Spinal cord-projecting vasopressinergic neurons in the rat paraventricular hypothalamus

Spinal Cord-Projecting VasopressinergicNeurons in the Rat Paraventricular

Hypothalamus

MARTIN HALLBECK* AND ANDERS BLOMQVIST

Division of Cell Biology, Department of Biomedicine and Surgery, Faculty of Health Sciences,University of Linkoping, S-581 85 Linkoping, Sweden

ABSTRACTThe paraventricular hypothalamic nucleus (PVH) is a key structure for the maintenance

of homeostasis. Homeostatic regulation includes modulation of signaling in the spinal cord.This may be exerted by neurons in the PVH with spinal projections. However, the PVH is not ahomogeneous structure, but consists of anatomically and functionally distinct subdivisions. Inthis study, we have analyzed the distribution of spinal cord-projecting PVH neurons thatexpress vasopressin, an important neuropeptide in autonomic regulation. Vasopressinergicneurons were identified with a radiolabeled riboprobe complementary to vasopressin mRNAcombined with immunohistochemical labeling of retrogradely transported cholera toxinsubunit b in spinally projecting neurons. More than 40% of the spinally projecting neurons inthe PVH of naive Sprague-Dawley rats were found to express vasopressin mRNA. The lateralparvocellular subdivision and the ventral part of the medial parvocellular subdivisioncontained the densest distribution of spinal cord-projecting vasopressin mRNA-expressingneurons. The magnocellular subdivisions displayed large numbers of vasopressin mRNA-expressing neurons, but very few of those projected to the spinal cord. The dorsal parvocellularsubdivision contained a large number of spinally projecting neurons, but very few of thoseexpressed vasopressin mRNA. These findings show that the PVH gives rise to a majorvasopressinergic projection to the spinal cord and that the spinal cord-projecting vasopressin-ergic neurons are parceled into anatomically distinct cell groups. This provides an anatomicalbasis for a selective activation of functionally different groups in the PVH as part of abehaviorally adaptive response, including modulation of autonomic activity and pain process-ing at the spinal level. J. Comp. Neurol. 411:201–211, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: in situ hybridization; retrograde labeling; subnuclei; parvocellular; mRNA

The paraventricular hypothalamic nucleus (PVH) hasbeen shown to be involved in many regulatory functionsand can be described as a homeostatic motor nucleus. Thefirst efferent projection to be detected was that to theposterior pituitary (Bargmann and Scharrer, 1951), afinding that demonstrated the route through which sev-eral of the long-known neurohormonal effects of the PVH(Oliver and Schafer, 1895; Dale, 1906; Du Vigneaud, 1954)could be exerted. Later it was shown that the PVH also isimportant for the control of the anterior pituitary, throughthe release of chemical messengers into the portal circula-tion from axons to the median eminence (Harris, 1948;Halasz et al., 1962; Burgus et al., 1970; Brazeau et al.,1973). Projections from the PVH to the brainstem andspinal cord were proposed in early studies (Beattie et al.,1930), but were not unequivocally demonstrated untilmuch later (Saper et al., 1976), giving an anatomicalexplanation of the long-known influence of the hypothala-

mus on the autonomic nervous system (see Swanson, 1987,for a review).

The PVH is not a homogenous structure, but consists ofseveral different subdivisions. These differ in cellularappearance (Kiss et al., 1991), peptide content (see Swan-son and Sawchenko, 1983, for a review), and projectionpatterns (Swanson and Kuypers, 1980). For example, themagnocellular subdivisions are the origin of the projectionto the neural lobe, the medial parvocellular subdivision(mp) gives rise to the projection to the median eminence,

Grant sponsor: Swedish Medical Research Council; Grant number: 7879;Grant sponsor: Swedish Society for Medical Research.

*Correspondence to: Martin Hallbeck, Division of Cell Biology, Department ofBiomedicine and Surgery, Faculty of Health Sciences, University of Linkoping,S-581 85 Linkoping, Sweden. E-mail: [email protected]

Received 12 November 1996; Revised 23 February 1999; Accepted 16March 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 411:201–211 (1999)

r 1999 WILEY-LISS, INC.

and the lateral (lp) and dorsal (dp) parvocellular subdivi-sions, as well as the mp, are the source of the descendingfibers to the brainstem and spinal cord. Furthermore, ithas been shown that these subdivisions of the PVH aredivided into several smaller parts (Swanson and Saw-chenko, 1983), which differ in intracellular messengers(Senba et al., 1993; Smith and Day, 1994; Hallbeck et al.,1996a) and response to different stimuli (Watts, 1992;Watts and Sanchez-Watts, 1995). Taken together, thesedata indicate that each distinct part of the PVH plays adistinct functional role.

