A study of axonal diameters and areas in lumbosacral roots and nerves in the rat

There has been debate as to whether there is a size difference between central and peripheral processes of dorsal root ganglion cells. In the present study, the mean areas of myelinated and unmyelinated fibers are measured as 27.8 micron2 and 0.55 micron2, respectively, in peripheral nerves and 13.72 micron2 and 0.14 micron2 in dorsal roots. Thus myelinated central processes of dorsal root ganglion cells have mean areas 50% less than the mean areas of the myelinated sensory axons in the same peripheral nerves, and the mean diameters of the central myelinated axons are 30% less than the peripheral myelinated axons. The mean areas of the unmyelinated sensory axons in the dorsal roots are 25% of the mean areas of the unmyelinated sensory unmyelinated axons are 50% of the mean diameters of the unmyelinated sensory axons in the same peripheral nerves. These data indicate that both myelinated and unmyelinated central processes of dorsal root ganglion cells are smaller than the peripheral processes of these same cells for lumbosacral segments in the rat. It is shown that axonal tapering is not responsible for these striking differences. Finally, documentation of differences in myelinated fiber histograms from dorsal roots of different segments in the rat is provided.     

Unmyelinated primary afferent fibers in dorsal funiculi of cat sacral spinal cord

The present study tests the hypothesis that there are numerous unmyelinated primary afferent fibers in cat posterior funiculi. The animals have unilateral dorsal rhizotomies from L6 to Ca3. One week later the axons of both S2 dorsal funiculi are counted. The data indicate that there are approximately 22,500 myelinated and 8,500 unmyelinated axons on the unoperated side and 11,000 myelinated and 3,900 unmyelinated axons on the operated side. On this basis, we suggest that 51% of the myelinated and 54% of the unmyelinated axons in cat dorsal funiculi arise from dorsal root ganglion cells and thus are primary afferent axons. If this is correct, then 71% of the primary afferent axons in the cat dorsal funiculus are myelinated and 29% are unmyelinated. The function of this large group of previously unsuspected fine sensory axons remains to be determined.     

Numbers of myelinated and unmyelinated axons in the dorsal, lateral, and ventral funiculi of the white matter of the S2 segment of cat spinal cord

The present work determines the numbers of myelinated and unmyelinated axons in the dorsal, lateral, and ventral funiculi of the S2 segment of the cat spinal cord. The major finding is that unmyelinated axons are almost as numerous as myelinated axons in these pathways. The myelinated axons tend to be distributed uniformly, although there is a slight concentration of these fibers in the dorsal part of the lateral funiculus. By contrast, the unmyelinated fibers, although found in significant numbers in all parts of these funiculi, concentrate in the dorsal part of the lateral funiculus and in the dorsal funiculus. Of particular note are the unmyelinated fibers in the dorsal funiculus, because it is highly likely that some of these are sensory. The findings in this study will serve as a basis for experimental studies to determine the numbers, locations, and types of unmyelinated fibers in the white matter of the mammalian cord.     

Unmyelinated axons in the ventral roots of the cat lumbosacral enlargement

The ventral roots L₇ and S₁ of the cat spinal cord were examined with the light and electron microscopes. Differences in the morphology of Schwann cells associated with large myelinated fibers and with small myelinated or unmyelinated fibers were observed. The blood vessels were largely encircled by pericytes. The most noteworthy finding was that 29% of the axons in these roots were unmyelinated. These unmyelinated axons were greatly reduced in number proximal but not distal to a ventral rhizotomy. Furthermore, they were reduced in number following dorsal root ganglionectomy, but not after dorsal rhizotomy, sympathectomy or peripheral nerve section. It is concluded that the ventral roots of the lumbosacral enlargement contain a large population of unmyelinated fibers originating from dorsal root ganglion cells.     

Myelo- and cytoarchitectonics of the cat pallidum]

600 thick serial frontal and sagittal sections of the cat cerebrum were made. Using the new controlled impregnation technique, it was shown that the pallidum is crossed by numerous transit axons bundles (TAB) consisting of thin myelinated and unmyelinated axons each 0.2-0.8 micron in diameter. TAB pass through the dorsal part of the putamen and the whole pallidum parallel to the sagittal plane and perpendicular to the frontal plane. Such TAB contain not less than 10 million axons in the pallidum of a hemisphere. The TAB are mainly related to the capsula interna and to the sensory-motor cortex. The cyto- and synaptoarchitectonics of the pallidum is described.     

