quantitative and neurogenic analysis of the total population and subpopulations of neurons defined...

Quantitative and Neurogenic Analysis ofthe Total Population and Subpopulationsof Neurons Defined by Axon Projection in

the Superficial Dorsal Horn of the RatLumbar Spinal Cord

TULSI N. BICE AND JOHN A. BEAL*Department of Cellular Biology and Anatomy, Louisiana State University Medical Center

in Shreveport, Shreveport, Louisiana 71130

ABSTRACTThe total neuron population of the superficial dorsal horn (SDH), i.e., laminae I and II,

was quantitated in Nissl preparations of spinal segment L1 in the rat. Subpopulations of theSDH, defined by axon projection, were quantitated following strategic intraspinal injections ofdual retrograde tracers (Fluoro-Gold and true blue). These methods were used in conjunctionwith [3H]thymidine (delivered in utero) autoradiography for neurogenic pattern analysis.Following stereological correction, each dorsal horn in spinal segment L1 contained 11neurons in lamina I and 42.6 neurons in lamina II per 10-µm transverse section. Neurons withlong projections, i.e., neurons with projections rostral to spinal segment T5, were only slightlymore numerous in lamina I than in lamina II. These neurons made up 34% of the total neuronpopulation in lamina I and 7.0% in lamina II. Most of these neurons did not demonstratedescending connections, and many (presumed supraspinal projection neurons) did notdemonstrate short, ascending, intersegmental connections. Neurons with short propriospinalprojections, i.e., neurons with connections caudal to spinal segment T5, made up approxi-mately half of the total neuron population in both lamina I and lamina II: 55% and 52%,respectively. Of these, 79% had both short ascending and descending projections; theremaining 21% had only descending projections. Neurons that were not labeled withretrograde tracers (presumed local circuit cells) represented 11% of the neurons in lamina Iand 41% in lamina II. Neurogenesis in the SDH proceeded along an axon-length gradient,whereby neurons with the longest axons completed neurogenesis first, and those with theshortest completed neurogenesis last. The generation of both propriospinal and supraspinalprojection neurons began on embryonic day 13 (E13). Nearly equal numbers of neurons in thisgroup were generated in laminae I and II through E14. On E15, neuron production slowed inlamina I and accelerated in lamina II as local circuit neurons and the remaining propriospinalneurons were generated. Neuron production ceased simultaneously in both lamina I andlamina II on E16. J. Comp. Neurol. 388:550–564, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: sensory systems; propriospinal; pain; autoradiography; retrograde transport

Lamina I (marginal layer) and lamina II (substantiagelatinosa) of Rexed (1952) make up the superficial dorsalhorn (SDH). The SDH contains primarily wide dynamicrange, nociceptive-specific, and thermoreceptive neurons(Christenson and Perl, 1970; Willis et al., 1974; Giesler etal., 1976; Kumazawa and Perl, 1978; Price et al., 1978,1979; Cervero et al., 1979a; Bennett et al., 1980; Fox et al.,1980; Menetrey et al., 1980, 1984; Woolf and Fitzgerald,1983; Craig and Kniffki, 1985; Ferrington et al., 1987;Yezierski et al., 1987; Villanueva et al., 1988; Light et al.,

1993). The SDH receives nociceptive input, modulates itvia a system of local circuit and propriospinal neurons, andultimately transmits it to higher brain centers via supra-

Grant sponsor: National Science Foundation; Grant number: BNS-8908601.

*Correspondence to: John A. Beal, PhD, Department of Cellular Biologyand Anatomy, Louisiana State University Medical Center, 1501 KingsHighway, Shreveport, LA 71130. E-mail: [email protected]

Received 15 December 1995; Revised 28 May 1997; Accepted 5 June 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 388:550–564 (1997)

r 1997 WILEY-LISS, INC.

spinal projection neurons. SDH neurons with supraspinalprojections in the rat, including spinothalamic neurons(Granum, 1986; Kemplay and Webster, 1986; Lima andCoimbra, 1988; Hylden et al., 1989; Burstein et al., 1990;Beal and Bice, 1994), spinomesencephalic neurons (Swettet al., 1985; Hylden et al., 1989; Lima and Coimbra, 1989),and spinomedullary neurons (Lima and Coimbra, 1991a,b),have been quantitatively assessed. No precise quantita-tion of the entire neuron population of the SDH based onprojection, however, has been undertaken.

The aim of the present study is to quantitate differenttypes of SDH neurons in spinal segment L1 classifiedaccording to projection. Determination of the number,proportion, and distribution of the various cell types thatmediate pain transmission in the SDH should provide abetter understanding of the circuitry involved. Also, acomparison of SDH cell ratios between lumbar and otherlevels of the cord should provide insight into functionalvariations between spinal segments.

In the present study, the total number of neurons in theSDH was determined in Nissl preparations. Subpopula-tions of SDH neurons that were labeled strategically withfluorescent retrograde tracers were quantitated accordingto projection site in two separate analyses. In subpopula-tion analysis I, neurons with both ascending and descend-ing axons, as well as neurons with only ascending axons,and neurons with only descending axons were quantitated.In subpopulation analysis II, neurons with long ascendingprojections (supraspinal and long propriospinal neurons),neurons with short ascending projections (short propriospi-nal neurons), and neurons with both long and shortascending projections were quantitated. An estimation ofthe number of local circuit neurons was made by subtract-ing the number of neurons with ascending projections plusthe number of neurons with only descending projectionsfrom the total number of SDH neurons.

A second aim of the present study was to determine theneurogenic pattern in both the total population and sub-populations of SDH neurons in order to understand thekinetics and mechanisms leading to the quantitative andspatial distribution of neurons in the adult. The temporalneurogenic pattern was assessed by using [3H]thymidineautoradiography in conjunction with Nissl and retrogradelabeling procedures.

Previous studies in this laboratory, which employed asingle retrograde tracer in combination with [3H]thymi-dine autoradiography, showed that, within each spinallamina, neurons with supraspinal projections were gener-ated prior to those with propriospinal projections (Nandi etal., 1990, 1993). These results suggested that the durationof the neurogenic period within each spinal lamina isrelated inversely to the distance between the nerve cellbody and its projection site. However, because only a singletracer was used in the latter studies, it was not possible toseparate neurons with only propriospinal projections fromsupraspinal projection neurons with propriospinal connec-tions. The present study used two retrograde tracers incombination with [3H]thymidine autoradiography to distin-guish the neurogenic patterns of neurons with only longprojections from those with long projections plus shortpropriospinal connections and those with only short pro-priospinal connections. Spinal segment L1 was selected forexamination in order to correlate the results with previousneurogenic and projection studies from this laboratory

performed on this spinal segment (Nandi et al., 1990,1991, 1993; Beal and Bice, 1994).