The present study is the first in a series that aims atanalyzing the peptide expression in PVH neurons thatsend their axons to the spinal cord, by using modern,sensitive methods. The results are interpreted in thecontext of the present knowledge of the fine anatomicalorganization of the PVH, in order to provide a basis forfuture functional studies on the role of the differentpeptide-expressing populations in the regulation of spinalcord activity. The present report deals with the vasopressin-ergic population. Fibers immunoreactive to vasopressinmake up an abundant terminal network in spinal cord,both in the superficial dorsal horn and in the intermediolat-eral cell column (Buijs, 1978; Nilaver et al., 1980), andspinal vasopressin has been associated with several auto-nomic mechanisms (Porter and Brody, 1986; Malpas andCoote, 1994;), as well as with pain modulation (e.g.,Watkins et al., 1986). Although many different sources forthe vasopressin fibers in the spinal cord have been sug-gested (see, e.g., Caffe and van Leeuwen, 1983; Caffe et al.,1985; Sofroniew, 1985; Kai-Kai et al., 1985, 1986; Kai-Kaiand Che, 1995), the bulk of evidence points to the PVH asthe main, if not the sole source for these fibers (e.g.,Sawchenko and Swanson, 1982; De Vries and Buijs, 1983;Lang et al., 1983). The latter findings are corroborated bythe demonstration that dorsal root ganglion cells do notsynthesize vasopressin (Hallbeck et al., 1996c) and thatthe PVH seems to be the only cell group with significantspinal projection that express VP mRNA (Hallbeck et al.,1999), the transcript encoding for the precursor moleculeof vasopressin.

Spinal cord-projecting neurons in PVH were labeled byretrograde transport of cholera toxin subunit b (CTb;Ericson and Blomqvist, 1988), which was visualized byimmunohistochemistry. Vasopressin-expressing neuronswere detected by using in situ hybridization with longcRNA probes against VP mRNA. Spinal cord-projectingneurons that expressed VP mRNA were visualized by

using a double-labeling protocol that combines retrogradetracing with CTb and in situ hybridization with radiola-beled riboprobes (Hermanson et al., 1994). The results havebeen reported in abstract form (Hallbeck et al., 1996b).

MATERIALS AND METHODS

The present study was performed on adult, male Spra-gue-Dawley rats, 300–600 g (B&K Universal, Sollentuna,Sweden). All animals were maintained on a 12:12 hourslight:dark cycle and were given food and water ad libitum.All experiments were approved by the Animal Care andUse Committee at the University of Linkoping. All chemi-cals used were from Merck (Darmstadt, Germany) if nototherwise stated.

Tissue preparation

A total of six rats were anesthetized with 6% chloralhydrate (0.5 ml/100 g). Pressure injections of 0.2–0.4 µl of1% CTb (List, Campbell, CA) were made into the cervicalspinal cord at the C2 level using a glass micropipette (o.d.40µm). The micropipette was lowered into the cord at thedorsal root entry zone and its tip placed in the dorsolateralfuniculus, where the injections were made. After 5–18days, the animals were reanesthetized with sodium pento-barbital (100 mg/kg; Apoteksbolaget, Umea, Sweden) andperfused transcardially with 100 ml 0.9% NaCl at roomtemperature, followed by 500 ml 4% paraformaldehyde(PFA) in 0.1 M phosphate-buffered saline (PBS; pH 7.4) at4°C. The brain and upper cervical spinal cord were re-moved and placed in 20% sucrose and 4% PFA in 0.1 MPBS overnight at 4°C, and then cut at 20 µm on a freezingmicrotome. The brain sections were collected in fouradjacent one-in-four series. One series of sections was usedfor double labeling, and two adjacent series were used forsingle labeling with immunohistochemistry or in situhybridization, respectively. The last series was stainedwith thionin and used for anatomical demarcation ofnuclear groups. From the cervical spinal cord, every eighthsection was selected for immunohistochemistry.