Numbers of axons in lateral and ventral funiculi of rat sacral spinal cord

The present study determines the numbers of myelinated and unmye-linated axons in the ventral and lateral funiculi of rat sacral spinal cord. On average, there are 55,000 myelinated and 110,000 unmyelinated axons in the lateral funiculus and 26,000 myelinated and 9,000 unmyelinated axons in the ventral funiculus at these levels. These figures combined with data from earlier studies of the posterior funiculus and the tract of Lissauer give approximate figures of 88,500 myelinated and 131,500 unmyelinated axons for the entire white matter of one side of the rat sacral spinal cord. Thus unmyelinated axons predominate in the white matter of the rat sacral spinal cord. The majority of axons, particularly the unmyelinated axons, are located in the lateral funiculus. The axons are concentrated in the dorsalateral part of the lateral funiculus, and so the dorsal part of the lateral funiculus, of ten referred to as the dorsolateral funiculus, contains more than half the fibers in the white matter of the spinal cord. A small nick in the dorsal and lateral part of the lateral funiculus, which is often done for various experimental reasons, could thus remove 40% of the axons in the white matter of rat sacral spinal cord. The data reported in the present paper will serve as a basis for future studies on the white matter of the isolated spinal cord.     

Primary afferent axons in the tract of Lissauer in the cat.

The present study is concerned with the numbers of primary afferent axons in the tract of Lissauer in the cat. The data show that approximately 27% of the axons in mid-thoracic and lumbosacral tracts are primary afferent fibers from the segment in question and another 20% of the axons are primary afferent fibers that come from nearby segments. In addition the data show that approximately 80% of the axons in the tract are unmyelinated and that there is a slightly higher proportion of unmyelinated as opposed to myelinated primary afferents. There is also a higher proportion of primary afferents in the medial as opposed to lateral parts of the tract, but there are significant numbers of primary afferents in lateral parts of the tract. Thus it seems clear that the tract contains more primary afferent fibers than was previously believed and if these data are confirmed, the conclusions will have a bearing on considerations of the primary afferent input into the dorsal horn.     

Regional variation in the abundance of axonal cytoskeletal proteins.

The relative abundance of several axonal cytoskeletal proteins was determined by immunoassay at various sites in the peripheral and central nervous systems of adult rats. Within the peripheral nervous system, the ratio of tubulin to neurofilaments was greatest for nerves composed of unmyelinated axons and least for nerves with large myelinated axons. MAP1 protein was more prominent in unmyelinated fibers; conversely tau proteins were relatively more abundant in large myelinated axons. An immunochemical index of neurofilament phosphorylation was less for unmyelinated fibers than for myelinated ones. In the fimbria-fornix, pyramidal tract, and superior cerebellar peduncle, similar trends were observed: small axons had more MAP1, less tau, and a greater ratio of tubulin to neurofilament proteins. The phosphorylation index was greatest for the superior cerebellar peduncle, the tract with the largest axons. The immunochemical index of neurofilament phosphorylation was greater for the optic nerve than for axonal tracts in the brain proper. These results suggest that development of large myelinated axons is associated with greater neurofilament content, neurofilament phosphorylation, and with greater abundance of tau proteins in the CNS and the PNS; however, quantitative aspects of these relationships differ in the PNS and the CNS.     

Trigeminal alveolar nerve of the lower jaw in the cichlid Tilapia mariae: evidence for continual axon generation and presence of exceptionally small myelinated axons

Cross sections from the trigeminal alveolar nerve of the lower jaw in the cichlid Tilapia mariae were examined by electron microscopy. The nerve fibers are arranged in groups with a core of unmyelinated and small myelinated axons, surrounded by myelinated axons of varying sizes. The core contains large bundles of unmyelinated axons collectively ensheathed by circumferentially located Schwann cells, as well as smaller bundles of unmyelinated axons partly separated from each other by Schwann cell processes. Among the unmyelinated axons, occasional scattered profiles resembling growth cones are seen. The total number of axons in this tooth-related nerve increases from approximately 1,500 to 5,000, as the animals grow in length from 4.5 to 21.5 cm. Some 24-49% of the axons are unmyelinated. The myelinated axons have maximum diameters of 1.0-3.0 micron, depending on body size. Most myelinated axons have diameters less than 1.0 micron and the smallest ones reach down to 0.3 micron. These results show that there is a continual addition of axons to the alveolar nerve of the lower jaw in Tilapia mariae and that the critical diameter for myelination in this peripheral nerve is similar to that typically found in the mammalian CNS.     

