John A. Beal

Quantitative and neurogenic analysis of the total population and subpopulations of neurons defined by axon projection in the superficial dorsal horn of the rat lumbar spinal cord

The 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 the SDH, defined by axon projection, were quantitated following strategic intraspinal injections of dual retrograde tracers (Fluoro‐Gold and true blue). These methods were used in conjunction with [3H]thymidine (delivered in utero) autoradiography for neurogenic pattern analysis. Following stereological correction, each dorsal horn in spinal segment L1 contained 11 neurons in lamina I and 42.6 neurons in lamina II per 10‐μm transverse section. Neurons with long projections, i.e., neurons with projections rostral to spinal segment T5, were only slightly more numerous in lamina I than in lamina II. These neurons made up 34% of the total neuron population in lamina I and 7.0% in lamina II. Most of these neurons did not demonstrate descending connections, and many (presumed supraspinal projection neurons) did not demonstrate short, ascending, intersegmental connections. Neurons with short propriospinal projections, i.e., neurons with connections caudal to spinal segment T5, made up approximately 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; the remaining 21% had only descending projections. Neurons that were not labeled with retrograde tracers (presumed local circuit cells) represented 11% of the neurons in lamina I and 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 the shortest completed neurogenesis last. The generation of both propriospinal and supraspinal projection neurons began on embryonic day 13 (E13). Nearly equal numbers of neurons in this group were generated in laminae I and II through E14. On E15, neuron production slowed in lamina I and accelerated in lamina II as local circuit neurons and the remaining propriospinal neurons were generated. Neuron production ceased simultaneously in both lamina I and lamina II on E16. J. Comp. Neurol. 388:550–564, 1997.

Quantitative and neurogenic analysis of neurons with supraspinal projections in the superficial dorsal horn of the rat lumbar spinal cord

Dual retrograde axonal tracers, Fluoro‐Gold (FG) and true blue (TB), were used in conjunction with [3H]thymidine autoradiography to determine the number and neurogenic pattern of neurons with supraspinal projections in the superficial dorsal horn (SDH), i.e., laminae I and II, in spinal segment L1 of the rat. FG was injected into rostral brain centers (dorsal thalamus and midbrain), and TB was injected into the caudal brainstem (medulla) in young adult rats previously administered [3H]thymidine in utero. Following stereological correction, each dorsal horn had an average of 1.22 neurons in lamina I and 0.24 neurons in lamina II that had supraspinal projections per 10‐μm transverse section. In the SDH, 52% of the neurons with supraspinal projections were found to project to rostral brain centers alone, 3.0% only to the caudal brainstem, and 45% to both areas. There was no significant difference in the percentage distribution of each of the three groups of neurons between lamina I and lamina II. Cell counts in the present study, in conjunction with previous observations in the literature, suggest that the majority of supraspinal projection neurons in the SDH fall into two groups: 1) spinomesencephalic neurons with collaterals to the medulla and 2) spinothalamic neurons with collaterals to the midbrain. The neurogenesis of supraspinal projection neurons in the SDH proceeded along an axon‐length gradient, whereby neurons with the longest axons, those with projections to rostral brain centers, completed neurogenesis prior to neurons with shorter axons, those with projections only to the caudal brainstem. The generation of all SDH neurons with supraspinal projections was completed on embryonic day 14 (E14), 2 days prior to the completion of neurogenesis for SDH neurons with intraspinal projections. J. Comp. Neurol. 388:565–574, 1997.

The ventral dendritic arbor of marginal (lamina I) neurons in the adult primate spinal cord

The ventral (gelatinosal) dendritic arbor of marginal neurons taken from lumbosacral cord was examined in Golgi preparations from adult Macaque monkey and adult Squirrel monkey where neurons were filled with horseradish peroxidase (HRP) via secondary diffusion. Based on morphological criteria two types of marginal neurons were found which give rise to a substantial ventral dendritic arbor. These dendrites penetrate the depth of lamina II and some extend well into lamina III. The cell bodies are medium to large in size and confined to the medial half of lamina I. Marginal neurons located in the lateral half of the marginal layer lack gelatinosal dendrites and are characterized instead by numerous interstitial dendrites.