MATERIALS AND METHODS

All animal protocols described in this paper, includinginjections, anesthesia, surgical procedures, postoperativecare, and killing, were approved by the Animal CareCommittee at the Louisiana State University MedicalCenter.

Laminar boundaries

Tissue used in this study was taken from the middleone-third of spinal segment L1 in 80- to 100-day-old maleand female albino rats of the Wistar strain. The bound-aries and dimensions of laminae I and II were determinedfrom camera lucida drawings of tissue fixed in osmiumtetroxide, stained with toluidine blue, embedded in plastic,and sectioned at 3.0 µm in accordance with the cytoarchi-tectonic study of the rat spinal cord by Molander et al.(1984). The dimensions obtained from these sections wereused to determine laminar borders in the retrogradetracing studies.

Total population analysis

Tissue processing. Three animals were anesthetizedwith an injection of ketamine hydrochloride (120 mg/kg)and xylazine (10 mg/kg) and were intracardially perfusedthrough the left ventricle with 200 ml of warm phosphate-buffered saline, pH 7.2, containing heparin (200 units/ml)and 1.0% procaine hydrochloride, followed by 400 ml of4.0% paraformaldehyde in 0.1 M phosphate buffer. Spinalcords were removed and immersed in fixative for 1 week.Tissue was dehydrated, embedded in paraffin, seriallysectioned in the transverse plane at 10 µm, mounted onglass slides, deparaffinized, stained with cresyl violet, thencleared in xylene, and coverslipped with cytoseal (VWR,West Chester, PA).

Cell counts. Camera lucida drawings, rather thanphotomicrographs, were used for cell counts, so that theobserver could focus through each section and ensurepositive identification of cell types. Drawings of the SDHwere made from ten serial transverse sections per animalby using a 340 objective at a total magnification of 3630.Nuclei of SDH neurons were counted on both the right andthe left sides of the cord in each section. Neurons weredistinguished from glial and endothelial cells on the basisthat nerve cell nuclei in the spinal gray of the adult rat, asshown by Bjugn et al. (1993), are larger, contain distinctnucleoli, and have marked indentations in the nuclearmembrane. Because a single neuronal nucleus may appearon more than one section, stereological correction was usedto obtain an accurate count. Two stereological methodswere used: 1) the classic method of Abercrombie (1946) and2) the unbiased dissector method (Sterio, 1984). By usingthe method of Abercrombie, the true number of cells (P)was calculated according to the formula:

P 5 A(M/L 1 M),

where A 5 the number of nuclei counted, M 5 the tissuethickness, and L 5 the mean length of a nucleus at rightangles to the plane of section, i.e., in the sagittal plane. Toobtain L, the mean rostrocaudal diameter of 900 neuronalnuclei taken from cresyl violet preparations of sagittal

POPULATION ANALYSIS OF SUPERFICIAL DORSAL HORN 551

sections from the lateral, middle, and medial one-thirds ofthe SDH and drawn at 3630 was calculated. By using thedissector method on the same set of transverse sections toobtain the Abercrombie factor, the true or corrected num-ber of cells was calculated by counting neuronal nuclei(‘‘tops’’), which appeared in the section analyzed but not inthe next adjacent serial section (Sterio, 1984).

Neurogenesis. The neurogenic pattern of the totalpopulation of SDH neurons was determined by using[3H]thymidine autoradiography. Rats that weighed 250–300 g were mated overnight. On the following morning, anintravaginal rinse of phosphate-buffered saline was exam-ined for sperm. The presence of sperm marked the date ofconception, which was designated as embryonic day 1 (E1).Pregnant rats were anesthetized by using metofane inha-lation and were injected with [3H]thymidine on 1 daybetween E13 and E16, the period during which neurogen-esis occurs in the lumbar cord. To ensure that all proliferat-ing cells were labeled, each dam was given two intraperito-neal injections of [3H]thymidine of equal dosage (1.0 ml)6–8 hours apart. [3H]thymidine was purchased from ICNPharmaceuticals, Inc. (Costa Mesa, CA) and had a specificactivity of 6.7 Ci/mMol with 1.0 mCi dissolved in 1.0 mlsterile water. Total dosage was 5.0 µCi/g body weight(Nornes and Das, 1974). Animals were divided into fourgroups. The first group was administered [3H]thymidineon E13, the second on E14, the third on E15, and the fourthon E16. Pups of each dam were raised until they were80–100 days of age; then, five animals from each groupwere anesthetized and intracardially perfused with fixa-tive, as described above. The middle one-third of spinalsegment L1 was serially sectioned at 10 µm in the trans-verse plane, mounted on glass slides, and deparaffinized.Slides were coated with Ilford K.5 nuclear emulsion (Poly-science, Niles, IL), exposed in the dark for 50 days at 4°C,developed in Kodak D-19 for 4 minutes, fixed in aqueous24% sodium thiosulfate for 4 minutes, then stained withcresyl violet. Slides were cleared in xylene and cover-slipped with cytoseal (VWR).

The neurogenic pattern was assessed in each animal bycounting the number of neuronal nuclei labeled and thenumber not labeled with [3H]thymidine in ten serialsections. Nuclei were considered labeled if they containedthree or more silver grains above the background distribu-tion. Neurons that were not labeled were considered tohave been generated prior to [3H]thymidine administra-tion. The percentage of neurons labeled with [3H]thymi-dine was determined in each animal, and the mean wascalculated for each group.

Subpopulation analysis

Retrograde tracer selection. Because this study wasdesigned to identify SDH neurons with multiple projectionsites, it was necessary to use two visually distinguishabletracers. Other criteria were minimal diffusion from injec-tion sites, minimal loss of fluorescence following autoradio-graphic processing and paraffin embeddment, and consis-tent high-yield labeling. A variety of fluorescent retrogradetracers, including Carboxyl-modified latex (CML,) redfluorospheres (Molecular Probes, Eugene, OR), Rhoda-mine B dextran (Sigma, St. Louis, MO), fast blue (Sigma),Diamidino yellow dihydrochloride (Sigma), propidium io-dide (Sigma), true blue (TB; Sigma), and Fluoro-Gold (FG;Molecular Probes) in various concentrations were testedby using the protocol of the present study.

FG plus TB proved to be the most effective combinationin meeting the above criteria. However, pilot studies inthis laboratory showed that fewer cells were labeled whenTB was injected at a site rostral to the FG injection site.The mechanism involved in this phenomenon is unknown,but it has been observed previously when FG was usedwith other retrograde tracers (Akitunde and Buxton,1992). For optimal results, therefore, FG was injected intothe most rostral site in each experiment in the presentstudy.