Immunohistochemical processing

The double-labeling protocol used here follows Herman-son et al. (1994). The sections were first incubated inanti-CTb antiserum (1:9,000; List) for 72 hours at 4°C, andthen in secondary antibody (1:100, rabbit anti-goat; Da-kopatts, Copenhagen, Denmark) and peroxidase-anti-peroxidase complex (1:150, PAP-goat; Dakopatts) for 1hour each at room temperature; all antibodies were di-luted in sterile PBS containing bovine serum albumin(BSA, 1%, Cohn fraction V; Sigma, St. Louis, MO) andheparin (500 IU/ml; Kabi Pharmacia, Uppsala, Sweden).For double labeling, RNAsin (20 U/ml; Promega, Madison,WI) was added to the primary antibody. Sections intendedfor double labeling with situ hybridization (see below)were presoaked with 1% cobalt acetate in 0.1 M sodiumacetate buffer (NaAc; pH 6.0), processed with 3,3’-diaminobenzidine tetrahydrochloride (DAB; 0.035%;Sigma), ammonium chloride (0.04%), glucose (0.2%), andglucose oxidase (0.07 U/ml; Sigma) in 0.1 M NaAc over-night at 4°C, and mounted on poly-L-lysin-coated slides insterile PBS. The slides were stored at -70°C until furtheruse. For single labeling, sections were processed in 0.035%DAB, 2.5% ammonium nickel sulfate, and 0.01% H2O2 for6–8 minutes before being mounted and coverslipped.

Abbreviations

am anterior magnocellular subdivisionap anterior parvocellular subdivisionCTb cholera toxin subunit bdp dorsal parvocellular subdivisionf fornical subdivisionlp lateral parvocellular subdivisionlpl lateral lpmm medial magnocellular subdivisionmp medial parvocellular subdivisionmpdd dorsal part of the dorsalmpdv ventral part of the dorsal mpmpv ventral mppm posterior magnocellular subdivisionpv periventricular regionPVH paraventricular hypothalamic nucleusVP mRNA (prepro)vasopressin mRNA3v third ventricle

202 M. HALLBECK AND A. BLOMQVIST

In situ hybridization

The slides were treated with 4% PFA in 0.1 M phosphatebuffer for 30 minutes, prehybridized with 0.001% protein-ase K (Boehringer Mannheim, Mannheim, Germany) in asolution of 0.1 M Tris buffer (pH 8.0) and 0.05 M ethylene-diaminetetraacetic acid (EDTA; Sigma) for 30 minutes at37°C, dehydrated with graded ethanol, and vacuum dried.The probe was prepared from a Bluescript KS1 plasmid,containing a 0.6 kb insert complementary to VP mRNA(Rehbein et al., 1986), that was linearized with BamHI(Promega, Madison, WI). Anti-sense probe synthesis andlabeling were performed utilizing T3 polymerase (Pro-mega) and [35S]-labeled UTP (Amersham, Amersham,UK). The probe was placed in hybridization buffer (50%formamide, 10% dextran sulfate, 13 Denhardt’s solution,0.3 M NaCl, 10 mM Tris, and 1 mM EDTA) containingtRNA (0.5 mg/ml, Boehringer Mannheim) and dithiothrei-tol (0.1 M, Sigma), and the specific activity was adjusted to5 3 106 cpm ml-1. The hybridization was performed at 58 61°C for 18 hours (single labeling) or 48 hours (doublelabeling). After the hybridization, the slides were rinsed in43 standard saline citrate (SSC), before digestion with0.002% RNase A (Boehringer Mannheim) in a 0.5 M NaCl,10 mM Tris, and 1 mM EDTA solution (30 minutes at37°C). The slides were then rinsed in falling grades of SSCbuffers before being heated to 75°C in 0.13 SSC for 30minutes. Dithiothreitol (1 mM) was added to all buffers.After dehydration and delipidization the slides were dippedin photographic emulsion (Kodak NTB2 [Kodak, Roches-ter, NY], diluted 1:1 in distilled water), stored at -20°C for4–7 days, developed in Kodak D-19 (Kodak, Paris, France),fixed, and coverslipped.

Controls

Controls included hybridization with probes complemen-tary to other mRNAs (preproenkephalin and preprooxyto-cin) and with sense strand probes, and hybridization oftissue that had been pretreated with RNase (0.002%RNase A, 37C, 60 minutes) prior to the PFA and proteinaseK steps. Cross-hybridization with preprooxytocin mRNAwas also examined by using dot blot hybridization. Thesecontrols, which are described in detail in the accompany-ing paper (Hallbeck et al., 1999), showed no significantcross hybridization between the VP probe and oxytocinmRNA.

