size and the pattern of their axonal projections. Whereas small pacemaker neurons possessed ramified axons that contacted ipsilateral motor circuits, large pacemaker neurons had unbranched axons that crossed the midline and ascended rostrally in the contralateral white matter. Recordings from identified spino-parabrachial and spino-periaqueductal gray neurons indicated the presence of pacemaker activity within neonatal lamina I projection neurons. Overall, these results show that lamina I pacemakers are positioned to regulate both the level of activity in developing motor circuits and the ascending flow of nociceptive information to the brain, thus highlighting a potential role for pacemaker activity in the maturation of pain and sensorimotor networks in the central nervous system. Our study relied heavily on previous work from the following paper, which is a useful reference on PRV-152: > **[The Use of PRV-Bartha to Define Premotor Inputs to Lumbar Motoneurons in the...](https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0011743)** > > Background The neonatal mouse has become a model system for studying the locomotor function of the lumbar spinal cord. However, information about the synaptic connectivity within the governing neural network remains scarce. A neurotropic pseudorabies... Also, it’s worth mentioning that the PRV vectors described in this paper are attenuated but replication competent. They will move transsynaptically in the retrograde direction and (in our experience) will eventually kill the animal around the time the infection reaches the brain. Also, the health of infected neurons is not great unless you catch them very soon after they start expressing the GFP. A subsequent publication describes a PRV-cre vector (Becker strain) which is replication deficient, improving safety and animal health, and limiting gene expression to only the primarily infected neurons and not their presynaptic partners. > > > Brain regions contain diverse populations of neurons that project to different long-range targets. The study of these subpopulations in circuit function and behavior requires a toolkit to characterize and manipulate their activity in vivo. We have developed a novel set of reagents based on Pseudorabies Virus (PRV) for efficient and long-term genetic tagging of neurons based on their projection targets. By deleting IE180, the master transcriptional regulator in the PRV genome, we have produced a mutant virus capable of infection and transgene expression in neurons but unable to replicate in or spread from those neurons. IE180-null mutants showed no cytotoxicity, and infected neurons exhibited normal physiological function more than 45 days after infection, indicating the utility of these engineered viruses for chronic experiments. To enable rapid and convenient construction of novel IE180-null recombinants, we engineered a bacterial artificial chromosome (BAC) shuttle-vector system for moving new constructs into the PRV IE180-null genome. Using this system we generated an IE180-null recombinant virus expressing the site-specific recombinase Cre. This Cre-expressing virus (PRV-hSyn-Cre) efficiently and robustly infects neurons in vivo and activates transgene expression from Cre-dependent vectors in local and retrograde projecting populations of neurons in the mouse. We also generated an assortment of recombinant viruses expressing fluorescent proteins (mCherry, EGFP, ECFP). These viruses exhibit long-term labeling of neurons in vitro but transient labeling in vivo. Together these novel IE180-null PRV reagents expand the toolkit for targeted gene expression in the brain, facilitating functional dissection of neuronal circuits in vivo. In our hands this vector did not result in detectable cre expression at either the site of injection or the spinal projection neurons we were attempting to label. However, this may not reflect the capabilities of this vector in a R26-CAG-LSL-reporter mouse where only a small amount of cre is necessary to enable strong expression of a reporter. We were using this vector in neonatal rat, in conjunction with a second cre-dependent AAV, so it is also possible that our lack of success was due to the cre-dependent vector and not the PRV-cre. ---
**Author:** ![achamess](https://yyz2.discourse-cdn.com/flex030/user_avatar/forum.painresearcher.net/achamess/32/534_2.png) [@achamess](https://forum.painresearcher.net/u/achamess)\ **Post date:** [April 9, 2018, 2:30pm UTC](https://forum.painresearcher.net/t/pseudorabies-virus-prv-for-drg-retrograde-labeling/414/3 "2018-04-09T14:30:51Z")
Wow. Thanks @liz! This is extremely helpful. Thanks for sharing. I may pass on PRV. The quest continues for a good anterograde transsynaptic tracer that can start with DRG neurons…