Two subpopulations of SDH neurons were labeled withretrograde tracers and counted: 1) neurons with ascendingand/or descending projections and 2) neurons with longand/or short ascending projections. For each subpopula-tion, six offspring were taken from dams treated with[3H]thymidine on E14, and six were taken on E15, the twodays when the majority of SDH neurons are known to begenerated (Nandi et al., 1993). Animals were allowed tosurvive until 80–100 days of age, as in the total populationanalysis; then, prior to retrograde tracer administration,each received an intramuscular injection of ketaminehydrochloride (40–80 mg/kg body weight) and xylazine(5–10 mg/kg). Each animal was then placed on a heatingpad to maintain its body temperature throughout thesurgical procedure and was secured in a small-animalstereotaxic instrument (Kopf, Tujunga, CA). Retrogradetracer injections of 1.0–2.0 µl of 2.0% FG or 2.0% TB insterile water were made over a period of 5–10 minuteseach with a micropipette (30–40 µm tip diameter), whichwas connected to a Hamilton syringe (Reno, NV) and wasdriven with a Stoelting (Wood Dale, IL) motorized microsy-ringe pump. All surgery was performed under asepticconditions. Postsurgically, each animal was maintained onprocaine penicillin (40,000 units/kg body weight at 48-hour intervals) prior to killing.

Ascending and descending projections. For this sub-population analysis, laminectomies were performed overspinal segments T5, T12, and L3. To label neurons withlong ascending projections (supraspinal and long proprio-spinal), a hemisection was performed on the right side atspinal segment T5, and a gelfoam pledgett soaked with FGwas placed between the severed ends of the cord. To labelneurons with short ascending projections, FG was pres-sure injected into both the right and left dorsal horn atspinal segment T12. To label neurons with descendingprojections, TB was pressure injected into both the rightand left dorsal horn at spinal segment L3.

Long and short ascending projections. For this sub-population analysis, laminectomies were performed overspinal segments T5 and T12. To label neurons with longascending projections, a hemisection was performed on theright side at spinal segment T5, and a gelfoam pledgettsoaked in FG was placed between the severed ends of thecord. To label neurons with short ascending projections,TB was pressure injected into the head of the dorsal hornon the right and left sides at spinal segment T12.

Tissue processing. Animals were maintained postsur-gically for 7 days, then anesthetized, and intracardiallyperfused with fixative, as described for the total populationanalysis. Spinal cords were removed and immersed infixative for 1 week. The middle one-third of spinal segmentL1 as well as injection and hemisection sites were dehy-drated, embedded in paraffin, serially sectioned in thetransverse plane at 10 µm, then mounted on glass slides.The L1 sections were processed for autoradiography, and

552 T.N. BICE AND J.A. BEAL

all sections were treated and coverslipped as describedabove.

Cell counts. Cells labeled with FG, TB, and FG plusTB were counted under a fluorescence microscope (At-tofluor; Zeiss, Thornwood, NY) with an excitation filter BP365/12, beam splitter FT 395, and barrier filter LP 397.Cells were counted in 30 serial sections from each animal,and the mean was calculated for each group.

Neurogenesis. The number and percentage of neu-rons labeled with [3H]thymidine and the number of neu-rons not labeled with [3H]thymidine were determined ineach animal, and the mean was calculated for each group.

Statistical analysis

Nonparametric or distribution-free statistical analyses,where populations do not have to be distributed normally,were employed. The Wilcoxon signed-rank test was used totest the significance of two sets of observations on the samegroup of animals, e.g., cells labeled with FG vs. TB inE14-treated animals. To test for the significance of measure-ments in two different groups of animals, e.g., E14 vs. E15[3H]thymidine-treated animals, the Wilcoxon rank-sum(Mann-Whitney) test was used. Data were consideredstatistically significant at P , 0.05. Deviations from themean are expressed as standard errors throughout thetext.

RESULTS


Cell counts. By far, the majority of neuron cell bodiesin both lamina I and lamina II were relatively small andmeasured less than 14 µm in diameter (Figs. 1, 2). Viewedin osmium tetroxide-fixed, toluidine blue-stained plasticsections of spinal segment L1, lamina I is seen as a layer ofneurons that caps the head of the dorsal horn on its dorsaland lateral surface. Lamina I is further characterized bynumerous small, myelinated fibers that run parallel to therostrocaudal axis. The cells in lamina I are generallyslightly larger than those in lamina II. The medial one-third of lamina I is composed of a single layer of cells, andthe lateral two-thirds are composed of one to two layers. Afew cells are located interstitially. Lamina II, which islocated immediately ventral to lamina I, is composed offive to six irregularly arranged layers of neurons and can

be divided into outer and inner zones of nearly equalthickness. The outer zone is peppered with small, longitu-dinally arranged, myelinated fibers, whereas the innerzone is nearly devoid of myelin. The lamina II-III border isirregular but distinct, because lamina III contains numer-ous myelinated fibers. The approximate dorsal-to-ventralextents, or thicknesses, of laminae I and II in the medialhalf of spinal segment L1 were 15 µm and 80 µm,respectively, and, in the lateral half, they were 10 µm and60 µm, respectively (Figs. 1, 2).

By using Nissl preparations, it was determined that 43%of the neurons in lamina I were located in the lateralone-third, 36% were in the middle one-third, and 21% werein the medial one-third. In lamina II, neurons weredistributed more equally; 35% were in the lateral one-third, 36% were in the middle one-third, and 29% were inthe medial one-third. The number of neuronal nucleicounted per 10-µm section in each dorsal horn was 105.1 62.7: 21.6 6 1.0 in lamina I and 83.5 6 2.5 in lamina II.

The Abercrombie (1946) factor (M/L1M) was equal to10/9.7 1 10 or 0.51. The stereologically corrected SDH cellcount, then, was equal to (0.51 3 105.1) or 53.6: 11 cells inlamina I and 42.6 cells in lamina II (Table 1). The correctedcount using the dissector method (57.4 6 4.5 per 10-µmsection) was not significantly different from that obtainedby using the Abercrombie method.

Neurogenesis. On E13 and E14, nearly equal num-bers of neurons were generated in laminae I and II.However, the percentage of neurons generated by E14 wasgreater in lamina I (47%) than in lamina II (20%). On E15,there was a slight increase in the number of neuronsgenerated in lamina I and a fourfold increase in the num-ber generated in lamina II. The percentage of neurons gen-erated by E15, on the other hand, was nearly the same inboth laminae. By E16, 88% of the neurons in lamina I and98% in lamina II had been generated. The few remainingSDH neurons labeled with [3H]thymidine were present innearly equal numbers in laminae I and II (Figs. 3, 4).