Substance P-immunoreactive nerves in the rat kidney

An indirect immunohistochemical method in which an avidin-biotinylated horseradish peroxidase complex is bound to the secondary antibody was used to visualize substance P-immunoreactive (SPI) nerves in the rat kidney. Rats were perfused with 2% paraformaldehyde + 0.15% picric acid in 0.1 M phosphate buffer, then transferred to the buffer. After 24-48 h, the kidneys were sectioned with a Vibratome at 200 or 300 micron and incubated in the primary antiserum for 18 h at room temperature. A dense plexus of SPI nerves innervates the rat renal calyx. A small proportion of intrarenal SPI axons innervates interlobular arteries and afferent arterioles, but most perivascular SPI axons terminate on interlobar and arcuate arteries. The densest plexuses are located on segments of interlobar arteries near the hilus of the kidney. Some of these axons probably are nociceptive; others may be chemo- or baroreceptive.

Characterization of Long Ascending Tract Projection Neurons and Non-Tract Neurons in the Superficial Dorsal Horn (SDH)

Using the Golgi technique, Ramon y Cajal (1909) showed that the marginal and substantia gelatinosal (SG) layers of the dorsal horn, now collectively known as the superficial dorsal horn (SDH), contained at least four distinct neuronal cell types; these were the marginale of the marginal zone and the transverseux., limitrophe., and centrale cells of the SG. Several investigators in subsequent Golgi studies have described the morphology of a variety of additional types of cells in the SDH (Pearson, 1952; Scheibel and Scheibel, 1968; Matsushita, 1969; Gobel, 1975, 1978a,b, Sugiura, 1975; Mannen and Sugiura, 1976; Beal and Cooper, 1978; Beal, 1979, 1983; Price et al., 1979; Bicknell and Beal, 1981a,b, 1984; Schoenen, 1982; Abdel-Maguid and Bowsher, 1984, 1985; Beal and Bicknell, 1984; Bowsher and Abdel-Maguid, 1984; and Lima and Coimbra, 1986). These studies had been performed on a variety of different species at different levels of the neuraxis and there was considerable disagreement between investigators on the various cell types present and on the terminology used. Also, although it is known that the SDH is comprised of both projection and non-projection neurons, these two groups have never been completely distinguished from one another on a morphological basis.

Structure and development of central arborizations of hair follicle primary afferent fibers

SummaryThe present study describes the structure and development of the flame-shaped central arborizations of hair follicle (HF) afferents in the lumbosacral spinal cord of the rat. Tissue was processed according to the rapid Golgi method at successive stages of development from embryonic day 17 through postnatal day 30. Collaterals of most HF afferents were found to enter the dorsal horn via a characteristic U-shaped pathway which often parallels the vascular pattern. The HF collaterals can first be identified at embryonic day 19 and by postnatal day 5 they have established the dorso-ventral and rostro-caudal limits of their field of arborization. Dorsally the arbors extend no further than the inner zone of lamina II (IIi) at any stage of development. Short aberrant branches were observed on some HF collaterals during the prenatal period but none of these developed synaptic terminals or contributed branches to other arbors. Each HF collateral formed a single well defined flame-shaped arbor with a distribution and branching pattern which could be distinguished from that of other afferents throughout the postnatal developmental period. Two types of HF collaterals were observed. Structure and distribution patterns suggest that type I collaterals are derived from G and T HF afferents while collaterals in the type II category are probably derived from both G and T as well as D (A-delta) HF afferents. Type I collaterals divide into well defined medial and lateral collateral branches which arborize mainly in lamina III with a few branches to lamina IV. Type II collaterals are characterized by a delicate arbor which is more vertically oriented than type I arbors. These arbors also have a few branches in lamina IV but distribute mainly to laminae III and IIi.

