neuroinvasiveness of pseudorabies virus injected intracerebrally is dependent on viral concentration...

Neuroinvasiveness of Pseudorabies Virus

Injected Intracerebrally Is

Dependent on Viral Concentration

and Terminal Field Density

J. PATRICK CARD,1,2* LYNN W. ENQUIST,3 AND ROBERT Y. MOORE1,2,4

1Department of Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 152602Department of Psychiatry, University of Pittsburgh, Pittsburgh, Pennsylvania 152603Department of Molecular Biology, Princeton University, Princeton, New Jersey 085444Department of Neurology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

ABSTRACTPseudorabies virus (PRV), a neurotropic swine a herpesvirus, has been used extensively

for transneuronal analysis of multisynaptic circuitry after peripheral injection. In the presentanalysis, we examined the influence of viral concentration and neuronal architecture on theinvasiveness, replication, and transynaptic passage of an attenuated strain of PRV (PRV-Bartha) injected into rat striatum. Different concentrations of PRV-Bartha were injected intothe striatum at a constant rate of infusion (10 nl/minute), and animals were killed 50 hourslater. Viral concentration was manipulated by either altering the volume of the inoculum (100,50, 20 nl) or by diluting the inoculum within a constant volume of 100 nl. Immunohistochemi-cal localization of infected neurons revealed dramatic differences in the progression ofinfection that were dependent directly on the concentration of injected virus. In every case, thepattern of infection was consistent with preferential uptake of virions by axon terminals andretrograde transynaptic passage of virus from the injection site. The known topographicallyorganized corticostriatal projections permitted a precise definition of the zone of viral uptake.This analysis demonstrated that the ‘‘effective zone of viral uptake’’ (i.e., the zone withinwhich viral uptake led to productive replication of virus) varied in relation to the concentra-tion of injected virus, with the highest concentration of PRV invading terminals within a 500µm radius of the canula. Concentration-dependent changes in the progression of retrogradetransynaptic infection also were observed. The highest concentration of virus produced themost extensive infection. The distribution of infected neurons in these cases included thosewith known afferent projections to striatum as well as those that became infected byretrograde transynaptic infection. Lesser concentrations of PRV-Bartha produced an increas-ingly restricted infection of the same circuitry within the same postinoculation interval. It isnoteworthy that neurons known to elaborate dense striatal terminal fields were less sensitiveto reduction in viral concentration than those giving rise to terminal fields of lesser density.Collectively, the data indicate that the onset of viral replication after intracerebral injection ofPRV is directly dependent on virus concentration and terminal field density at the site of virusinjection. J. Comp. Neurol. 1999;407:438–452. r 1999 Wiley-Liss, Inc.

Indexing terms: transynaptic tracing; neurotropic virus; striatum

Neurotropic a herpesviruses have become used increas-ingly to analyze the organization of multisynaptic neuro-nal circuits (for reviews, see Strick and Card, 1992; Cardand Enquist, 1994; Loewy, 1995; Ugolini, 1995; Enquist etal., 1998). This approach, which is based on the ability ofvirus to pass transneuronally, has become particularlypopular for defining the organization of visual and auto-

Grant sponsor: National Institutes of Health; Grant numbers: MH53574,NINDS33506, and NS16304.

*Correspondence to: J. Patrick Card, Ph.D., Department of Neuroscience,446 Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260.E-mail: [email protected]

Received 31 July 1998; Revised 1 December 1998; Accepted 17 December1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 407:438–452 (1999)

r 1999 WILEY-LISS, INC.

nomic circuitry, because these systems can be infected byperipheral inoculation. These viruses also have been usedto characterize polysynaptic circuits after direct injectioninto the central nervous system (CNS; McLean et al., 1989;Norgren and Lehman, 1989; Zemanick et al., 1991; Bar-nett et al., 1993; Hoover and Strick, 1993; Lynch et al.,1994; Middleton and Strick, 1994, 1996; Jasmin et al.,1997; O’Donnell et al., 1997). These studies have demon-strated the feasibility of this approach and have contrib-uted to a literature demonstrating that the direction ofviral transport through a circuit depends on the strain ofvirus (Zemanick et al., 1991; Barnett et al., 1993; Hooverand Strick, 1993; Sun et al., 1996; Card et al., 1998).Nevertheless, little is known regarding the factors thatinfluence uptake, replication, and transynaptic passage ofvirus through a circuit after direct injection into brainparenchyma. This is due in large part to the fact that avariety of strains of virus have been used in these investi-gations, and there has been no systematic parametricanalysis of viral uptake in this paradigm. In this report, weexamine the invasiveness of an attenuated strain of pseu-dorabies virus (PRV-Bartha), a virus commonly used fortransynaptic analysis of CNS circuitry after peripheral orcentral injection. The genomic organization of this virus iswell characterized (for review, see Enquist et al., 1998),and isogenic strains have been used previously to definethe molecular basis of PRV invasiveness of the visualsystem (for review, see Card, 1998). Most recently, we havedemonstrated that a wild type laboratory strain of PRV istransported bidirectionally (anterograde and retrograde)after injection into prefrontal cortex, whereas PRV-Barthais transported only retrogradely after identical injection(Card et al., 1998). The data from the present studydemonstrate that the onset of viral replication and trans-port through multisynaptic circuits innervating striatumis directly dependent on the concentration of injected virusand the density of axonal arbors at the site of injection.

MATERIALS AND METHODS

Animals

Adult male Sprague-Dawley rats (n 5 33) weighing200–300 grams at the time of injection were used in thisstudy. Animals were maintained in a 12:12-hour photope-riod (light on at 0700) with free access to food and water.All experiments conformed to guidelines stipulated in theNational Institutes of Health Guide for the Care and Useof Laboratory Animals, and the protocol was approved bythe University of Pittsburgh Institutional Animal Careand Use Committee. Animals were inoculated, housed,and killed in a biosafety level 2 (BSL-2) laboratory ap-proved for the use of class 2 infectious agents. The safetyprocedures mandated by this experimental approach havebeen published (Strick and Card, 1992; Card and Enquist,1994; Enquist and Card, 1996).

Virus

The Bartha strain of PRV (Bartha, 1961) was used in allexperiments. Detailed information about the preparation,characterization, and use of this strain in our laboratoryhas been published (Enquist and Card, 1996). A singlestock of virus was used for all experiments. The titer of thisstock, determined on PK15 cells, was 1.4 3 109 plaqueforming units (pfu)/ml. The total number of pfu injectedper animal is indicated in Table 1. Aliquots of virus (100

µl/vial) were stored at 280°C, and single vials werethawed immediately prior to injection. Each experimentincluded three animals (Table 1). All animals in a singleexperiment were injected with virus from the same vial,and excess virus was inactivated with Clorox and dis-carded.

Antisera

Rabbit polyclonal antisera (Rb 133 and Rb134) raisedagainst acetone inactivated PRV were used at a dilution of1:10,000 to localize virally encoded proteins in tissuesections. These antisera recognize major capsid and enve-lope proteins and have been characterized in prior investi-gations (Card et al., 1990). Fluorescence double-labelingstudies also were conducted to localize viral antigens andtyrosine hydroxylase (TH) immunoreactivity in sectionsthrough the striatum and substantia nigra (SN). In thesestudies, the rabbit anti-PRV polyclonal was used at adilution of 1:2,000, and TH immunoreactivity was identi-fied by using a mouse monoclonal antiserum (Chemicon;Temecula, CA) diluted to a final concentration of 1:5,000.

Experimental paradigms

Two sets of experiments were conducted to determinethe effect of viral concentration on the onset and progres-sion of viral replication through multisynaptic circuitsinnervating the striatum. Animals included in the firstexperimental paradigm received an intrastriatal injection(for details, see Intracerebral injections, below) of 20, 50,or 100 nl of the same aliquot of virus. Four experimentswere conducted in this group, and each experiment in-volved three animals that were injected with differentvolumes of virus and killed 50 hours later. The same basicexperimental approach was used in the second paradigm,with one important exception. Rather than injecting in-creasingly smaller aliquots of virus, the injection volumewas standardized to 100 nl, and the concentration wasaltered by diluting the inoculum with tissue culture me-dium (Dulbecco’s modified Eagle’s medium). Seven sets ofanimals were used in this analysis, and all animals werekilled 50 hours postinoculation.