Subpopulation analysis I: Ascendingand descending projections

Retrograde tracer injection sites. To determine thenumber of SDH neurons with only descending projections,

Fig. 1. Photomicrograph of the head of the dorsal horn in a 10-µmtransverse section used in total population analysis showing laminae Iand II in spinal segment L1. Arrows demarcate ventral border of thesuperficial dorsal horn (SDH). Nissl preparation. Scale bar 5 100 µm.

Fig. 2. Photomicrographs taken from the center of the head of thedorsal horn demonstrating laminae I, II, and III of spinal segment L1.Lamina II is divided into an outer zone (oz) and an inner zone (iz).Osmium-fixed, 3-µm plastic section (A) has few myelinated fibers(small black profiles) in the iz. Nissl-stained, 10-µm section (B) showsexamples of neuronal nuclei (arrowheads) and glial and endothelialnuclei (arrows). Scale bar 5 50 µm.


it was necessary to distinguish them from neurons withascending projections and neurons with both ascendingand descending projections. Neurons with long projections(supraspinal and/or long propriospinal neurons) were la-beled with FG via a right hemisection at spinal segmentT5. Neurons with short ascending propriospinal projec-tions were labeled with FG via bilateral injections into thedorsal horn at spinal segment T12. Neurons with descend-

ing projections were labeled with TB via bilateral injec-tions into the dorsal horn at spinal segment L3. TheFG-stained necrotic zone at the T5 hemisection site de-stroyed the gray matter and severed the dorsal and lateralwhite columns and a portion of the ventral white columnon the right side of the cord. Bilateral injections of FG andTB into the dorsal horn at spinal segments T12 and L3,respectively, produced a central necrotic zone and a halo-stained area that spread into the dorsolateral fasciculusand slightly into the fasciculus proprius (Fig. 5).

Cell counts. Neuron cell bodies and, in some cases,portions of dendritic arbors were labeled with retrogradetracers. In spinal segment L1, cells labeled only with FGhad a golden yellow fluorescence, with varying amounts ofyellow granules. Cells labeled only with TB displayed ahomogenous, nongranular, light-blue fluorescence. Cellslabeled with FG plus TB had a pale-blue backgroundpeppered with FG granules. The FG granules had a silverfluorescence against the blue background of the cell (Fig. 6).

A total of 55,148 SDH neurons labeled with retrogradetracers were analyzed in this experiment. Of these, 14,424neurons (26%) were in lamina I, and 40,724 (74%) were inlamina II. There was no significant difference in thenumber of SDH neurons labeled with retrograde tracersbetween the right and left dorsal horn (27,642 cells vs.27,506 cells, respectively). The majority of SDH neuronswere labeled with FG plus TB; therefore, they presumablyhad both ascending and descending projections. Therewere three times as many neurons with ascending plusdescending projections (FG- plus TB-labeled cells) in laminaII as in lamina I, but they made up the same percentage(,75%) of the population in both laminae (Fig. 7A,B).

SDH neurons with only ascending projections (FG-labeled cells) were distributed in nearly equal numbers inlaminae I and II. In lamina I, the number of neurons withonly ascending projections (FG-labeled cells) was nearlyequal to the number of neurons with only descendingprojections (TB-labeled cells); these made up 15% and12%, respectively, of the tracer-labeled population in laminaI. In lamina II, neurons with only ascending projectionsmade up 7.0% of the labeled population, and those withonly descending projections made up 19% of the labeledpopulation (Fig. 7A,B).

The percentage of cells labeled only with FG neverdipped below 2.0% of the tracer-labeled population in anyof the 12 animals in either lamina I or lamina II. Thepercentage of cells labeled only with TB was never below2.0% in lamina I or below 6.0% in lamina II. There was nostatistically significant difference between the right andleft sides in the percentage of SDH neurons within any ofthe three projection groups (FG-, FG- plus TB-, or TB-

TABLE 1. Total and Subpopulation Cell Counts

Lamina I Lamina II

Cell count1Stereological

corrected cell countPercent

of total population Cell count1Stereological

corrected cell countPercent

of total population

Total population cell count 21.6 6 1.0 11 100 83.5 6 2.5 42.6 100Subpopulation cell count

Long ascending projections 7.4 6 1.4 3.8 34 5.7 6 1.3 2.9 6.6Short ascending propriospinal 9.3 6 1.0 4.7 43 32.3 6 2.6 16.5 39Descending propriospinal 2.5 6 0.5 1.3 12 10.5 6 1.5 5.3 13

Total subpopulation cell count 19.2 6 2.9 9.8 89 48.5 6 5.4 24.7 59Unlabeled population (local circuit?) 2.4 (0–6)2 1.2 11 35 (27–43)2 17.9 41

1Number of neurons counted per 10 µm section in each dorsal horn.2Numerical range.

Fig. 3. Histograms from total population analysis showing thepercentage of the total population of neurons in laminae (LAM) I andII labeled with [3H]thymidine (A) and the number of neurons gener-ated in laminae I and II on embryonic days 13–16 (E13–E16; B).


labeled cells) in either lamina I or lamina II. The numberand percentage of SDH neurons with only descendingprojections, as determined on the right side of the cord, are

Fig. 4. Autoradiographs of laminae I and II from animals treatedwith [3H]thymidine on E13–E16. On E13 (A), nearly all neuronalnuclei are labeled with [3H]thymidine (black granules). On E14 (B), afew neurons are not labeled with [3H]thymidine (arrowheads). On E15(C), large numbers of neurons are not labeled with [3H]thymidine. OnE16 (D), only a few neurons remain labeled with [3H]thymidine.Nissl-stained, 10-µm section. Scale bar 5 50 µm.

Fig. 5. Drawing of retrograde tracer injection sites taken from asingle animal from subpopulation analysis I (ascending and descend-ing projections). Fluoro-Gold (FG) was administered at the hemisec-tion on the right side at spinal segment T5 and was injected bilaterallyinto the dorsal horns at segment T12. True blue (TB) was injectedbilaterally into the dorsal horns at segment L3. Solid dark regionsindicate necrotic areas, and hatched regions indicate halo-stainedareas following retrograde tracer administration. Straight arrowsindicate direction of travel for retrograde transport of tracers frominjection sites to neuron cell bodies in spinal segment L1.


Fig. 6. Autoradiographs of retrograde tracer-labeled SDH neuronstaken from subpopulation analysis I (A–C) and II (D–F). Neuronsillustrated are labeled with FG (gold cells) in lamina I (A) and laminaII (C,E), TB (blue cells) in lamina II (C,F), and FG plus TB (silver-blue

cells) in lamina I (B,D). All neurons shown are labeled with [3H]thymi-dine (black granules) except for the FG-labeled cell (A) and the FG-plus TB-labeled cell (B). Animals were treated with [3H]thymidine onE14 (D,E) and E15 (A–C,F). Scale bar 5 25 µm.

shown in Table 1. This determination was based onlabeling via tracer injections into both the right and leftdorsal horns and is equal to the total number of neurons ineach dorsal horn that have descending projections, regard-less of whether the projections are ipsilateral, contralat-eral, or bilateral.