Analysis

Sections were examined under brightfield and darkfieldillumination. Cell counts were performed under brightfieldillumination at a final magnification of 3300. Neuronsdisplaying a brown, granular reaction product in thecytoplasm were identified as retrogradely labeled. Theidentification of VP mRNA-expressing neurons was basedon the presence of silver grains in the overlaying emulsionlayer, and was related both to their density in relation tobackground labeling and their distribution. Backgroundmeasured over the thalamus, which does not contain VPmRNA, was on average 0.6 grains/100µm2, and randomclustering of grains within an area corresponding to theaverage size of spinally projecting PVH neurons did notexceed 4 grains. However, background labeling in thehypothalamus, measured outside the VP mRNA-express-ing cell groups, was 1.3 grains/100µm2. Since no differencebetween the thalamus and hypothalamus was seen follow-ing hybridization with sense probe, we ascribe the higher

‘‘background’’ in the hypothalamus to the presence of VPmRNA in the neuronal processes in this brain region.Because of this, a detection level corresponding to .20times the true background, equaling .10 times the ‘‘back-ground’’ in the hypothalamus, within an area between40µm2 and 250µm2 was chosen, in order to exclude overes-timation of the number of VP mRNA-expressing cellbodies.

The distribution of labeled neurons was plotted with acamera lucida equipment. Nuclear boundaries were tracedin adjacent Nissl-stained sections and were superimposedon the plots by using transversely cut capillaries aslandmarks. Subdivisions of the PVH were delineated andnamed according to Swanson and Sawchenko (1983). Cellcounting was performed on the plots. Errors due to doublecounting of profiles were corrected for (Abercombie, 1946),by using different mean cell sizes for each subdivision ofthe PVH (Kiss et al., 1991).

RESULTS

Retrograde labeling

In all except one case (see below), the area covered by theinjected tracer substance was localized to one side of thespinal cord (Fig. 1A). The injected area encompassed bothgray matter and white matter; it always involved thedorsal horn and the dorsolateral funiculus, where thefibers descending from the PVH are primarily located(Swanson and Sawchenko, 1983; Liposits, 1993). In fourcases, the injected area also involved the intermediategray matter and the ventrolateral funiculus. In one case itinvolved the dorsal funiculus and encroached slightly onthe contralateral dorsal funiculus.

All injections resulted in retrogradely labeled neurons inthe ipsilateral PVH. They were identified by the presenceof dense granular labeling in the cell body and proximaldendrites (Fig. 1). The identification of retrogradely la-beled neurons was generally unequivocal, since back-ground staining was absent, as in the single-labeledsections (Fig. 1), or only faint, as in the double-labeledsections (Figs. 2, 3). The contralateral PVH containedscattered, but distinctly stained, CTb-immunoreactive neu-rons, showing the presence of a small contralateral spinalcord-projecting population. The large difference in numberof labeled neurons between the PVH on the two sides,taken together with a restricted retrograde labeling inother parts of the hypothalamus (primarily CTb-positiveneurons in the lateral hypothalamic area on the ipsilateralside), provided a control for the specificity of the CTbimmunohistochemistry.

The retrogradely labeled neurons were not uniformlydistributed throughout the PVH, but preferentially lo-cated to some of its subdivisions (Fig. 1) in a patternconsistent across the investigated animals. In sectionsthrough the caudal part of the PVH, the lateral parvocellu-lar subdivision (lp) contained many retrogradely labeledneurons (Fig. 1B); however, its lateral portion (lpl) and thefornical area (f) were only sparsely labeled. Further ros-trally, the retrogradely labeled neurons were split into twodensely labeled populations (Fig. 1C), corresponding to thedorsal parvocellular subdivision (dp) and the ventral partof the medial parvocellular subdivision (mpv). Some la-beled neurons were also present in the dorsal component ofthe dorsal mp (mpdd), whereas the ventral component of

SPINALLY PROJECTING VP NEURONS IN RAT PVH 203

the dorsal mp (mpdv) and the posterior magnocellularsubdivision (pm) were only sparsely labeled. Still furtherrostrally, the two labeled groups disappeared and onlyscattered labeled neurons were seen. At this level, theanterior parvocellular subdivision (ap) contained a smallnumber of labeled neurons (Fig. 1D), whereas the medialmagnocellular (mm) and the anterior magnocellular (am)subdivisions were virtually unlabeled.

To obtain an estimate of the total number of neuronsprojecting to the spinal cord, all labeled profiles werecounted in the ipsilateral PVH. In one-in-four series fromthree rats, an average of 499 (SEM 641) retrogradelylabeled neurons was found. Correction for double counting(Abercombie, 1946) yielded a total of about 1,300 neuronsprojecting to the spinal cord (Table 1).