Categories of axons in the inferior cardiac nerve of the cat

Myelinated and unmyelinated axons in the inferior cardiac nerve of the cat were examined to determine how many axons were (1) sensory, (2) preganglionic sympathetic, and (3) postganglionic sympathetic. In one group of cats, a segment was removed from the middle of the inferior cardiac nerve as a control, and the proximal and distal stumps of the nerve were examined one week later. In another group of cats, the control segment of nerve was removed and the first thoracic white ramus communicans and sympathetic trunk were cut proximal to the stellate ganglion, followed in one week by examination of the proximal and distal stumps of the inferior cardiac nerve. In still another group of cats, the first five thoracic spinal nerves were cut just distal to the dorsal root ganglion. The counts of myelinated and unmyelinated axons after these surgical procedures indicated that, in the cat inferior cardiac nerve, all or almost all of the approximately 30,000 unmyelinated axons and 10 percent of the myelinated axons are postganglionic sympathetic fibers, and that approximately 90 percent of the myelinated axons are sensory.     

C-fiber structure varies with location in peripheral nerve

Recent advances in regeneration and pain research have revealed gaps in the understanding of normal C-fiber anatomy. In the rat PNS, C-fiber axons assemble into Remak bundles, but beyond this, features of C-fiber organization are not defined. Systematic sampling and quantitation reveals that Remak bundles exiting from the L5 dorsal root ganglion (DRG) contain large numbers of axons, for example, 56% of unmyelinated axons were in bundles of >20 axons. This is different from distal nerve segments such as the hindpaw plantar nerve where the median number of axons per bundle is 3. The cross-sectional area of unmyelinated axons in dorsal root was homogeneous near the DRG but variability in axonal area increased near the spinal cord (p = 0.00001) and the mean axonal area was unchanged. Unmyelinated axons in peripheral nerve were almost always isolated from one another by Schwann cell processes; however, in dorsal root 7% to 9% of unmyelinated axons were immediately adjacent within pockets containing 2 or more axons. Remak bundles in the distal peripheral nerve clustered with other Remak bundles. We observe that multiple unmyelinated axons are juxtaposed within the C-fiber/Remak bundle and that the close association of afferent axons may have important functional implications.     

Unmyelinated and myelinated axon membrane from rat corpus callosum: differences in macromolecular structure

The macromolecular structure of unmyelinated and myelinated internodal axon membrane was examined with freeze-fracture electron microscopy. Unmyelinated axons exhibited a gradient of axonal diameters, generally ranging from 0.1 to 0.5 micron, with some unmyelinated axons of up to 0.7 micron diameter. Myelinated fibers also displayed a range of axonal diameters, with axons generally 0.3-1.0 micron. The overlap in diameters, between unmyelinated and myelinated fibers, permitted a comparison of membrane structure in myelinated and unmyelinated axons of the same diameter. Small (less than 0.5 micron) diameter unmyelinated axons exhibited a moderate density (approximately 700/micron2) of P-face intramembranous particles (IMPs), while large (greater than or equal to 0.5 micron) caliber unmyelinated axons displayed a significantly greater P-face IMP density (approximately 1100/micron2). Internodal membrane of both small (less than 0.5 micron) and large (greater than or equal to 0.5 micron) diameter myelinated fibers exhibited densities of P-face particles (approximately 1400/micron2) that were similar to each other, but significantly different from unmyelinated fibers. These results demonstrate that there are differences in membrane structure between unmyelinated and myelinated axons of similar diameter. These findings also demonstrate that membrane structure of unmyelinated axons is not invariant for all unmyelinated fibers within a given CNS tract but, on the contrary, is related to diameter.     

Reexamination of the dorsal root projection to the spinal dorsal horn including observations on the differential termination of coarse and fine fibers.