Neurogenesis. Of the neurons labeled with retrogradetracers, there was a significantly greater percentage la-beled with [3H]thymidine in lamina II than in lamina I ineach of the three neuronal projection groups in animalstreated with [3H]thymidine on E14 and in those treated onE15. In both lamina I and lamina II, neurons with onlydescending projections (TB-labeled cells) had the greatestpercentage of [3H]thymidine-labeled cells. These werefollowed, in decreasing order of magnitude, by neuronswith ascending plus descending projections (FG- plusTB-labeled cells), then by neurons with only ascendingprojections (FG-labeled cells). The difference in the percent-age of [3H]thymidine-labeled neurons was statisticallysignificant between each projection group within eachlamina, with the exception of neurons in lamina II withascending plus descending projections (FG- plus TB-labeled cells) vs. those with only descending projections(TB-labeled cells). In laminae I and II, neurons in both ofthe ascending projection groups (FG-labeled and FG- plusTB-labeled cells) exhibited a statistically significant de-crease in the percentage of [3H]thymidine-labeled cellsbetween E14 and E15, whereas those in the group withonly descending projections (TB-labeled cells) did not(Fig. 7C).

Although there were statistically significant differencesbetween lamina I and lamina II in the percentage oftracer-labeled neurons that were labeled with [3H]thymi-dine in both E14- and E15-treated animals, there was nosignificant difference between lamina I and lamina II inthe number of tracer-labeled neurons generated on E14.By E15, the number of tracer-labeled neurons generatedwas only slightly (15%) greater in lamina II than in laminaI (Fig. 8).

Subpopulation analysis II: Long and shortascending projections

Retrograde tracer injection sites. The number ofSDH neurons with long ascending projections (supraspi-nal and long propriospinal neurons), labeled with FG via aright hemisection at spinal segment T5, and the numberwith short ascending projections, labeled with TB viabilateral injections at T12, were determined in this experi-ment. The FG-stained necrotic zone at the T5 hemisectionsite destroyed the gray matter, severed the dorsal andlateral white columns and a portion of the ventral whitecolumn, and was confined to the right side of the cord. Thebilateral TB injections at T12 produced a necrotic zone ineach dorsal horn and in a portion of the dorsolateralfasciculus and produced a fluorescent halo that spread intothe remainder of the dorsolateral fasciculus and the fascicu-lus proprius (Fig. 9).

Cell counts. Neurons in spinal segment L1, as insubpopulation analysis I, were labeled with FG, TB, or FG

Fig. 7. Histograms from subpopulation analysis I (ascending anddescending projections) showing the number of neurons labeled withretrograde tracers (A), the percentage of retrogradely labeled neuronsthat were labeled with each retrograde tracer (B), and the percentageof tracer-labeled neurons that were labeled with [3H]thymidine inanimals administered [3H]thymidine on E14 and E15 (C). The numberof tracer-labeled neurons shown is the mean number counted in 3010-µm sections per animal on the side ipsilateral to the hemisection.Neurons with ascending projections are labeled with FG, neurons withdescending projections are labeled with TB, and neurons with bothascending and descending projections are labeled with FG plus TB.


plus TB (Fig. 6). A total of 39,440 SDH nerve cells labeledwith retrograde tracers were analyzed in this experiment.Of these, 10,904 (28%) were located in lamina I and 28,536(72%) in lamina II. To determine the total number of SDHneurons with long ascending projections, (i.e., those thatproject to or beyond spinal segment T5, located in eachdorsal horn, the number of SDH neurons labeled with FGand FG plus TB on both the right side and the left side ofthe cord was determined. The total number of SDHneurons with long ascending projections located in eachdorsal horn was assumed to be equal to the number ofneurons with axons that project rostrally (FG-labeled andFG- plus TB-labeled cells) on the same or ipsilateral sideplus the number of neurons with axons that projectrostrally via the contralateral side of the cord, or, simply,

FGipsi 1 FG plus TBipsi

1

FGcontra 1 FG plus TBcontra.

By using this formula, neurons with bilateral projections(i.e., with axons that ascend on both ipsilateral andcontralateral sides) would have been counted twice. Thistheoretical, but possible, source of error should be kept inmind when evaluating the data. The number of SDHneurons with short ascending projections was determinedon both right and left sides of the cord. On the sideipsilateral to the hemisection, the number of neurons withshort ascending projections was determined by countingthe TB-labeled cells on that side. FG- plus TB-labeledneurons on the contralateral side were then subtractedfrom this figure, because the latter neurons are included inthe cell population labeled only with TB on the ipsilateralside, as follows:

TBipsi 2 FG plus TBcontra.

Conversely, the number of neurons with short ascendingprojections determined for the contralateral side was:

TBcontra 2 FG plus TBipsi.

Neurons with long ascending projections (FG-labeled orFG- plus TB-labeled cells) were distributed in nearly equalnumbers in laminae I and II and represented 44% of theretrograde tracer-labeled population in lamina I but only15% of the tracer-labeled population in lamina II. Approxi-mately 64% of the SDH cells that were labeled with FGwere also labeled with TB; therefore, they had both longascending projections and short propriospinal collateralsto the dorsal gray in spinal segment T12 (Fig. 10A,B, Table1). In lamina II, there was a greater percentage of neurons

Fig. 8. Histogram from subpopulation analysis I (ascending anddescending projections) showing the total number of neurons labeledwith retrograde tracers that were generated (not labeled with [3H]thy-midine) in laminae I and II on E14 and E15. Numerical valuesrepresent the mean number of neurons counted in 30 10-µm sectionsper animal on the side ipsilateral to the spinal hemisection.

Fig. 9. Drawing of retrograde tracer injection sites taken from asingle animal from subpopulation analysis II (long and short ascend-ing projections). FG was administered at the hemisection on the rightside at spinal segment T5, and TB was injected bilaterally into thedorsal horns at segment T12. Solid dark regions indicate necroticareas, and hatched regions indicate halo-stained areas followingretrograde tracer administration. Straight arrows indicate direction oftravel for retrograde tracers from injection sites to neuron cell bodiesin spinal segment L1.


with long projections located in the inner zone (63%) thanin the outer zone (37%).