Vasopressin mRNA labeling

Neurons expressing VP mRNA were identified by thepresence of an accumulation of silver grains in the emul-sion layer over the cell body (Fig. 2). Neurons sectionedthrough the nucleus showed a nuclear clearing. The distri-bution of labeled neurons corresponded to that describedin the accompanying paper (Hallbeck et al., 1999). Thus,areas containing dense labeling involved the lp, f, mpdd,mpv, and pm. The dp, mpdv, and ap only contained a few VPmRNA-expressing neurons.

Double labeling

Double-labeled neurons were characterized by a brown-ish, granular immunohistochemical staining of the cellbody and an accumulation of silver grains in the overlay-ing emulsion layer. The criteria for the identification ofdouble-labeled neurons are illustrated by the high-powermicrographs in Figure 2. Because the plane of focus was onthe emulsion layer, the CTb labeling may appear some-what blurred, but, as demonstrated by Figs. 2 A,A8, distinctCTb-labeled profiles were visualized by changing the focusto the tissue layer.

As shown in Figure 2, the classification of neurons assingle- or double-labeled was generally unequivocal. Thus,most double-labeled neurons displayed dense accumula-tions of silver grains over a CTb-stained cell body, and,when the neurons were cut equatorially, the distribution ofthe silver grains followed the extent of the cytoplasm,leaving a nuclear clearing (e.g., Fig. 2C, G). Obviously,identification of VP mRNA-positive neurons was easiest inareas with scattered neurons (e.g., Fig. 2E,F,H), wherelittle VP mRNA-expression was present in the neuropil,but generally it was also unambiguous in densely packedregions (e.g., Fig. 2B). However, to avoid false-positiveidentifications, the criteria used for considering a profile asdouble-labeled were conservative, requiring a grain den-

Fig. 1. Tracing of projections from the paraventricular hypothalamicnucleus to the spinal cord using cholera toxin subunit b (CTb).A: Extent of the CTb injection into the C2 segment. B–D: Retrogradelylabeled neurons in the ipsilateral paraventricular nucleus. B is caudal

and D is rostral. The sections are separated by 240µm. Midline is tothe left, and dorsal is upwards. Annotations indicate subdivisions ofthe paraventricular hypothalamic nucleus. Scale bar (shown in D) 5500 µm in A; 120 µm in B–D.


sity .20 times background (see Materials and Methods),in addition to a grain distribution matching that of theunderlying profile. When the distribution of silver grains

did not match that of the underlying CTb-labeled neurons,that neuron was considered unlabeled. For example, theCTb-labeled neuron to the right in Figure 2D displays

Fig. 2. High-power microphotographs from the paraventricular hypo-thalamic nucleus (PVH), showing examples of single- and double-labeled neurons following immunohistochemical detection of retro-gradely transported cholera toxin subunit b (CTb) and in situhybridization for preprovasopressin mRNA (VP mRNA). Retrogradelylabeled neurons contain a granular brownish reaction product in thecytoplasm, and VP mRNA-expressing cells display an accumulation ofsilver grains in the overlaying emulsion layer. A and A’ are from thesame section with focus on the emulsion layer and the tissue plane,

respectively. In B–H, focus is on the emulsion layer. Arrows point atdouble-labeled neurons, arrowheads show single-labeled CTb-stainedneurons, and open arrows show single-labeled VP mRNA-expressingneurons. A: Dorsal parvocellular subdivision (dp) of the PVH.B–D: Lateral parvocellular subdivision (lp). E: Ventral part of medialparvocellular subdivision (mpv). F: Posterior portion of lp. G: Ventralpart of the dorsal medial parvocellular subdivision (mpdv). H: Posteriorportion of lp. Scale bar (shown in H) 5 12 µm in A and D; 9 µm in B andF; 9.5 µm in C; 11 µm in E and G; and 10 µm in H.


silver grains over the lower part of the cell body, but theirdistribution points to an origin from the adjacent VPmRNA-positive profile. The differences in cell shape alsohelped in distinguishing overlapping profiles lying atdifferent planes of the sections, as shown by unambiguousidentification of the single- and double-labeled neurons at

the top of Fig. 2B, and of the two single-labeled neuron atthe top of Figure 2D.