Primary afferent fibers in the lumbar, sacral, and caudal spinal segments of several mammals (rat, cat, monkey) were stained by applying horseradish peroxidase to the proximal part of cut dorsal rootlets and reacting the tissue histochemically after several hours of survival. The stained fibers' pattern of termination in the dorsal horn was similar in all three species, with many bouton-like enlargements in the ipsilateral marginal zone, substantia gelatinosa, and nucleus proprius, as well as a few projections at each level to the dorsal commissure and contralaterally to the ventral border of the nucleus proprius. Partial lesions of dorsal rootlets in monkey revealed that the thin fibers comprising the lateral division end principally in the marginal zone and substantial gelatinosa, while the thick fibers of the medial division terminate in the nucleus proprius and deeper regions, contributing little to the substantia gelatinosa and marginal zone. On the basis of the termination patterns observed for whole and partly sectioned rootlets, the superficial dorsal horn can be divided into at least four regions. (1) The marginal zone (lamina I of cat) appears to receive terminations from intermediate (smaller myelinated) fibers; (2) the outer substantia gelatinosa (outer lamina II) receives many terminations from the very finest afferent fibers; (3) the inner substantia gelatinosa (inner lamina II) receives endings from some of the finest fibers and also from intermediate (smaller myelinated) fibers; and (4) the superficial part of the nucleus proprius (lamina III) receives endings from intermediate and large diameter dorsal root fibers.     

GABA and Glycine in Synaptic Glomeruli of the Rat Spinal Dorsal Horn

The superficial dorsal horn of rat spinal cord contains two types of synaptic glomerulus, which are centred around the terminals of unmyelinated and myelinated primary afferents respectively. Both types of glomerulus contain GABAergic axons and dendrites, which are thought to originate from local inhibitory interneurons. Some of the dendrites contain synaptic vesicles, and may be presynaptic to the central axon at dendroaxonic synapses. In order to determine whether GABAergic structures in the two types of glomerulus are derived from different populations of interneurons, a quantitative post-embedding immunogold study of GABA- and glycine-immunoreactivity in rat dorsal horn was performed. In type I glomeruli, all of the peripheral axons and most vesicle-containing dendrites were GABA-immunoreactive, but only one of 32 axons and none of the vesicle-containing dendrites was glycine-immunoreactive. In contrast, most of the peripheral axons and some of the vesicle-containing dendrites in type II glomeruli possessed both GABA- and glycine-immunoreactivity. This strongly suggests that different types of inhibitory interneuron in the dorsal horn are associated with the two types of glomerulus. It is therefore likely that different populations of interneurons mediate presynaptic inhibition of unmyelinated and myelinated primary afferents.     

Structural changes in kittens' ventral and dorsal roots L7 after early postnatal sciatic nerve transection

The number and size distribution of myelinated and unmyelinated axons were studied in spinal roots L7 of 19 kittens, 8 to 200 days after early postnatal left sciatic nerve transection. Ventral and dorsal roots on the side of transection were compared with corresponding contralateral roots. Three normal kittens were used as additional controls. On the control side the proportion of unmyelinated ventral root axons increased from about 15 to 30% between 3 and 7 months postnatally. In the ventral roots on the lesion side there was a loss of myelinated axons of all sizes (total loss 15 to 25%). The loss seemed to be somewhat greater in the gamma population. The number of unmyelinated ventral root axons increased markedly through sprouting. This increase was similar at different root levels. The persistence of such axonal sprouts in the proximal stump after ventral root division in one kitten indicated that they originate proximally in the ventral root or within the central nervous system. The dorsal roots on the lesion side showed a 30% deficit of both myelinated and unmyelinated axons. Signs of axonal sprouting were not observed. Both in ventral and dorsal roots the size spectra of myelinated axons were markedly shifted to the left on the lesion side due to a growth retardation of larger axons. With respect to the unmyelinated axons the size distribution was expanded toward larger sizes in the ventral roots and remained largely unaltered in the dorsal roots.     

Axonal bifurcation in the dorsal root ganglion of the cat: a light and electron microscopic study

Bifurcation of the axon of unipolar neurons in the dorsal root ganglion of the cat has been studied by light and electron microscopy. Osmic acid, Golgi and silver preparations have revealed two types of bifurcation: (1) of myelinated fibers characterized by constriction at the nodal region with peripheral and central branches of equal diameter; (2) of small fibers characterized by a broad triangular expansion at the junctional region with a much thicker peripheral as compared to the central branch. These differences in bifurcation of unmyelinated and myelinated axons can be related to the velocity of conduction in the peripheral nerve and dorsal roots. The ultrastructure of the nodal region at the bifurcation resembles the node of Ranvier of a peripheral nerve fiber. The node of Ranvier at the bifurcation consists of three cells of Schwann and adjacent cells are closely apposed and interdigitated. Neurofilaments and microtubules are prominent structures within the axoplasm at the nodal regions. They group into two streams as the unipolar process bifurcates, entering the peripheral and central branch respectively. At the junctional region within the axoplasm, numerous mitochondria and scattered multivesicular bodies are always observed.     