By far, the majority of SDH neurons labeled withretrograde tracers were labeled only with TB. These

neurons are presumed to have short ascending propriospi-nal projections that terminate caudal to the T5 level.There were over three times as many TB-labeled neuronsin lamina II as in lamina I. TB-labeled neurons repre-sented 56% of the tracer-labeled population in lamina Iand 85% in lamina II. There was no statistically significantdifference in the mean number of neurons with shortascending projections (TB-labeled cells) between the rightand left sides of the cord. Counts taken from the right side,shown in Figure 10A and Table 1, represent the totalnumbers of neurons in each dorsal horn that have shortascending projections, regardless of whether the projectionwas ipsilateral, contralateral, or bilateral.

Neurogenesis. Like the findings in subpopulation I,there was a significantly greater percentage of [3H]thymi-dine-labeled neurons in lamina II than in lamina I in eachof the three tracer-labeled projection groups in animalstreated with [3H]thymidine on E14 and in those treated onE15. There were statistically significant differences in thepercentage of SDH neurons labeled with [3H]thymidinebetween each of the three retrogradely labeled cell groups.The SDH neurons with short ascending propriospinalprojections (TB-labeled cells) had the greatest percentageof [3H]thymidine-labeled neurons. These were followed, indecreasing order of magnitude, by neurons with longprojections and short propriospinal collaterals (FG- plusTB-labeled cells) and then by neurons with long projec-tions without collaterals (FG-labeled cells; Fig. 10C). Therewas a statistically significant decrease in the percentage of[3H]thymidine-labeled neurons between E14 and E15 inthe long projection groups (FG-labeled and FG- plusTB-labeled cells) in laminae I and II and the short projec-tion group (TB-labeled cells) in lamina I but not lamina II(Fig 10C).

Combined total and subpopulation analysis

Cell counts. Interpolation of the total population andsubpopulation cell counts, as shown in Table 1, indicatesthat SDH neurons with long projections, i.e., those withsupraspinal projections plus those with long propriospinalprojections, make up 34% of the total population in laminaI and 6.6% in lamina II; neurons with short ascendingpropriospinal projections and descending projections makeup 43% of the total population in lamina I and 39% inlamina II; and neurons with only descending projectionsmake up 12% of the total population in lamina I and 13% inlamina II. The sum of all of the retrograde-labeled neuronstaken from the subpopulation mean cell counts equals19.2 6 2.9 cells in lamina I and 48.5 6 5.4 cells in lamina IIper 10 µm transverse section. The total population cellcount determined in cresyl violet preparations, on theother hand, is 21.6 6 1.0 cells in lamina I and 83.5 6 2.5cells in lamina II per 10 µm section. This leaves a

Fig. 10. Histograms from subpopulation analysis II (long and shortascending projections) showing the number of neurons labeled withretrograde tracers (A), the percentage of retrogradely labeled neuronsthat were labeled with each tracer (B), and the percentage of tracer-labeled neurons that were also labeled with [3H]thymidine on E14 andE15 (C). The number of tracer-labeled neurons shown is the meannumber counted in 30 10-µm sections per animal and extrapolated forone side of the cord. Neurons with long ascending projections arelabeled with FG, neurons with short ascending projections are labeledwith TB, and neurons with both long and short ascending projectionsare labeled with FG plus TB.


remainder of 2.4 cells in lamina I and 35 cells in lamina IIper 10 µm section, i.e., 11% of the neurons in lamina I and41% in lamina II that were not labeled with either FG orTB in the subpopulation studies. These cells are presumedto be local circuit neurons that were not labeled withretrograde tracer, because the axons of these neurons areconfined to local intrasegmental connections. Based on thestandard errors derived in the total and subpopulationstudies, the mathematical range for the number of localcircuit neurons before stereological correction is 0–6 cellsin lamina I and 27–43 cells in lamina II per 10 µmtransverse section.

Neurogenesis. In animals treated with [3H]thymidineon E14, the number of retrogradely labeled neurons thatwere generated in subpopulation analysis I, which in-cluded all neurons with ascending and/or descendingprojections, was 10 cells in lamina I and 10 cells in laminaII per 10 µm section, whereas the number of neuronsgenerated in the total population analysis was 12 cells inlamina I and 16 cells in lamina II per 10 µm section.Results indicate, then, that the majority of SDH neuronsgenerated by E14 were long axon (Golgi type I) neuronswith supraspinal or propriospinal connections.

The neurogenic pattern of neurons with long projections(FG-labeled and FG- plus TB-labeled cells) in subpopula-tion II approximates most closely that of neurons with onlyascending projections (FG-labeled cells) in subpopulation Iand suggests that a large percentage of the neurons withlong ascending projections does not have descending projec-tions (compare Fig. 7C with Fig. 10C). Also, the neurogenicpattern as well as the number of neurons with shortascending propriospinal projections (TB-labeled cells) insubpopulation analysis II approximate most closely thoseof neurons with both ascending and descending projections(FG- plus TB-labeled cells) in subpopulation analysis I.The similarities suggest that many of the same neuronswere labeled in both groups, leading to the conclusion thatthe majority of neurons with short ascending propriospi-nal connections also have descending collaterals (compareFig. 7A,C with Fig. 10A,C).

DISCUSSION


The stereological method of Abercrombie (1946) wasfound to be more versatile than the dissector method ofSterio (1984), because the Abercrombie correction factordetermined in the total population analysis could also beused in the subpopulation analyses. This was possible,because the same region of the spinal cord and the sametissue section thickness were used in all of the experi-ments. However, the accuracy of the Abercrombie (1946)method has recently been questioned because of its assump-tion (‘‘bias’’) that all nuclei counted are similar in length(Coggeshall, 1992). Fortunately, neuronal nuclei in theSDH, as measured in sagittal sections in the present study,exhibited little variation in diameter, and the results ofboth the Abercrombie method and the unbiased dissectormethod were nearly identical. The stereological correctionfactor of Abercrombie (1946), therefore, could be appliedwith confidence throughout the present study.

Subpopulation analyses

The accompanying paper (Bice and Beal, 1997) showsthat only 11% of the neurons in lamina I and 0.6% of the

neurons in lamina II project to supraspinal centers. MostSDH neurons, then, must be either propriospinal or localcircuit neurons. Although data on propriospinal neurons inthe SDH are rather sparse compared with the informationavailable on supraspinal neurons, studies using the Golgiimpregnation method in several species have shown thatsome neurons in lamina I have axons that enter either thedorsolateral fasciculus of Lissauer or the fasciculus pro-prius (Ramon Y Cajal, 1909; Pearson, 1952; Szentagothai,1964; Scheibel and Scheibel, 1968; Beal et al., 1981). Thesetwo tracts are thought to convey mainly intersegmentalpropriospinal projections. Cervero et al. (1979b), by usingelectrophysiological techniques, found that approximatelytwo-thirds of the neurons in lamina I of the cat could beantidromically activated from the dorsolateral fasciculus,and most projected no farther than two segments. Otherinvestigators, by using degeneration and ablation tech-niques (Molenaar and Kuypers, 1975, 1978; Chung andCoggeshall, 1983), retrograde tracers (Burton and Loewy,1976; Chung et al., 1984; English et al., 1985; Menetrey etal., 1985; Verburgh and Kuypers, 1987; Verburgh et al.,1990; Yezierski and Mendez, 1991; Nandi et al., 1993), andanterograde tracers (Craig, 1991), have also demonstratedpropriospinal neurons in lamina I in a variety of mamma-lian species.