To check that the double-labeling procedure did notresult in an underestimation of the number of labeledneurons due to attenuation of the immunohistochemical orautoradiographic signals, the labeling in the double-

Fig. 3. Photomicrographs of sections through the paraventricularhypothalamic nucleus (PVH) processed for cholera toxin subunit b(CTb) immunohistochemistry and in situ hybridization for preprovaso-pressin mRNA (VP mRNA). The peroxidase reaction product is seen asa dark brown staining of the cytoplasm, which is slightly blurred sincethe focus is on the overlaying emulsion layer. Silver grains representVP mRNA. A: Overview corresponding approximately to the levelshown in Figure 4E. B–F: Examples of single and double-labeledneurons in different portions of the PVH. B is from the posterior

portion of lateral parvocellular subdivision (lp); C and F show themiddle region of lp, and at upper left, part of dorsal parvocellularsubdivision (dp); D is from the ventral part of the dorsal medialparvocellular subdivision (mpdv); and E is from the rostral portion oflp. Arrows indicate double-labeled neurons, arrowheads show singlelabeled CTb-stained neurons, and open arrows show single-labeled VPmRNA-expressing neurons. Scale bar (shown in F) 5 90 µm in A; 40µm in B–F.


labeled sections was compared with the labeling present inadjacent single-labeled sections. Such a comparison didnot show any significant differences between the sections.Furthermore, comparison of VP mRNA expression be-tween the ipsilateral and the contralateral PVH demon-strated a similar distribution and labeling density, irrespec-tive of the large differences in the number of retrogradelylabeled neurons.

Cell counts showed that on average 42% of the retro-gradely labeled neurons in the PVH also expressed VPmRNA (Table 1). However, the distribution of spinallyprojecting vasopressinergic neurons varied between thesubdivision of the PVH (Figs. 3, 4). The densest accumula-tion of double-labeled neurons was seen in lp proper and inmpv, where 59% and 49%, respectively, of the spinallyprojecting neurons expressed VP mRNA (Fig. 4 and Table1). The mpdd also contained a significant population ofdouble-labeled neurons, but this was small in comparisonwith either of the single-labeled populations (Table 1). Thedp contained many retrogradely labeled neurons but few ofthose (less than 10%) were double-labeled. Some areas,such as pm, f, and lpl, contained very few retrogradelylabeled neurons; however, these neurons were generallydouble-labeled (Table 1).

Although the total number of labeled neurons differedbetween animals, the differences were generally small.Across animals, the labeling pattern was very similar.

Other nuclei expressing VP mRNA were also analyzedfor double labeling. These included the bed nucleus of striaterminalis, the medial amygdaloid nucleus, the suprachi-asmatic nucleus, and the supraoptic nucleus including itsretrochiasmatic part. None of these nuclei contained anyretrogradely labeled neurons (data not shown).

DISCUSSION

The present study shows that over 40% of the spinallyprojecting neurons in the PVH express VP mRNA anddemonstrates that these neurons are localized to specificparts of the PVH. Thus, a large proportion of the spinallyprojecting neurons in the lp proper and mpv were vaso-pressinergic. In contrast, very few of the large number ofthe spinally projecting neurons in dp expressed VP mRNA.The present study also confirms earlier observations point-ing out the PVH as the most important source of VP in thespinal cord (Sawchenko and Sanson, 1982; De Vries andBuijs, 1983; Lang et al., 1983), since no spinally projectingneurons could be found in other vasopressinergic nucleiexamined.

The present findings extend previous observations onspinally projecting vasopressinergic neurons in the PVH(Sawchenko and Swanson, 1982; Sawchenko, 1987; Ce-chetto and Saper, 1988). Thus, such neurons were demon-strated in colchicine-treated animals using double-label-ing immunohistochemistry, but the proportions ofvasopressinergic neurons within the spinal cord-projectingPVH population was found to be much smaller thansuggested by the present study. Several explanations forthis difference may be considered, as discussed below.

The number of retrogradely labeled neurons could havebeen underestimated in the present study, resulting in ahigh proportion of spinally projecting vasopressinergicneurons. However, this is not the case, since the estimatednumber of spinally projecting neurons in the present study(1,323) was larger than the number obtained in previouswork (955; Swanson and Sawchenko, 1980).

Alternatively, the injection site chosen in the presentstudy (the C2 segment) could have labeled a larger propor-tion of vasopressinergic neurons than the injection site(thoracic spinal cord) chosen in the study by Swanson andSawchenko (1980). However, this is also unlikely, becauseCTb is taken up both by axon terminals and by fibers ofpassage (Ericson and Blomqvist, 1988). Hence, the injec-tions in the present study, which in all cases encompassthe dorsal horn and the dorsolateral funiculus, will labeldescending fibers from the PVH both to and past the C2segment including the fibers to the thoracic spinal cordwhere most of the vasopressinergic fibers are known toterminate (Buijs, 1978; Nilaver et al., 1980).