Injection of the three animals in each experiment wascompleted within 1 hour, and, as noted above, all animalswere injected with virus from the same aliquot of virusthat was stored on ice during this period. In prior unpub-lished studies, we determined that the titer of PRV-Barthastored in this manner does not decrease over the course of12 hours. However, to ensure that this was not a consider-

TABLE 1. Experimental Paradigms1

Paradigm

Amount of injected virus

High Intermediate Low

I. Injection of different vol-umes (100, 50, 20 nl) ofsame aliquot of virus 1.4 3 105 pfu (4) 7 3 104 pfu (4) 2.8 3 104 pfu (4)

II. Injection of different con-centrations of virus in thesame volume (100 nl) 1.4 3 105 pfu (7) 7 3 104 pfu (7) 3.5 3 104 pfu (7)

1The concentration of virus injected into the striatum in different experimentalparadigms is indicated. Each experiment included three animals, each of which wasinjected with high, intermediate, or low concentrations of virus. Concentration of viruswas determined by either injecting different volumes of the same aliquot of pseudorabiesvirus-Bartha (paradigm I) or injecting different concentrations in the same volume(paradigm II). All animals were killed 50 hours postinoculation. Concentration of virusis indicated in plaque forming units (pfu), and the number of animals included in eachexperiment is indicated in parentheses. Other relevant details regarding the conduct ofthe experiment are described in Materials and Methods.

NEUROINVASIVENESS OF PSEUDORABIES VIRUS 439

ation in the present analysis, we changed the sequence ofinjection in different experiments. That is, some experi-ments involved the consecutive injection of high, interme-diate, and low concentrations of virus into three animals,and the sequence was reversed in others. The effects ofviral concentration on the onset of infection were the sameirrespective of this manipulation.

Intracerebral injections

Viral injections were conducted in the following manner.Animals were anesthetized by intraperitoneal injection ofketamine and xylazine (60 mg/kg ketamine; 7 mg/kgxylazine) and mounted in a sterotaxic frame (Stoelting,Wood Dale, IL). The dorsal surface of the cranium wasexposed by making an incision in the scalp, and the area ofinjection was located by using the following sterotaxiccoordinates derived from Paxinos and Watson (1986):anteroposterior 5 10.5 mm from Bregma, mediolateral 52.5 mm from the sagittal suture, tooth bar at 23.3 mm. Ahole was drilled in the skull at this location, and the durawas reflected to expose the underlying cortex. A 1-µlHamilton syringe (Reno, NV) equipped with a 26-gaugebeveled needle was then loaded with virus, and the tip ofthe canula was lowered 5.0 mm from the surface of theskull. The inoculum was injected at 10 nl per minute, andthe barrel of the syringe was left in situ for 5 minutesfollowing completion of the injection to reduce reflux of theinoculum along the needle tract. The needle was thenremoved, the hole in the cranium was filled with Gelfoam,the scalp incision was closed with wound clips, and theanimals were placed under a heat lamp to recover. Uponrecovery, animals were transferred to cages in a hepafil-tered animal isolation unit and kept in the BSL-2 labora-tory for the balance of the experiment.

Tissue processing

Rats were reanesthetized with an overdose of ketamineand xylazine 50 hours after injection and were killed bytranscardiac perfusion fixation with saline followed bybuffered aldehyde fixative, pH 7.4. These solutions wereinfused at a controlled pressure using a peristaltic pump(Cole-Palmer, Vernon Hills, IL). The saline wash (30–50ml) was used to clear the vasculature of blood and wasfollowed by infusion of 300 ml of paraformaldehyde-lysine-periodate fixative (PLP; McLean and Nakane, 1974). Thebrain was then removed, postfixed in PLP for 1 hour at4°C, and cryoprotected by overnight immersion in a 20%phosphate-buffered sucrose solution, pH 7.4, also at 4°C.The brain was sectioned at 35 µm/section using a freezingmicrotome, and sections were collected sequentially inwells of 10 mM sodium phosphate-buffered saline (PBS),pH 7.4. Each well of tissue contained a one-in-six series ofsections (frequency of 210 µm) through the rostrocaudalextent of the forebrain and brainstem. One well of tissuewas processed immediately for immunohistochemical local-ization of infected neurons (see below), and the others weretransferred to cryoprotectant (Watson et al., 1986) forstorage at 220°C.

Immunoperoxidase localization of virally encoded pro-teins in fixed tissue was used to determine the distributionof infected neurons in all experimental animals. Tissuesections were incubated in one of the rabbit polyclonalanti-PRV antisera diluted to a final concentration of1:10,000 with PBS, Triton X-100, and normal serum for24–48 hours at 4°C. Thereafter, sections were washed inseveral changes of PBS prior to and following incubation in

an affinity-purified biotinylated donkey anti-rabbit immu-noglobulin G (Jackson ImmunoResearch Laboratories,Inc., West Grove, PA) and prepared for immunoperoxidaselocalization of viral antigens by using Vectastain Elitereagents (Vector Laboratories, Burlingame, CA). Pro-cessed sections were mounted on gelatin-coated slides,dehydrated with a graded ethanol series, cleared in xylene,and coverslipped with Permount. Details of these proce-dures, as applied in our laboratory, have been published(Card and Enquist, 1994).

Sections through the striatum and SN also were pro-cessed for dual localization of PRV and TH immunoreactiv-ity. This was accomplished by simultaneous incubation ofthe tissue in Rb133 and in the mouse monoclonal anti-THdiluted to final concentrations of 1:2,000 and 1:5,000,respectively. The tissue was incubated in the primaryantibodies for 48 hours at 4°C and washed thoroughly inPBS prior to and following incubation in affinity-purifiedsecondary antibodies raised in donkey and conjugated toCY2 and CY3 (Jackson ImmunoResearch Laboratories,Inc.). The incubations in these species-specific secondaryantibodies were conducted simultaneously for 1 hour atroom temperature. Sections were mounted on gelatin-coated slides, dehydrated with ethanol, cleared with xy-lene, and coverslipped with Cytoseal60 (VWR Scientific,Pittsburgh, PA). The tissue was analyzed and photographedwith a Zeiss Axioplan photomicroscope (Thornwood, NY)equipped with epifluorescence and the appropriate filters.

Analysis of tissue

Comparative analysis of the degree of infection in eachanimal was accomplished in three ways. First, 12 coronalplanes that accurately reflected the extent of infection inanimals infected with the highest concentration of viruswere selected from templates from the brain maps pub-lished by Swanson (1992). A subset of these templates isillustrated in Figure 5.

The bilateral distribution of infected neurons in theseanimals was mapped on the templates, taking care topreserve the relative cellular density and differentialdistribution of immunoreactive neurons within topographi-cally organized regions of the neuraxis. Second, preciserecords of the distribution of infected neurons in tissueprocessed with the immunoperoxidase method were ob-tained by digitizing images of regions that project tostriatum using a MTI 3CCD camera and a Simple32 imageanalysis system (C-Imaging Systems, Cranberry Town-ship, PA). Third, quantitative measures of the number ofinfected neurons in frontal cortex, striatum, globus palli-dus, thalamus (intralaminar and ventromedial nuclei), SN(pars compacta and reticulata), as well as the mesence-phalic reticular and dorsal raphe nuclei were collected foreach animal. Comparisons were standardized betweenanimals by selecting comparable planes of section andgrouping cases that had equivalent injection sites. In somecases, more than one section was taken from the sameregion in order to insure complete analysis of areas withcomplex cytoarchitecture and/or topographically orga-nized projections. Plates from the atlas of Swanson (1992)corresponding to the levels sampled for each of theseregions are as follows: prefrontal cortex, plate 8; globuspallidus, plates 20 and 22; thalamus, plate 29; SN, plates36–40; dorsal raphe and mesencephalic reticular nucleus,plate 45.