Investigators using the Golgi impregnation technique inseveral species have shown that some neurons in laminaII, like those in lamina I, are propriospinal and have axonsthat enter the dorsolateral fasciculus as well as thefasciculus proprius (Ramon Y Cajal, 1909; Pearson, 1952;Szentagothai, 1964; Scheibel and Scheibel, 1968; Beal andCooper, 1978). By using the isolated spinal segment tech-nique, Szentagothai (1964) showed that propriospinalneurons in lamina II have axons that ascend up to twosegments and that descend three to four segments. Localcircuit neurons, defined by Ramon Y Cajal (1909) asinterneurons with axons that form a terminal plexus in thevicinity of the cell body, have also been described in laminaII (Scheibel and Scheibel, 1968; Gobel, 1978; Bennett et al.,1980; Bicknell and Beal, 1984; Light and Kavookjian,1988; Beal et al., 1989).

Results of the present study provide a more precise andcomplete assessment of the number and types of SDHneurons than has been demonstrated previously. Themajority of SDH neurons labeled with retrograde tracerswere short propriospinal neurons. Because these neuronswere retrogradely labeled with TB via spinal segment T12but were not retrogradely labeled with FG that wasdelivered at the T5 hemisection in subpopulation analysisII, it is concluded that these neurons have axons thatascend no less than two and no more than eight spinalsegments. Although specific functions of propriospinalneurons have yet to be ascertained, Sandkuhler et al.(1993) have provided some evidence that propriospinalneurons play a role in the modulation of the backgroundactivity of projection neurons. In addition, Craig (1991)has shown that some propriospinal neurons in lamina Iproject to the intermediolateral cell column and maymediate somatosympathetic reflexes in thermoregulation.It has also been suggested that some propriospinal neu-rons may be involved in a multisynaptic pain pathway(Basbaum, 1973).

The present study also showed that most SDH neurons,as demonstrated in subpopulation analysis I, have bothascending and descending projections, but some have only


descending projections. It is possible that some of theneurons with only descending projections have long ascend-ing axons that were not labeled with FG, because theaxons ascended on the side contralateral to the hemisec-tion. This seems unlikely, however, because neurons withlong ascending axons and those with only descendingaxons have disparate neurogenic patterns (see Fig. 7C). Itis also possible that the neurons with only descendingaxons had ascending axons that either did not reach orsomehow bypassed the bilateral FG injection sites in thedorsal horn. However, because these neurons were ob-served in substantial numbers in both lamina I and laminaII in all animals examined, they must tentatively beconsidered as a distinct cell type. From a functionalstandpoint, neurons with only descending projections inspinal segment L1 would be ideally located to mediateconcerted sensory and motor functions confined to the hindlimb.

Although they were not specifically demonstrated in thepresent study, neurons with only short ascending projec-tions may also be present in the SDH. These neuronswould be labeled only with FG in subpopulation analysis I.However, the [3H]thymidine labeling pattern of the FG-labeled neurons exemplifies that of neurons with long, notshort, projections. This, coupled with the fact that thereare few neurons in the FG-labeled group, suggests thatthere are few, if any, neurons with only short ascendingprojections.

Cell counts. Clear mathematical relationships werefound in the distribution of propriospinal neurons betweenlamina I and lamina II. Subpopulation analysis II showedthat neurons with short ascending propriospinal projec-tions, like those with only descending projections demon-strated in subpopulation analysis I, were found in nearlyequal proportions in both laminae (Table 1). Neurons withlong propriospinal projections, but not supraspinal projec-tions, on the other hand, were found in nearly equalnumbers in both laminae. This determination was madeby subtracting the number of neurons with supraspinalprojections (2.4 6 0.3 in lamina I and 0.5 6 0.1 in lamina IIper 10 µm section), determined in the accompanying paper(Bice and Beal, 1997), from the number of neurons withlong projections (7.4 6 1.4 in lamina I and 5.7 6 1.3 inlamina II per 10 µm section), determined in subpopulationanalysis II of the present study (Table 1). The numbers oflong propriospinal neurons, then, would be 5.0 6 1.1 inlamina I and 5.2 6 1.2 in lamina II, or, after stereologicalcorrection, 2.6 and 2.7 cells per 10 µm, respectively, inlaminae I and II.

Nandi et al. (1993), by using the retrograde tracer FG,also labeled neurons with long propriospinal projections inspinal segment L1, and their cell counts in lamina I werecomparable to those in the present study: 4.3 6 0.7 cells vs.5.0 6 1.1 cells per 10 µm section, respectively. However, inlamina II, only 1.4 6 0.6 cells with long propriospinalprojections per 10 µm section were counted by Nandi et al.compared with 5.2 6 1.2 cells per 10 µm section in thepresent study. Differences in the lamina II cell countsbetween these two studies are probably due to differencesin the spinal levels at which the retrograde tracers weredelivered. Nandi et al. injected FG into spinal segment C5,whereas, in the present study, FG was delivered at spinalsegment T5. The combined results of these two studiessuggest that there are nearly equal numbers of neuronswith long propriospinal projections in laminae I and II,

but, in general, those in lamina I have axons that projectgreater distances than those in lamina II.

The present study showed that there were approxi-mately twice as many neurons with long projectionslocated in the inner zone (63%) as in the outer zone (37%)of lamina II. Because neurons with supraspinal projec-tions were found to be distributed equally between theouter and inner zones, as demonstrated in the accompany-ing paper (Bice and Beal, 1997), it is concluded thatneurons with long propriospinal projections are distrib-uted unequally.

Connectivity. Analysis of the retrograde tracer datain conjunction with the [3H]thymidine autoradiographydata provides some insights into the connectivity of SDHneurons. The fact that the neurons with ascending, but notdescending, projections in subpopulation analysis I andthe neurons with long projections determined in subpopu-lation analysis II demonstrated similar neurogenic pat-terns suggests that many of the neurons with long projec-tions (supraspinal and long propriospinal) do not havedescending collaterals. These results are in agreementwith previous studies in the rat, which found that mostsupraspinal projection neurons in the SDH of the cervicalspinal cord do not have descending projections (Verburghand Kuypers, 1987; Verburgh et al., 1990), and only 3.0% ofall spinomesencephalic neurons in segments T9–T13 havedescending collaterals to lower lumbar segments (Yezier-ski and Mendez, 1991).