Because different tracers were used in the differentstudies (Fast blue: Sawchenko and Swanson, 1982; CTb,present study), uptake and transport by various types offibers could have differed, resulting in the labeling ofdifferent populations of cell. Although this cannot beexcluded, there is no evidence for such selectivity inuptake and retrograde transport for either Fast blue orCTb, when utilized for tracing in the central nervoussystem. Both tracers have been used extensively, and theyseem to be sensitive tools for tract tracing in all systemsstudied (e.g., Blomqvist and Ericson, 1988; Skirboll et al.,1989; Luppi et al., 1990).

Accordingly, the methods used to identify the vaso-pressinergic neurons most likely account for the differencebetween this and previous work regarding the size of thespinal cord-projecting vasopressinergic population in thePVH. Thus, it is now well established that in situ hybrid-ization, particularly the employment of long cRNA probes,is a more sensitive technique than immunohistochemistryfor the detection of peptidergic cell bodies (see, e.g.,Simmons et al., 1989; Hermanson et al., 1995). In addition,because the in situ hybridization technique, in contrast toimmunohistochemistry, does not require the use of colchi-cine for the visualization of cell bodies, possible neurotoxin-induced changes in gene transcription (e.g., Ceccatelli etal., 1991; Rethelyi et al., 1991) are avoided.

Although the present method for combined immunohis-tochemical and hybridization histochemical localizationhas been reported and evaluated in several previousreports (e.g., Hermanson et al., 1994, 1998), a few techni-cal issues also deserve comment here. These include theconsideration that hybridization signals attributable to VPmRNA-containing elements lying at different planes ofsection than the retrogradely labeled cells may havecontributed to false-positive identification. However, this

TABLE 1. Mean Number (6SEM) of Neurons in Subdivisions of theParaventricular Nucleus (n 5 3)1

Subdivision VP mRNA CTb DBL % DBL

lp 599 6 37 505 6 66 298 6 33 59lp1 60 6 8 28 6 4 15 6 3 54f 42 6 8 17 6 12 11 6 6 65dp 49 6 12 283 6 17 23 6 6 8mpdd 376 6 10 235 6 9 81 6 10 34mpdv 111 6 23 44 6 15 15 6 10 34mpv 197 6 42 180 6 18 88 6 14 49pm 535 6 40 31 6 8 24 6 6 77Total 1,970 6 119 1,323 6 107 556 6 54 42

1VP mRNA, all neurons containing preprovasopressin mRNA; CTb, all neurons retro-gradely labeled with CTb; DBL, neurons double-labeled with VP mRNA and CTb; %DBL, the percentage of the neurons projecting to the spinal cord that expressed VPmRNA. See Materials and Methods for details on the method of counting. For otherabbreviations, see list.


Fig. 4. A–J: Drawings of sections through the paraventricular hypotha-lamic nucleus showing the distribution of vasopressinergic (crosses),spinally projecting (open circles), and double-labeled (filled circles)neurons. Each symbol represents one labeled profile. A is caudal and J

is rostral. The sections are separated by 80 µm. Note the accumulationof double-labeled neurons in the lp and caudal mpv. Note also that thedp contains many retrogradely labeled, but preprovasopressin mRNA-negative neurons. Scale bar 5 250 µm.

was not a significant problem in the present study, sincethe identification of double-labeled neurons was assistedby a distribution of silver grains corresponding to theshape of the underlying profile, as visualized by thecytoplasmic CTb staining (Figs. 2, 3). If anything, becauseof the energy profile of the b-emission from the 35S-isotopes(Brady and Finlan, 1990), it is more likely that the numberof double-labeled profiles was underestimated, since radio-labeled profiles in the lower part of the section may havegiven rise only to a weak autoradiographic staining in theoverlying emulsion layer. Because the background signalin the hypothalamus was somewhat elevated, the criterionfor identification of vasopressinergic neurons was conser-vative (labeling density .20 times the general back-ground), implying that some neurons with a low expres-sion of VP mRNA may have been excluded from the cellcount.

When assessing the present findings, it is also importantto recall that the present experiments were carried out onnaive animals. It is possible that neurons that are unla-beled in the naive animal will express VP mRNA underother physiological or pathological conditions, which couldresult in other estimates of the proportion of vasopressin-ergic neurons. Thus, increased cell counts have beenshown for VP- and other peptide-expressing hypothalamicneurons, for example, after salt loading and adrenalec-tomy (Sawchenko et al., 1984; Swanson and Simmons,1989; Watts, 1992; Watts and Sanchez-Watts, 1995).