Statistical comparisons of the number of infected neu-rons in striatum, prefrontal cortex, globus pallidus, thala-

440 J.P. CARD ET AL.

mus, SN, dorsal raphe, and mesencephalic reticular nucleuswere made using one-way analysis of variance (ANOVA)and Student’s t-test for independent groups. Numbers ofcells in each area from the three groups of animals injectedwith different concentrations of virus were analyzed ini-tially with ANOVA to determine whether there werestatistical differences between groups. Student’s t-test wasthen used to determine whether there were statisticaldifferences between individual groups.

Preparation of figures

All figures were assembled from digitized images, asdescribed above. Plates were assembled and labeled byusing Adobe Photoshop software (Adobe Systems Inc.,Mountain View, CA). The brightness and contrast ofindividual images were adjusted slightly so that all plateswere of uniform density. Color slides of material used inFigure 4 were digitized with a SprintScan35 Slide Scanner(Polaroid Corporation, Rochester, NY). These images wereassembled and labeled by using Adobe Photoshop, but thecolor, brightness, and contrast of the images was notaltered.

RESULTS

Our experiments were designed to determine the effectof viral concentration on the onset of viral replicationwithin permissive neurons. They were based on the hypoth-eses that axon terminals are the principal site of viral

invasion after intracerebral injection of PRV-Bartha andthat the onset of viral replication occurred earlier inneurons with denser afferent projections to the injectedarea. The latter hypothesis assumes that initiation of aproductive infection is concentration-dependent and pre-dicts that neurons with denser projections would be moreresistant to reduction of viral concentration than neuronswith sparser projections. Also, because the enveloped PRVvirion is approximately 200 nm in diameter and containsenvelope glycoproteins that bind tightly to extracellularmatrix proteins, such as heparin sulfate proteoglycan, wealso expected that virus would not diffuse extensively fromthe injection site.

We found that the extent of infection 50 hours postinocu-lation did vary with the concentration of injected virus. Forsome neurons, reducing the concentration produced onlyminor effects upon the progression of viral infection throughmultisynaptic circuitry innervating striatum, whereas in-fection of other neurons was severely compromised byreduction in viral concentration. Quantitative analysis ofthe data demonstrated that concentration was the mostimportant variable. There was no statistical differencewhen the same concentration of virus was injected at acontrolled rate (10 nl/minute) in different volumes.

Injection sites

All of our determinations of the extent of viral infectionwere made 50 hours after injection of PRV-Bartha. This issufficient time for several cycles of virus growth and

Fig. 1. The regions of the striatum subjected to quantitativeanalysis and the effect of viral concentration on the magnitude ofneuronal infection 50 hours after injection of virus into striatum areillustrated. Counts of infected striatal neurons made in the fourcoronal planes (A–D) form the basis for the statistical analysispresented in Figure 2. E–G illustrate infected neurons in striatumafter injection of different concentrations of virus into the regiondemarcated by the box in B. The circles in E–G are 500 µm in diameterand are centered around the densest region of infection. All concentra-tions of pseudorabies virus (PRV) infected a circumscribed populationof neurons in the immediate vicinity of the canula tract. The largest

number and broadest distribution of infected neurons were achievedby injection of the highest concentration [1.4 3 105 plaque-formingunits (pfu)] of virus (F). Lower numbers of infected neurons were foundwithin a smaller radius of the canula after injection of intermediate(7 3 104 pfu; F) and low (3.5 3 104 pfu; G) concentrations of virus. Theschematic diagrams in A–D are taken from plates 10, 14, 18, and 22,respectively, in Swanson’s (1992) brain maps. The heavy lines indicatesection borders and the borders of brain ventricles, shaded areas markthe positions of myelinated fiber tracts, and dashed lines define thelamination of cortex or cytoarchitecturally defined regions of theforebrain. Scale bars 5 200 µm in E–G.


transynaptic passage (for review, see Enquist et al., 1998).It is critical to note that we did not observe necrotizingspread of viral infection from the site of injection (seeFig. 1). Thus, we conclude that nonsynaptic spread of viralinfection is not a confounding variable in our studies.

Eight sets of animals (three rats per set) that receivedinjections of different volumes (n 5 3) or different concen-trations of virus in the same volume (n 5 5) had compa-rable canula placements. In all of these cases, the numberof infected neurons in the striatum decreased in parallelwith the reduction in concentration of the inoculum.Examples of the results obtained after injection of differentconcentrations of virus in a constant volume of 100 nl areshown in Figures 1E–G. After injection of the highestconcentration of virus, PRV-immunopositive neurons weredensely concentrated along the course of the canula tract,with the highest number found in a circumscribed regionimmediately adjacent to the opening on the beveled sur-face of the canula (Fig. 1E). The average cross-sectionaldiameter of the region adjacent to the canula that con-tained the densest concentration of infected neurons was538 µm, but scattered infected neurons were also observedwithin a 1,500-µm radius of the injection site (Fig. 1E).Injection of lower concentrations of virus resulted in theinfection of fewer neurons within a smaller radius of thecanula opening. After injection of an intermediate concen-tration of virus, the zone of dense infection adjacent to thecanula was similar to that observed after injection of thehigh concentration of virus. However, the number of cellsoutside of this zone was substantially less and rarelyextended beyond a 1,000-µm radius of the canula (Fig. 1F).Only small numbers of infected striatal neurons werefound after injection of the lowest concentration of virus.The majority of these cells were within a 500-µm radius ofthe canula opening, but scattered infected neurons werefound as far as 1,000 µm of the canula opening (Fig. 1G).

The qualitative observations described above were vali-dated by quantitative analysis (Fig. 2). The average num-ber of PRV-infected neurons in the four planes of sectionillustrated in Figure 1A–D was 218 6 13 after injection of1.4 3 105 pfu of virus in 100 nl of vehicle. In contrast,identical injection of intermediate (7 3 104 pfu) or low (2.8or 3.5 3 104 pfu) concentrations of virus infected 151 6 8and 76 6 7 cells, respectively (Fig. 2A,B). One-way ANOVAdemonstrated that the differences between these groupswas significant (F(2,21) 5 23.13; P , 0.0001). It is alsoimportant to note that injecting different volumes ordiluting the concentration of PRV-Bartha in a constantvolume of 100 nl produced very similar results.

The position of the canula also influenced the distribu-tion of infected striatal neurons. In two cases, the canuladiverged laterally and anteriorly to an area immediatelyadjacent to the corpus callosum. Even though the inocu-lum contained high viral titer (1.4 3 105 pfu), infectedstriatal neurons were few in number (51 and 81 vs. theaverage of 218 6 13 typically observed when this titer ofvirus was injected into the center of striatum) and wereconfined to a circumscribed region between the opening onthe beveled surface of the canula tip and the myelinatedfibers of the corpus callosum. The use of sharpened canu-las with an opening on the beveled surface also influencedthe disposition of infected neurons with respect to thecanula tract. In every instance, the largest number ofinfected neurons were observed adjacent to the canulaopening on the beveled surface of the canula.