Neurogenesis of spinothalamic and spinocerebellar tract neurons in the lumbar spinal cord of the rat

The temporal and spatial neurogenic patterns of spinothalamic and spinocerebellar neurons were determined in spinal cord segment L1 of the rat. Neurogenic patterns were demonstrated with [3H]thymidine administered to fetal rats during the period when neurons with supraspinal projections are known to be generated, i.e. on one of embryonic (E) days E13, E14, or E15. The animals were allowed to survive 50 to 100 days postpartum, then neurons with spinothalamic and spinocerebellar projections were retrogradely filled with fluorescent axonal tracers, Fluoro-Gold or True blue, which were pressure injected into the dorsal thalamus and cerebellum in various combinations in the same and in separate animals. Neurons labeled with each retrograde tracer and [3H]thymidine and neurons labeled with retrograde tracers alone were counted in spinal cord segment L1 in each of the animals. Spinothalamic and spinocerebellar neurons were found to be separate and distinct populations. Statistical analysis of the data showed that spinothalamic and spinocerebellar neurons also have distinctly different patterns of neurogenesis which suggest early determination in each cell line. The temporal neurogenic pattern followed a projection-distance gradient, such that spinothalamic neurons, which have longer axons than spinocerebellar neurons, completed neurogenesis prior to spinocerebellar neurons in each region of the spinal gray.

Neurogenesis of ascending supraspinal projection neurons: ipsi- versus contralateral projections☆

The present study tests the hypothesis that contralaterally projecting supraspinal projection neurons (SPNs) are generated prior to ipsilaterally projecting SPNs. Neuronal time of origin was determined by injecting pregnant rats with tritiated thymidine on one of embryonic (E) days E12 through E15. In mature offspring of thymidine-treated dams, SPNs in the lumbar cord were retrogradely labelled with True Blue delivered at the site of a hemisection in spinal segment C3. Ipsi and contralaterally projecting SPNs in laminae I, VII and VIII and the lateral spinal nucleus, which are known to give rise to long sensory pathways, were generated simultaneously throughout their neurogenic period (E12-E14), while ipsilaterally projecting SPNs in lamina IV and the nucleus dorsalis, which give rise to short sensory pathways, completed neurogenesis one day later (E15). Results suggest that the projection target and its distance from the nerve cell body of origin are more consistent correlates of the duration of the neurogenic period than the course of the axon.

Classification of aberrant primary afferents in the substantia gelatinosa of the rat following neonatal capsaicin treatment

Administration of capsaicin to newborn rats results in a loss of a large percentage of primary afferent C fibers many of which terminate in the substantia gelatinosa (SG). Using the Golgi silver impregnation technique, the present study shows that the loss of C fibers results in an invasion of aberrant myelinated primary afferents in the SG by 10 days after birth. The aberrant afferents, identified on the basis of their distinctive collateral arborizations, are derived from hair follicles and slowly adapting type I mechanoreceptors.

Neurogenic period of ascending tract neurons in the upper lumbar spinal cord of the rat

Although the neurogenic period for neurons in the lumbar spinal cord has been clearly established (Days 12 through 16 of gestation), it is not known when the neurogenesis of ascending tract neurons is completed within this period. The purpose of the present study was to determine the duration of the neurogenic period for projection neurons of the ascending tracts. To label neurons undergoing mitosis during this period, tritiated thymidine was administered to fetal rats on Embryonic (E) Days E13 through E16 of gestation. Ascending tract neurons of the lumbar cord were later (Postnatal Days 40-50) labeled in each animal with a retrograde tracer, Fluoro-Gold, applied at the site of a hemisection at spinal cord segment C3. Ascending tract neurons which were undergoing mitosis in the upper lumbar cord were double labeled, i.e., labeled with both tritiated thymidine and Fluoro-Gold. On Day E13, 89-92% of the ascending tract neurons were double labeled; on Day E14, 35-37%; and on Day E15, 1-4%. Results showed, then, that some ascending tract neurons were double labeled through Day E15 and were, therefore, proliferating in the final one-third of the neurogenic period. Ascending tract neurons proliferating on Day E15 were confined to laminae III, IV, V, and X and the nucleus dorsalis. Long tract neurons in the superficial dorsal horn (laminae I and II), on the other hand, were found to have completed neurogenesis on Day E14 of gestation. Results of the present study show that spinal neurogenesis of ascending projection neurons continues throughout most of the neurogenic period and does not completely follow the well-established ventral to dorsal gradient.