Results of the present study also indicate that approxi-mately one-third of the SDH neurons with long ascendingprojections (cells labeled with FG only), demonstrated insubpopulation analysis II, do not have short ascendingcollaterals to thoracic segment T12 (Fig. 10A). The numberand neurogenic period of the cells in this group are nearlyidentical to those of supraspinal projection neurons de-scribed in the accompanying paper (Bice and Beal, 1997).Results suggest, then, that many, if not most, neurons withsupraspinal projections have neither short ascending nordescending collaterals and, thus, probably do not directlyinfluence pain transmission or modulation at other spinallevels. It should be pointed out, however, that neuronswith long projections may have collaterals to spinal seg-ments that were not examined in this study, includinglocal intrasegmental collaterals that have been demon-strated on some neurons in lamina I (Light et al., 1979;Beal et al., 1981; Bennett et al., 1981).

Some neurons with long projections in subpopulationanalysis II did demonstrate putative short collaterals tospinal segment T12, and the possibility that the TB tracerinjected into the dorsal horn at spinal segment T12 waspicked up by long projection fibers of passage, rather thanby intersegmental collaterals to the dorsal horn, needs tobe considered. Although this possibility cannot be ruledout completely, there are arguments against it. First, thefact that the SDH neurons labeled with FG plus TBexhibited a neurogenic pattern that was significantlydifferent from that of neurons labeled only with FGstrongly suggests that each is a distinct group of neurons.Second, although TB can be picked up by fibers of passagefrom a central necrotic zone of a TB injection site, it is notpicked up from the surrounding halo region (Skagerberg etal., 1985). Furthermore, neither TB nor FG is transportedtransneuronally (Skagerberg et al., 1985; Schmued andFallon, 1986; Payne, 1987), and neither exhibits extracellu-lar diffusion (Nandi et al., 1993; Beal and Bice, 1994).


Nevertheless, because the TB injection sites do show that aportion of the dorsolateral fasciculus was located in thenecrotic zone penetrated by the micropipette, it is possiblethat there were some fibers that passed through this areathat were stained with TB but did not have collaterals tothe dorsal horn at T12. This possible source of error shouldbe kept in mind when interpreting the data. However, itshould be added that axons of SDH neurons that ascend inthe dorsolateral fasciculus are known to be relatively shortand have multiple intersegmental collaterals (Ramon YCajal, 1909; Szentagothai, 1964; Scheibel and Scheibel,1968; Cervero, 1979b).

Neurogenesis. [3H]thymidine studies on the rat haveshown that the generation of dorsal horn neurons in thelumbar spinal cord occurs between E13 and E16 (Nornesand Das, 1974; Altman and Bayer, 1984; Nandi et al., 1990,1993). Results of the present study show that fluctuationsin the rates of neuronal production within laminae I and IIduring this period are related to the generation of specifictypes of neurons. This production proceeds, as suggestedby Nandi et al. (1993), along an intralaminar axon-lengthgradient. Lamina I, which has a greater proportion ofneurons with long projections than lamina II, has arelatively early peak production period. Conversely, laminaII, which has a larger proportion of neurons with shortaxons, has a later peak production period. This temporalpattern of neurogenesis, whereby long axon (Golgi type I)neurons complete neurogenesis prior to local circuit (Golgitype II) neurons, is similar to that seen in most subcorticalcenters, including the cerebellum (Uzman, 1960; Mialeand Sidman, 1961; Fujita, 1964, 1967; Fujita et al., 1966;Das and Nornes, 1972; Altman and Bayer, 1978, 1985),auditory nuclei (Taber Pierce, 1967), olfactory bulb (Hinds,1968a,b; Bayer and Altman, 1987), lateral geniculate body(Hitchcock et al., 1984), locus coeruleus (Steindler andTrosko, 1989), and the hippocampus (Rakic and Nowa-kowski, 1981).

Why neurogenesis follows the axon-length gradient isopen to speculation. Certainly, from the standpoint ofconnectivity, it would be advantageous for neurons withsupraspinal projections to complete neurogenesis early,because they sprout axons that will travel the longestdistances. However, close examination of the neurogenicpattern of the subpopulations of neurons retrogradelylabeled in the present study shows that all types of Golgitype I long axon neurons, supraspinal projection as well aspropriospinal neurons, are generated very early in theneurogenic period. In fact, cell counts reveal that, by E14,prior to completion of the neurogenesis of SDH neuronswith supraspinal projections, more short propriospinalneurons are generated than neurons with supraspinalprojections. It might also be argued that the neurogenicpattern in the SDH conforms to the axon-length gradient,because neurons with the longest axons in the SDH are thefewest in number. The generation of neurons with thelongest axons, as a result, would be completed earlier thanthat of neurons with shorter axons, because the latter aremore numerous and must pass through a greater numberof cell divisions. However, the validity of this argument isalso questionable, because the supraspinal projection neu-rons that have the longest projections (spinothalamic andspinomesencephalic neurons), as demonstrated in theaccompanying paper (Bice and Beal, 1997), complete neu-rogenesis prior to those with shorter projections (spinomed-ullary neurons), even though the spinomedullary group is

made up of a much smaller number of neurons. Theaxon-length gradient, then, appears to prevail, regardlessof the total number of neurons generated in each group.

Like the retrograde tracer analyses, a mathematicalrelationship between lamina I and lamina II can also beseen in the neurogenic pattern. The present study showsthat the generation of Golgi type I neurons occurs at thesame time and at approximately the same rate in bothlamina I and lamina II. For each Golgi type I neurongenerated in lamina I, one is also generated in lamina IIthrough E14. This one-to-one distribution pattern appar-ently continues until approximately E15, when productionslows in lamina I.

The neurogenesis of presumptive local circuit (Golgitype II) neurons, on the other hand, does not follow thisone-to-one mathematical distribution pattern. Local cir-cuit neurons, as shown in the present study, are generatedduring the second half of the neurogenic period andmigrate mainly into lamina II. However, neurogenesiscontinues to follow the ventral-to-dorsal gradient depictedby Nornes and Das (1974) and Altman and Bayer (1984),because some of the last SDH neurons generated on E16are located in lamina I. Although they are not conclusive,the facts that the genesis of this small group of neurons inlamina I is relatively late and that the number of thesecells is approximately the same as the number of lamina Ineurons that were not labeled with retrograde tracers inthe subpopulation analyses suggest that these late-dividing cells in lamina I are local circuit cells.

ACKNOWLEDGMENTS

The authors thank Wanda Green for her technicalassistance, Indrani Nandi for her statistical expertise, andAngela Swanton for her computer and word processingskills.

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quantitative and neurogenic analysis of the total population and subpopulations of neurons defined...

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