Effective zone of viral uptake

Because PRV has a very high affinity for axon terminals(Marchand and Schwab, 1987), it would be inappropriateto use the distribution of infected neurons as a measure ofthe zone of virion uptake. However, the topographic organi-zation of corticostriate inputs demonstrated by McGeorgeand Faull (1989) allows a precise means of determining thezone of axonal uptake of virus. This is demonstrated inFigure 3, which illustrates the patterns of infection inmedial prefrontal cortex (PFC) in cases in which thecanula placement varied only slightly or the concentrationof the injected virus differed. Figure 3A,C illustrates twocases in which the high concentration of virus (1.4 3 105

pfu in 100 nl) was injected into regions of striatum that,although they are close in proximity, are known to receivecorticostriatal projections that originate in different re-gions of cortex. In the case illustrated in Figure 3A, theprimary site of injection was confined largely to thedorsomedial region of striatum innervated by the medialmesocortex. This injection produced a dense infection ofthe anterior cingulate and prelimbic subdivisions of themedial mesocortex (Fig. 3B). This distribution contrastedwith that resulting from injection of the same concentra-

Fig. 2. Statistical analysis of the number of infected striatalneurons 50 hours following injection of different volumes of PRV-Bartha (A) or different concentrations of virus in a constant volume of100 nl (B) are illustrated. The amount of injected virus in B is shown inpfu. Table 1 lists the concentration of PRV found in the 20-nl, 50-nl,and 100-nl aliquots injected into animals that provided the data for A.Immunopositive neurons were counted in the four coronal planesillustrated in Figure 1A–D, and the data were subjected to statisticalanalysis by using a one-way analysis of variance (ANOVA) andStudent’s t-test for independent groups. Injection of decreasing concen-trations of virus produced a corresponding decrease in the number ofinfected striatal neurons that were statistically different when the twoexperimental paradigms were analyzed separately or as one group(F(2,21) 5 23.13; P , 0.0001).


Fig. 3. Influences of canula placement and viral concentration onfirst-order retrograde infection of the prefrontal cortex are illustrated.A,C: Cases in which animals received an infection with an equivalentconcentration of virus (1.4 3 105 pfu), but the canula placementdiffered slightly. Canula placement was equivalent in the casesillustrated in C and E, but different concentrations of virus wereinjected: One animal received 1.4 3 105 pfu (C,D), and the otherreceived 7 3 104 pfu in a volume of 100 nl (E,F). When the primary siteof infection, judged on the basis of the canula tract and the distributionof infected neurons, was in the dorsomedial quadrant of the striatum(A), first-order infection of layer V neurons in frontal cortex wasconcentrated in the medial mesocortex and medial component of the

agranular motor cortex (B). In contrast, when the same concentrationof virus was injected more laterally into the dorsolateral striatum,which is known to be innervated by sensorimotor cortex (C), layer Vneurons were prevalent in the agranular motor cortex but were absentin the medial mesocortex (D). Injection of a lower concentration ofvirus into the same region of dorsolateral striatum injected in the caseillustrated in Figure 4C infected a subset of the neurons observed inthat case (E,F). The presence of infected neurons in superficial layersof these regions of cortex is due to transynaptic passage of virusthrough intracortical circuitry. Magnification in A–F is equivalent;Scale bars 5 500 µm.


Figure 4

tion of PRV-Bartha dorsolaterally into the region inner-vated by sensorimotor cortex (Fig. 3C). In this case, largenumbers of infected neurons were present in secondarymotor cortex (Mos), and only scattered neurons werepresent in the medial mesocortex (Fig. 3D). The proximityof the two injection sites demonstrates that the zone ofviral uptake that leads to retrograde infection of specificzones of PFC is within a 500-µm radius of the canula tipwhen high concentrations of virus are injected in a 100 nlvolume infused at 10 nl/minute.

We also observed concentration-dependent influences onthe effective zone of virion uptake in cases in which thecanula placements were equivalent. The two animalsillustrated in Figure 3C,E had equivalent canula place-ments but were injected with either 1.4 3 105 pfu (Fig. 3C)or 7 3 104 pfu (Fig. 3E), both in 100 nl. Although both casesproduced an infection of Mos, the magnitude of infection inthe two cases differed substantially. The animal injectedwith the higher concentration of virus contained largenumbers of infected layer V neurons that were most densemedially but extended through the mediolateral extent ofMos (Fig. 3D). In contrast, a much smaller subset of thispopulation was infected in the animal injected with thelower concentration of virus (Fig. 3F). These data areconsistent with the interpretation that the zone of viraluptake by striatal afferents is smaller after injection oflower concentrations of virus.

The laminar distribution of infected cortical neuronsprovided further insight into the progression of infection.It is well established that corticostriatal afferents ariseprimarily from layer V pyramidal neurons in all regions ofthe rat cortical mantle (McGeorge and Faull, 1989). Accord-ingly, we observed infected neurons in this cortical layerirrespective of the concentration of virus. However, we alsoobserved infected neurons in superficial cortical laminaein these areas, and their number varied directly with theconcentration of virus injected into striatum. The largestnumbers of neurons were observed in cases in which

animals were injected with the highest concentrations ofvirus. The numbers decreased in parallel with reductionsin the concentration of the injected inoculum (Figs. 3B,D,F,6A2–A4) and the presence of infected neurons in otherregions of cortex (Fig. 5). These data suggest that the onsetof viral replication was earlier in animals injected withhigh concentrations of virus than in animals injected withlower concentrations. In addition, it is likely that appear-ance of infected neurons in superficial laminae occurs inresponse to retrograde transynaptic passage of virusthrough corticocortical connections.

Axon terminals as sites of virion invasion

The preceding data suggested that axon terminals arethe primary site of viral invasion. This was tested moredirectly by using immunofluorescence for simultaneouslocalization of PRV and TH immunoreactivity. Figure 4A,Billustrates striatal injection sites from animals that re-ceived 1.4 3 105 pfu (Fig. 4A) or 7 3 104 pfu (Fig. 4B) ofPRV-Bartha in a volume of 100 nl. In both cases, the dopamin-ergic afferents of the nigrostriatal projection revealed by THimmunoreactivity (red and yellow fluorescence) were distrib-uted uniformly through the region of infection. This demon-strates that injection of virus did not produce fiber loss in thisprojection system within the 50-hour postinoculation survivalinterval. TH-immunoreactive fibers in the area immediatelysurrounding the canula also contained immunofluorescencefor viral antigens (yellow fluorescence). These PRV-immuno-fluorescent axons occupied an area slightly smaller than thedistribution of infected neurons (green fluorescence). Examina-tion of the SN in these cases provided further evidence thatthis zone accurately reflects the region in which viral uptakeleads to retrograde infection. Injection of high (Fig. 4C,D),intermediate (Fig. 4E,F), or low (Fig. 4G,H) concentrations ofvirus (all in 100 nl) into the same region of striatum infected asubpopulation of dopaminergic neurons in the medial half ofthe nigra (yellow fluorescence). The distribution of these cellsconforms with the demonstrated topography of the nigro-striatal projection (Fallon and Moore, 1978; Beckstead etal., 1979; Swanson, 1982; Francois et al., 1994).

Progression of infection

We observed concentration-dependent differences in theprogression of infection, such as those observed in thefrontal cortex and SN, in all regions that project tostriatum. These effects occurred whether the concentra-tion of the inoculum was reduced by decreasing the volumeof virus (data not shown) or diluting the inoculum within aconstant volume of 100 nl (Fig. 5). The cases illustrated inFigure 5 are representative of all experiments in whichcanula placement was equivalent among animals injectedwith different concentrations of PRV-Bartha.