The present study extends previous observation not onlywith regard to the number and proportion of spinallyprojecting vasopressinergic neurons, but also with respectto their distribution. Previous studies reported that thespinally projecting vasopressinergic neurons were locatedin the lp and mp subdivisions of the PVH (Sawchenko andSwanson, 1982; Cechetto and Saper, 1988). However,using cytoarchitectonic criteria, these subdivisions of thePVH have been further subdivided into several smallerparts (Swanson and Sawchenko, 1983), which have beenshown to differ in projections (Swanson and Sawchenko,1980), intracellular messengers (Senba et al., 1993; Smithand Day, 1994; Hallbeck et al., 1996a), and response todifferent stimuli (Watts, 1992; Watts and Sanchez-Watts,1995). The present findings, which show that the spinallyprojecting neurons were localized to lp proper and mpv,provide further support for this extended parcellation ofthe PVH (see also Sawchenko, 1987).

This anatomical parcellation is likely to be the substratefor the selective and specific physiological responses of thePVH to various stimuli that influence the homeostasis ofthe organism. For example, different types of osmoticstimuli have been shown to elicit distinct response pat-terns that involve the different subdivisions in a selectiveway (Watts, 1992; Watts and Sanchez-Watts, 1995). Thus,the effect of spinally released vasopressin on differentautonomic functions could be exerted by different subdivi-sions of the PVH to enable a physiological adaptiveresponse. This could include such functions as cardiovascu-lar regulation (Porter and Brody, 1986) and the modula-tion of renal sympathetic nerve activity (Malpas andCoote, 1994), both of which have been shown to be influ-enced by vasopressin. Furthermore, tissue damage willalso disturb homeostasis, and tissue damaging (i.e., noci-ceptive) stimuli have been shown to activate the PVH(Anderson et al., 1989; Day and Sibbald, 1990). It isconceivable that such activation involves selective, func-

tionally different cell groups in the PVH, such as thespinally projecting vasopressinergic group, to produce aresponse adaptive to the behavioral situation that mayinclude the concerted modulation of the autonomic outflowand pain processing (cf. Craig, 1993). Vasopressin has beenshown to be involved in analgesia (Berntson and Berson,1980; Berkowitz and Sherman, 1982; Berson et al., 1983;Kordower and Bodnar, 1984; Watkins et al., 1986; Thurs-ton et al., 1988, 1992), an effect believed to be exerted atthe spinal level (Watkins et al., 1986; Thurston et al., 1988,1992), and the vasopressin-mediated analgesia seems tobe mediated by pathways separate from the endogenousopioid analgesia system (Berntson and Berson, 1980;Berkowitz and Sherman, 1982; Berson et al., 1983; Kor-dower and Bodnar, 1984; Thurston et al., 1992). The PVHis known to be one of the main sources of descending fibersto the superficial laminae of the spinal dorsal horn (Fieldsand Basbaum, 1994), where nociceptive signals from theperiphery are connected to centrally projecting neurons.Thus, the present anatomical findings, taken togetherwith previous functional observations, suggest a role forspecific subdivisions of the PVH in the control of spinallyelicited vasopressin analgesia.

In addition to vasopressin, several other peptides havebeen described in the parvocellular PVH, such as leucine-and methionine-enkephalin, dynorphin, oxytocin, CRF,somatostatin and vasoactive intestinal peptide (for review,see Swanson and Sawchenko, 1983). Of these peptides,oxytocin and the opioids have been shown to be present inspinally projecting neurons (Sawchenko and Swanson,1982; Code and Fallon , 1986; Cechetto and Saper, 1988).In the present study a large number of retrogradelylabeled neurons that did not express VP mRNA werefound. Hence, it is likely that at least some of theseneurons express oxytocin and/or opiates, especially sincemany of the VP mRNA-negative spinally projecting neu-rons were located in the dp and mpv, areas that previouslyhave been shown to contain oxytocin (Rhodes et al., 1981;Sawchenko and Swanson, 1982; Cechetto and Saper, 1988)and enkephalins (Cechetto and Saper, 1988), respectively.Studies elucidating the number and distribution of spinalcord-projecting PVH neurons that express opiates or oxyto-cin are under way in this laboratory (Hallbeck et al., 1997).

ACKNOWLEDGMENTS

We thank Dr. O. Hermanson for valuable discussions ontechnical aspects of the work, Dr. P.P. Sanna for thegenerous gift of the preprovasopressin cDNA, and Dr. D.Larhammar for preparation of the plasmid.

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Spinal cord-projecting vasopressinergic neurons in the rat paraventricular hypothalamus

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