The degree to which virus concentration affected sixregions that give rise to well-described projections tostriatum is shown in Figure 6. Cortical regions projectingto striatum, as noted above, characteristically exhibitedlaminar-specific patterns of infection that were concentra-tion-dependent. Both the medial prefrontal cortex (Fig.6A1–A4) and the perirhinal cortex (Fig. 6B1–B4) exhibitedlarge numbers of infected neurons 50 hours followinginjection of the highest concentration of virus and morecircumscribed distributions of infected neurons after injec-tion of intermediate or low concentrations of PRV-Bartha.Quantitative analysis of the number of infected neurons indifferent lamina of a 100-µm-wide column extending fromthe pial surface to the underlying white matter of the

Fig. 4. A–H: Dual localizations of viral and tyrosine hydroxylase(TH) immunoreactivity in the striatum and substantia nigra areillustrated. A and B illustrate this distribution in the striatum ofanimals injected with 1.4 3 105 pfu or 7 3 104 pfu of virus. THimmunofluorescence (red) reveals dopaminergic fibers densely distrib-uted among the myelinated fiber bundles (dark areas) that traversethe rat striatum. A subset of these fibers that are coextensive with thedensest concentration of infected striatal neurons (green fluorescence)also colocalize viral immunofluorescence, producing a yellow signal.The majority of these double-labeled fibers are found within a 400-µmzone surrounding the site of viral injection. C–H illustrate thedistribution of PRV-immunoreactive (green) and TH-immunoreactive(red) neurons in the substantia nigra and ventral tegmental area(VTA) after injection of different concentrations of virus into the sameregion of striatum. D, F, and H are higher magnification views of partsC, E, and G, respectively, in which colocalization of PRV immunofluo-rescence with dopaminergic nigral neurons is most extensive. Thearea of injection in each animal is equivalent to that illustrated inFigure 4A. Note that essentially all of the dopaminergic nigralneurons that are infected by retrograde transport of PRV-Bartha fromthe striatum (yellow fluorescence) are found in the medial half of parscompacta and that infected VTA neurons are few in number and arerelegated to the animal that was injected with the highest concentra-tion of virus (C,E,G). Note also that the pars reticulata of the nigracontains neurons (green) that have become infected by transynapticpassage of virus from the dopaminergic neurons of the immediatelyadjacent region of pars compacta and that the number of these cellsvaries with the concentration of injected virus. Scale bars 5 100 µm inB (also applies to A), 1 mm in G (also applies to C,E), 500 µm in H (alsoapplies to D,F).


Fig. 5. The distribution of infected neurons in forebrain and rostralmidbrain after injection of 1.4 3 105 pfu, 7 3 104 pfu, and 2.8 3 104 pfuof PRV-Bartha in a constant volume of 100 nl is illustrated. Thesemaps reflect the relative distribution and density of infected neuronsin three animals that were injected with virus from the same aliquotand were killed 50 hours after injection. They were adapted fromcamera lucida drawing from each case that were transferred onto

schematic diagrams of coronal sections taken from the brain maps ofSwanson (1992). The maps faithfully represent the distribution ofinfected neurons at each level, but no quantitative inferences shouldbe made regarding the number of cells within a given region. Thelocation of the injection sites in these animals approximates thatshown in Figure 4A.


medial PFC confirmed this observation. The only signifi-cant reduction in the number of infected neurons in eitherdeep (layers 5 and 6) or superficial (layers 2 and 3) laminaeof PFC occurred after injection of the lowest concentrationof virus (Fig. 7A,B). This contrasted with the effects thatviral concentration exerted on infection of the more spa-tially distant perirhinal cortex. Quantitative measureswere not made in this area, but qualitative examination ofthe patterns of infection revealed dramatic differences inthe extent of infection (Fig. 6B2–B4). Thus, transynapticpassage of virus through corticocortical connections wasinfluenced by both the concentration of injected virus andthe distance from the site of injection.

Quantitative analyses of other areas that project tostriatum also demonstrated regional differences in theeffect of viral concentration on the magnitude of infection.For example, the globus pallidus classically is consideredas a relay center through which striatal projections influ-ence thalamocortical input. However, the dorsal and ven-tral pallidum also project to the rat striatum (Staines etal., 1981; Beckstead, 1983; Walker et al., 1989; Kuo andChang, 1992) through a projection system that terminatesin dense patches separated by areas of sparse innervation(Rajakumar et al., 1994; Olive et al., 1997). Our datademonstrated that the pallidal projection was very sensi-tive to reductions in the concentration of the inoculum.Statistically significant reductions in the number of in-fected pallidal neurons were observed after intrastriatalinjection of intermediate or low concentrations of PRV-Bartha (Figs. 6C1–C4, 7; Table 2). This result differed fromthat observed in other areas, such as the thalamic intrala-minar nuclei (centromedial, paracentral, and centrolat-eral; Berendse and Groenewegen, 1990) and mesence-phalic reticular nucleus (Jones and Yang, 1985), thatproject to the striatum but also have prominent projectionsto cortical regions that were infected retrogradely byintrastriatal injection of PRV-Bartha. Neurons in both ofthese regions could have been infected directly by retro-grade transport of PRV from the striatum as well asindirectly through retrograde transynaptic passage ofvirus through other neurons. Accordingly, these areaswere more resistant to reduction of viral concentration,showing a statistically significant reduction in the numberof infected neurons only following injection of the lowestconcentration of virus (Figs. 6D1–D4,F1–F4, 7; Table 2).

Infection of the SN provided additional insights into thefactors that contribute to the onset and progression ofinfection. Quantitative analysis revealed that the densenigrostriatal dopaminergic projection arising form parscompacta of SN was affected least by reduction in viralconcentration (Figs. 4C–H, 6E2–E4). Counts of neurons infive representative coronal planes that completely sampledthe rostrocaudal extent of the SN demonstrated thatinjection of high or intermediate concentrations of PRV-Bartha into striatum infected an average of 383 6 58 and333 6 67 infected neurons, respectively. The only statisti-cally significant reduction in the number of infected neu-rons (190 6 23) was observed after injection of the lowestconcentration of virus (Fig. 7, Table 2).

Although cell counts did not reveal significant differ-ences in the number of infected SN pars compacta neuronsafter injection of high or intermediate concentrations ofvirus, the magnitude of infection in the pars reticulata ofSN suggested that the progression of transynaptic passagewas faster in the animals injected with the highest concen-

tration of PRV. This interpretation is supported by thefollowing observations: Infected reticulata neurons alwayswere distributed throughout the dorsoventral extent of thearea immediately adjacent to infected pars compactaneurons (Fig. 4), and the pattern of infection did notconform to the lamellar segregation shown for reticulataneurons that project to colliculus, thalamus, or tegmentum(Deniau and Chevalier, 1992). Thus, it is probable that thepopulation of reticulata neurons observed in our experi-ments represent the local circuit neurons previously shownto synapse on pars compacta dopaminergic neurons (De-niau et al., 1982; Grofova et al., 1982; Nitsch and Risen-berg, 1988; Bolam and Smith, 1990). Both the distributionand the temporal course of infection support this interpre-tation. The quantitative analysis of the number of theseneurons revealed a statistically significant reduction thatparalleled the reduction in the concentration of the inocu-lum (Fig. 7), supporting the interpretation that the onsetof viral replication in pars compacta neurons was concen-tration-dependent.

DISCUSSION

We have demonstrated reproducible, concentration-dependent patterns of infection resulting from retrogradetransynaptic passage of virus from the striatum. Ourfindings provide further support for the conclusion thatintracerebral injection of neurotropic viruses can be usedeffectively to examine polysynaptic circuitry and alsoprovide novel information important for interpreting thedata produced by using this approach. Of particular impor-tance is the demonstration that the onset of viral replica-tion within a circuit depends on the viral concentrationand terminal field density at the injection site. To providethe most rigorous test of this hypothesis, we focused ouranalysis on the most well-characterized striatal afferentsand polysynaptic connections that can be interpretedeasily based on known connections. A full description of themultisynaptic pathways and routes of viral transportrevealed in the more advanced infections was not a goal ofour study, and, although this material was mapped andanalyzed, in this report, we consider only connectionaldata that provide insight into factors that influence viralinvasiveness and onset of replication.

Virion invasiveness and the effective zoneof PRV uptake

When PRV is injected directly into the brain, it willinfect susceptible cells (those with appropriate receptors)and can replicate only in permissive cells (those capable ofsustaining virus replication). Defining the temporal se-quence in the synthesis of virally encoded proteins, as wehave done in this report, not only maps the progression ofviral spread through multisynaptic circuits but also re-veals insights into the nature of the injection site thatinfluences uptake and replication of virus. Virions injectedinto the brain encounter a complex milieu that can differsubstantially from one region to another. This milieu hasan important influence on the fate of viral particles. Theycan bind tightly to extracellular matrix through envelopeglycoprotein gC (Mettenleiter et al., 1990; Karger andMettenleiter, 1993; Sawitzky et al., 1993; Flynn and Ryan,1996) and can be taken up by nonpermissive cells (e.g.,microglia or astrocytes; Card et al., 1993; Rinaman et al.,1993). Nevertheless, to a first approximation, the extent of


Fig. 6. The distribution of infected neurons in six regions 50 hoursfollowing injection of high, intermediate, and low concentrations ofvirus into striatum are illustrated. In this experiment, the volume wasstandardized at 100 nl. The boxed regions of the schematics in leftcolumn define the fields illustrated in the photomicrographs to theright of each schematic. Each row of photomicrographs illustrates thedistribution of infected neurons 50 hours after intrastriatal injection

of high (column 2), intermediate (column 3), or low (column 4)concentrations of virus. The regions illustrated in each row include themedial prefrontal cortex (A1–A4), the perirhinal cortex (B1–B4), theglobus pallidus (C1–C4), the intralaminar and ventromedial thalamicnuclei (D1–D4), the substantial nigra (E1–E4), and the dorsal rapheand mesencephalic reticular nucleus of midbrain (F1–F4). Scalebars 5 250 µm in A,E, 500 µm in B–D,F.


infection must correlate with the number of susceptibleand permissive cellular profiles in the area of inoculation.

The number and distribution of infected neurons in thestriatum and throughout the neuraxis after intrastriatalinjection of PRV-Bartha provide strong evidence thatvirions preferentially, but not exclusively, invade axonterminals. Approximately 200 PRV-immunoreactive neu-rons were observed in striatum 50 hours after injection ofthe highest concentration of virus (1.4 3 105 pfu), whereasthousands of cells were infected through retrograde tran-synaptic passage of virus through striatal afferents at thesame postinoculation interval. After injection of the lowestconcentrations of virus (2.8 or 3.5 3 104 pfu), the totalnumber of infected striatal neurons was reduced anddiffered in distribution from that observed after injectionof higher concentrations of virus. The principal differencein this regard was the failure of low concentrations of virusto infect large numbers of neurons immediately adjacent tothe canula. Instead, scattered neurons that exhibitedmorphologic features of both cholinergic interneurons andmedium spiny projection neurons were infected within aradius of approximately 1,000 µm. Either of these cellgroups could have been infected through local projections(cholinergic interneurons) or recurrent collaterals (projec-tion neurons) that terminate within the zone of viraluptake. In contrast, it is probable that the dense concentra-tion of PRV-immunoreactive neurons immediately adja-cent to the canula in animals became infected by viralinvasion of terminals, axons, and perikarya. a-Herpesvi-ruses have affinity for all of these neuronal compartments,although the affinity for axons and perikarya is muchlower than for axon terminals (Marchand and Schwab,1987; Vahlne et al., 1978., 1980). The data thereforesuggest that neurons in areas of very high viral concentra-tion may become infected through their perikarya but that

the primary site of entry is through axon terminals. This isan important feature that dictates the patterns of retro-grade transynaptic passage of virus observed in thisinvestigation and others that have injected this strain ofvirus intracerebrally (Jasmin et al., 1997; O’Donnell et al.,1997; Card et al., 1998).

It stands to reason that virion affinities, includingextracellular matrix and nonneuronal cells, restrict diffu-sion of PRV to a circumscribed region surrounding thecanula and thereby define the zone of uptake that leads toproductive infection. Jasmin and colleagues estimatedthat injection of 100 nl of PRV-Bartha (1 3 108 pfu/ml) at20 nl/minute led to viral uptake within a 520 µm radius ofthe injection site and that this zone of diffusion wassmaller than that produced by injection of the b subunit ofcholera toxin (CTb; Jasmin et al., 1997). O’Donnell andcoworkers also reported that the zone of PRV-Barthadiffusion was less than CTb after injection into the ratmediodorsal nucleus and demonstrated that it was pos-sible to make restricted injections of an equivalent volumeof PRV-Bartha (1.4 3 109 pfu/ml) that were confined tosubdivisions of this thalamic nucleus (O’Donnell et al.,1997). Our findings demonstrate restricted uptake ofPRV-Bartha by striatal afferents that is concentration-dependent. This influence of viral concentration was ob-served in all areas that project to the striatum but wasparticularly apparent in the cortex. On the basis of topog-raphy in the corticostriate projection, we could demon-strate accurately that the zone of viral uptake was con-fined to a radius of approximately 500 µm of the canulaafter injection of 100 nl of the highest concentration ofvirus, a finding remarkably similar to that reported byJasmin and colleagues. These observations argue thatinjection of comparable concentrations of PRV-Bartha intoregions of neuropil that are not constrained by adjacent

Figure 6 (Continued)


myelinated fiber tracts or densely packed neuronal peri-karya will produce similar zones of viral diffusion, andthey make the point that cytoarchitecture should beconsidered carefully in the design of studies involvingintracerebral injection of a herpesviruses.

What influences the onset of replicationwithin a circuit?

Marked differences were noted in the onset of viralreplication within multisynaptic circuits after injection ofdifferent concentrations of virus into striatum. Such con-centration-dependent effects are not surprising in light ofprior studies that demonstrated that infectious dose is animportant consideration in the outcome of infection afterperipheral inoculation (Ugolini et al., 1987; Card et al.,1995; Sams et al., 1995). In the visual system, a minimumof 105 pfu must be injected into the vitreous body to achievereplication and transynaptic infection of retinorecipientneurons in 100% of experimental animals; reduction of

titer by 1 log unit reduces the efficiency of infection to 20%(Card et al., 1995). Similarly, reductions of viral concentra-tion compromise the ability of PRV or herpes simplex virusto invade and replicate within motor circuits innervatingthe tongue (Ugolini et al., 1987) or autonomic circuitsinnervating the anterior chamber of the eye (Sams et al.,1995). The present data demonstrate that 100% infectioncan be achieved after intracerebral injection of virus with aconcentration as low as 2.8 3 104 pfu but that the onset ofviral replication can vary considerably when the concentra-tion of the inoculum is varied within a relatively narrowrange (from 2.8 3 104 pfu to 1.4 3 105 pfu). This is mostassuredly related to the architecture of the systems thatwere analyzed. Efferent projections of somatic and auto-nomic neurons are dispersed over a large area, such thatreduction in the concentration of the inoculum wouldsubstantially reduce the ratio of virions to permissiveprofiles. This is not the case after intracerebral injection,in which virions are exposed to large numbers of axons and

Fig. 7. The numbers of infected neurons in the prefrontal cortex(PFC; superficial and deep layers), globus pallidus, thalamic intralami-nar nuclei, and substantia nigra (pars compacta and pars reticulata)50 hours after intrastriatal injection of high, intermediate, or lowconcentrations of PRV-Bartha are illustrated. Cells were counted inone or more coronal planes through each area [the numbers of sectionscounted and the criteria for selecting comparable planes of sectionbetween animals are defined in Materials and Methods]. Grouped datafrom 30 experimental animals with comparable injections were sub-

jected to statistical analysis with a one-way ANOVA and the Student’st-test for independent groups. In each case, the ANOVA revealed astatistical difference between groups of animals injected with differentconcentrations of virus (PFC deep layers: F(2,30) 5 8.22, P , 0.0014;PFC superficial layers: F(2,30) 5 7.08, P , 0.003; globus pallidus:F(2,30) 5 5.67, P , 0.0082; intralaminar nuclei: F(2,30) 5 4.55, P , 0.019;substantia nigra, pars compacta: F(2,30) 5 3.59, P , 0.039; substantianigra, pars reticulata: F(2,30) 5 6.83, P , 0.0036).


neuronal perikarya within a confined region, and the ratioof permissive neuronal profiles is substantially higher thatin the periphery.

An important issue that emerges from the above find-ings relates to the minimum concentration of virus that isnecessary to infect a permissive neuron. In theory, a singleviral particle should be sufficient to infect a neuron;however, in practice, this is not the case. Several factors, asdiscussed above, can compromise the ability of virus toelicit a productive infection. In addition to the probableloss of substantial numbers of particles because of virionbinding to the extracellular matrix or uptake by glial cells,other factors can affect adversely the ability of virions toinfect neurons. For example, the inoculum is produced byreplicating virus in PK15 cells in vitro. This process is notperfect, and defective particles are produced along withinfectious progeny (Locker and Frenkel, 1979; Frenkel etal., 1980). Similarly, virions may be physically damagedduring the harvesting and purification process that pre-cedes intracerebral injection. Particles that enter a suscep-tible neuron following injection are also subject to cellulardefense mechanisms that may lead to destruction of somevirions in lysosomes (Card et al., 1993). Consequently,induction of a productive infection in permissive neuronsthat have access to the site of injection is dependent oninjection of enough particles to surmount these potentialbarriers.

The findings of this study strongly support the conclu-sion that terminal field density exerts a pronounced influ-ence on a neuron reaching the threshold for productivereplication of virus. Neurons in the SN, pars compacta thatelaborate dense projections to the striatum were infectedreadily and were the least sensitive to reducing theconcentration of injected virus. On the other hand, infec-tion of neurons that elaborate sparse striatal projectionswas compromised by injecting lower concentrations ofPRV-Bartha. The reduced infectivity of the latter systemscannot be attributed to axon length, because most of theseregions (e.g., globus pallidus and perirhinal cortex) aremuch closer to the striatum than the nigral neurons.Furthermore, the progressive decrease in the number ofinfected neurons produced by identical injection of thethree concentrations of virus supports the interpretationthat terminal field density is responsible for the differ-

ences in the onset of infection in different systems. Thismay explain our failure to infect basolateral amygdalaneurons after injection of PRV-Bartha into the mediodor-sal nucleus in a prior investigation (O’Donnell et al., 1997).These neurons have demonstrated projections to the medio-dorsal thalamic nucleus, and we have demonstrated thatthey can be infected through other routes of inoculation(unpublished observations). Therefore, low terminal fielddensity is the explanation most parsimonious with thisresult. These observations emphasize the need for cau-tious interpretation of the patterns of infection producedby viral replication and transport and the need to combinethis approach with monosynaptic tracers to ensure that allfirst-order neurons have been infected. They also suggestthat a temporal analysis is essential for identifying allneurons that project to a region, because delayed onset ofviral replication within neurons with sparser projectionsmay preclude these neurons from being identified at asingle postinoculation interval.

CONCLUSIONS

These data demonstrate that viral concentration andterminal field density are important determinants of theonset of viral replication within a neuronal circuit afterintracerebral injection. The findings have important impli-cations for interpreting patterns of transneuronal passageproduced in studies in which PRV and other a herpesvi-ruses are used as transynaptic tracers as well as studies inwhich neurotropic a herpesviruses viruses are used asvectors for delivery of genes to the CNS.

ACKNOWLEDGMENTS

We gratefully acknowledge the expert technical assis-tance of Jihyun Park, Jen-Shew Yen, and Marlies El-dridge. An abstract of these data was presented at the1996 Society for Neuroscience meeting (Park et al., 1996).This work was supported by NIH RO1s MH53574 (J.P.C.),NINDS33506 (L.W.E.), and NS16304 (R.Y.M.).

LITERATURE CITED

Barnett EM, Cassell MD, Perlman S. 1993. Two neurotropic viruses, herpessimplex virus type 1 and mouse hepatitis virus, spread along differentneural pathways from the main olfactory bulb. Neuroscience 57:1007–1025.

Bartha A. 1961. Experimental reduction of virulence of Aujeszky’s diseasevirus. Magy Allatorv Lapja 16:42–45.

Beckstead RM. 1983. A pallidostriatal projection in the cat and monkey.Brain Res Bull 11:629–632.

Beckstead RM, Domesick VB, Nauta WJH. 1979. Efferent connections ofthe substantia nigra and ventral tegmental area in the rat. Brain Res175:191–217.

Berendse H, Groenewegen HJ. 1990. Organization of the thalamostriatalprojections in the rat, with special emphasis on the ventral striatum. JComp Neurol 299:187–228.

Bolam JP, Smith Y. 1990. The GABA and substance P input to dopaminergicneurones in the substantia nigra of the rat. Brain Res 529:57–78.

Card JP. 1998. Practical considerations for the use of pseudorabies virus intransneuronal studies of neural circuitry. Neurosci Biobehav Rev22:685–694.

Card JP, Enquist LW. 1994. Use of pseudorabies virus for definition ofsynaptically linked populations of neurons. In: Adolph KW, editor.Molecular virology techniques, part A, vol 4. San Diego: AcademicPress. p 363–382.

Card JP, Rinaman L, Schwaber JS, Miselis RR, Whealy ME, Robbins AK,Enquist LW. 1990. Neurotropic properties of pseudorabies virus: uptake

TABLE 2. Distribution of Infected Neurons1

Cell group 1.4 3 105 pfu 7 3 104 pfu 3.5 3 104 pfu

CortexPrefrontal cortex

Superficial layers (2 and 3) 100 6 24 124 6 25 19 6 9Deep layers (5 and 6) 215 6 37 182 6 30 59 6 14

Basal gangliaGlobus pallidus 120 6 31 76 6 12 29 6 5

ThalamusIntralaminar nuclei (centromedial,

paracentral, centrolateral) 103 6 22 113 6 26 34 6 8Ventromedial 95 6 40 64 6 20 25 6 12

MidbrainSubstantia nigra compacta 384 6 58 333 6 67 191 6 54Substantia nigra reticulata 101 6 22 47 6 12 25 6 5Dorsal raphe 19 6 6 19 6 8 4 6 2Mesencephalic reticular n. 35 6 10 26 6 9 6 6 2

1This table summarizes the distribution of infected neurons in different regions of theneuraxis following injection of different concentrations of virus into the striatum.Quantitative measures of the numbers of infected cells within each region followinginjection of different concentrations of virus are indicated to the right of each cell group.The cell counts, presented as the mean 6 S.E.M. for each area, were taken from animalsthat were killed 50 hours following injection of virus. A description of the method ofsampling in the quantitative analysis can be found in Materials and Methods, and thelocations of some of the more complex regions are illustrated in Figure 1.


and transneuronal passage in the rat central nervous system. JNeurosci 10:1974–1994.

Card JP, Rinaman L, Lynn RB, Lee B-H, Meade RP, Miselis RR, EnquistLW. 1993. Pseudorabies virus infection of the rat central nervoussystem: ultrastructural characterization of viral replication, transport,and pathogenesis. J Neurosci 13:2515–2539.

Card JP, Dubin JR, Whealy ME, Enquist LW. 1995. Influence of infectiousdose upon productive replication and transynaptic passage of pseudora-bies virus in rat central nervous system. J Neurovirol 1:349–358.

Card JP, Levitt P, Enquist LW. 1998. Different patterns of neuronalinfection after intra-cerebral injection of two strains of pseudorabiesvirus. J Virol 72:4434–4441.

Deniau JM, Chevalier G. 1992. The lamellar organization of the ratsubstantia nigra pars reticulata: distribution of projection neurons.Neuroscience 46: 361–377.

Deniau JM, Kitai ST, Donoghue JP, Grofova I. 1982. Neuronal interactionsin the substantia nigra pars reticulata through axon collaterals of theprojection neurons. Exp Brain Res 47:105–113.

Enquist LW, Card JP. 1996. Pseudorabies virus: a tool for tracing neuronalconnections. In: Lowenstein PR, Enquist LW, editors. Protocols for genetransfer in neuroscience. towards gene therapy of neurological disor-ders. Chichester: John Wiley & Sons. p 333–348.

Enquist LW, Husak PJ, Banfield BW, Smith GA. 1998. Infection and spreadof alpha herpesviruses in the nervous system. Adv Viral Res 51:237–347.

Fallon JH, Moore RY. 1978. Catecholamine innervation of the basalforebrain. IV. Topography of the dopamine projection to the basalforebrain and striatum. J Comp Neurol 180:545–580.

Flynn SJ, Ryan P. 1996. The receptor-binding domain of pseudorabies virusglycoprotein C is composed of multiple discrete units that are function-ally redundant. J Virol 70:1355–1364.

Francois C, Yelnik J, Percheron G, Fenelon G. 1994. Topographic distribu-tion of the axonal endings from sensorimotor and associative striatumin the macaque pallidum and substantia nigra. Exp Brain Res 102:305–318.

Frenkel N, Locker H, Vlazny DA. 1980. Studies of defective herpes simplexviruses. Ann NY Acad Sci 354:347–370.

Grofova I, Deniau JM, Kitai ST. 1982. Morphology of the substantia nigrapars reticulata projections neurons intracellularly labeled with HRP. JComp Neurol 208:249–262.

Hoover JE, Strick PL. 1993. Multiple output channels in the basal ganglia.Science 259:819–821.

Jasmin L, Burkey AR, Card JP, Basbaum A. 1997. Transneuronal labelingof a nociceptive pathway, the spino-(trigemino-)parabrachio-amygda-loid, in the rat. J Neurosci 17:3751–3765.

Jones BE, Yang T-Z. 1985. The efferent projections from the reticularformation and the locus coeruleus studied by anterograde and retro-grade axonal transport in the rat. J Comp Neurol 242:56–92.

Karger A, Mettenleiter TC. 1993. Glycoproteins gIII and gp50 play domi-nant roles in the biphasic attachment of pseudorabies virus. Virology194:654–664.

Kuo H, Chang HT. 1992. Ventral pallido-striatal pathway in the rat brain: alight and electron microscopic study. J Comp Neurol 321:626–636.

Locker H, Frenkel N. 1979. Structure and origin of defective genomescontained in serially passaged herpes simplex virus type 1 (Justin). JVirol 29:1065–1077.

Loewy AD. 1995. Pseudorabies virus: a transneuronal tracer for neuroana-tomical studies. In: Kaplitt MG, Loewy AD, editors. Viral vectors: genetherapy and neuroscience applications. San Diego: Academic Press. p349–366.

Lynch JC, Hoover JE, Strick PL. 1994. Input to the primate frontal eye fieldfrom the substantia nigra, superior colliculus, and dentate nucleusdemonstrated by transneuronal transport. Exp Brain Res 100:181–186.

Marchand CF, Schwab ME. 1987. Binding, uptake and retrograde axonaltransport of herpes virus suis in sympathetic neurons. Brain Res383:262–270.

McGeorge AJ, Faull RLM. 1989. The organization of the projection from thecerebral cortex to the striatum in the rat. Neuroscience 29:503–537.

McLean IW, Nakane PK. 1974. Periodate-lysine-paraformaldehyde fixa-tive. A new fixative for immunoelectron microscopy. J HistochemCytochem 22:1077–1083.

McLean JH, Shipley MT, Bernstein D. 1989. Golgi-like transneuronalretrograde labeling with CNS injections of herpes simplex virus type 1.Brain Res Bull 22:867–881.

Mettenleiter TC, Zsak L, Zuckermann F, Sugg N, Kern H, Ben-Porat T.1990. Interaction of gIII with a cellular heparinlike substance mediatesadsorption of pseudorabies virus. J Virol 64:278–286.

Middleton FA, Strick PL. 1994. Anatomical evidence for cerebellar andbasal ganglia involvement in higher cognitive function. Science 266:458–461.

Middleton FA, Strick PL. 1996. The temporal lobe is a target of output fromthe basal ganglia. Proc Natl Acad Sci USA 93:8683–8687.

Nitsch C, Risenberg R. 1988. Immunocytochemical demonstration of GAB-Aergic synaptic connections in the rat substantia nigra after differentlesions of the striatonigral projection. Brain Res 461:127–142.

Norgren R, Lehman MN. 1989. Retrograde transneuronal transport ofherpes simplex virus in the retina after injection in the superiorcolliculus, hypothalamus and optic chiasm. Brain Res 479:374–378.

O’Donnell P, Lavin A, Enquist LW, Grace AA, Card JP. 1997. Interconnectedparallel circuits between rat nucleus accumbens and thalamus revealedby retrograde transynaptic transport of pseudorabies virus. J Neurosci17:2143–2167.

Olive MF, Anton B, Micevych P, Evans CJ, Maidment NT. 1997. Presynapticversus postsynaptic localization of mu and delta opioid receptors indorsal and ventral striatopallidal pathways. J Neurosci 17:7471–7479.

Park J, Enquist LW, Moore RY, Card JP. 1996. The effect of viralconcentration upon invasiveness, replication, and transynaptic trans-port of pseudorabies virus injected into striatum. Soc Neurosci Abstr22:1730.

Paxinos G, Watson C. 1986. The rat brain in stereotaxic coordinates. NewYork: Academic Press.

Rajakumar N, Elisevich K, Fumerfelt BA. 1994. The pallidostriatal projec-tion in the rat: a recurrent inhibitory loop? Brain Res 651:332–336.

Rinaman L, Card JP, Enquist LW. 1993. Spatiotemporal responses ofastrocytes, ramified microglia, and brain macrophages to central neuro-nal infection with pseudorabies virus. J Neurosci 13:685–702.

Sams JM, Jansen ASP, Mettenleiter TC, Loewy AD. 1995. Pseudorabiesvirus mutants as transneuronal tracers. Brain Res 687:182–190.

Sawitzky D, Voigt A, Habermehl KO. 1993. A peptide-model for theheparin-binding property of pseudorabies virus glycoprotein gIII. MedMicrobiol Immunol 182:285–292.

Staines WA, Atmadja S, Fibiger HC. 1981. Demonstration of a pallido-striatal pathway by retrograde transport of HRP-labeled lectin. BrainRes 206:446–450.

Strick PL, Card JP. 1992. Transneuronal mapping of neural circuits withalpha herpesviruses. In: Bolam JP, editor. Experimental neuroanatomy.a practical approach. Oxford: IRL Press/Oxford University Press. p81–101.

Sun N, Cassell MD. Perlman S. 1996. Anterograde, transneuronal trans-port of herpes simplex virus type 1 strain H129 in the murine visualsystem. J Virol 70:5405–5413.

Swanson LW. 1982. The projections of the ventral tegmental area andadjacent regions: A combined fluorescent retrograde tracer and immuno-fluorescence study in the rat. Brain Res Bull 9:321–353.

Swanson LW. 1992. Brain maps: structure of the rat brain. Amsterdam:Elsevier Science Publishers BV.

Ugolini G. 1995. Transneuronal tracing with alpha-herpesviruses: a reviewof the methodology. In: Kaplitt MG, Loewy AD, editors. Viral vectors.gene therapy and neuroscience applications. San Diego: AcademicPress. p 293–318.

Ugolini G, Kuypers HGJM, Simmons A. 1987. Retrograde transneuronaltransfer of herpes simplex virus type 1 (HSV1) from motoneurons.Brain Res 422:242–256.

Vahlne A, Nystrom B, Sandberg M, Hamberger A, Lycke E. 1978. Attach-ment of herpes simplex virus to neurons and glial cells. J Gen Virol40:359–371.

Vahlne A, Svennerholm B, Sandberg M, Hamberger A, Lycke E. 1980.Differences in attachment between herpes simplex type 1 and type 2viruses to neurons and glial cells. Infection Immunity 28:675–680.

Walker RH, Arbuthnott GW, Wright AK. 1989. Electrophysiological andanatomical observations concerning the pallidostriatal pathway in therat. Exp Brain Res 74:303–310.

Watson RE, Wiegand ST, Clough RW, Hoffman GE. 1986. Use of cryopro-tectant to maintain long-term peptide immunoreactivity and tissuemorphology. Peptides 7:155–159.

Zemanick MC, Strick PL. Dix RD. 1991. Direction of transneuronaltransport of herpes simplex virus 1 in the primate motor system isstrain-dependent. Proc Natl Acad Sci USA 88:8048–8051.


neuroinvasiveness of pseudorabies virus injected intracerebrally is dependent on viral concentration...

Documents