Vasoactive intestinal polypeptide and substance P in primary afferent pathways to the sacral spinal cord of the cat

An analysis of vasoactive intestinal polypeptide immunoreactivity (VIP-IR) and substance P-IR in the cat spinal cord has revealed marked differences in the distribution of the two peptides. While substance P-IR was located at all levels of the cord, VIP-IR was most prominent in the sacral segments in Lissauer's tract and lamina I on the lateral edge of the dorsal horn. VIP-IR was also present in the sacral cord in (1) laminae V, VII, and X, (2) a thin band on the medial side of the dorsal horn, (3) the dorsal commissure, (4) the lateral band of the sacral parasympathetic nucleus, and (5) in a few animals in Onuf's nucleus. In other segments of the spinal cord VIP-IR was much less prominent but was present in Lissauer's tract and laminae I, II, and X. Substance P-IR was more uniformly distributed at all segmental levels in laminae I-III, V, VII, and X and in the dorsal commissure. In ventrolateral lamina I of the sacral spinal cord both VIP-IR and substance P-IR exhibited a distinctive periodic pattern in the rostrocaudal axis. The peptides were associated with bundles of dorsoventrally oriented axons and varicosities spaced at approximately 210-micron intervals center to center along the length of the spinal cord. The bundles in lamina I continued into lamina V where they further divided into smaller bundles that extended medially through laminae V and VII. The most prominent bundles of VIP axons passed ventrally from lateral laminae V and VII to enter lamina X and the ventral part of the dorsal gray commissure. On the other hand the majority of substance P axons in lamina V turned dorsally to join with axons on the medial side of the dorsal horn and to pass into the dorsal part of the dorsal gray commissure. Rostrocaudal VIP axons were present not only in Lissauer's tract but also in dorsolateral lamina I, in the lateral funiculus and in the ependymal cell layer of the central canal. Following unilateral transection of the sacral dorsal roots (2 weeks-22 months) the density of VIP axons and terminals was markedly reduced in ipsilateral Lissauer's tract and lateral laminae I and V; however, no change was detected in lamina X. Sacral deafferentation reduced substance P-IR in the dorsal gray commissure and in lateral laminae I and V.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of CaMKIIα in rat primary sensory neurons: increase in inflammation

This study investigates Ca(2+)/calmodulin kinase IIalpha (CaMKIIalpha) in primary sensory neurons. Immunohistochemical staining with a CaMKIIalpha antibody demonstrates 28% of dorsal root ganglion (DRG) cells are positively stained and have a diameter of 27 +/- 2.4 microm (mean +/- S.D.). Placement of tight ligatures around the sciatic nerve demonstrates a build up of immunoreaction product proximal to the ligatures indicating that CaMKIIalpha is transported into the peripheral processes of DRG cells. Immunostaining of lumbar dorsal roots at the electron microscopic level demonstrates reaction product in 15.4 +/- 2.1% of unmyelinated and 2.4 +/- 1.0% of myelinated axons, indicating that CaMKIIalpha is transported into the central processes of DRG cells. Electron microscopic analysis of normal digital nerves demonstrates CaMKIIalpha labeling in 3.3 +/- 0.3% of unmyelinated and 2.0 +/- 1.1% of myelinated cutaneous axons. These percentages increase significantly to 14.1 +/- 2.3% for unmyelinated and 5.1 +/- 1.4% for myelinated axons 48 h after complete Freund's adjuvant-induced inflammation of the hindpaw. The data indicate that CaMKIIalpha is present in small diameter primary sensory neurons, that it is transported into the peripheral and central processes of these cells and may play a role in processing noxious input, particularly in the inflamed state.     

Analysis of calcitonin gene-related peptide-like immunoreactivity in the cat dorsal spinal cord and dorsal root ganglia provide evidence for a multisegmental projection of nociceptive C-fiber primary afferents.

Recent studies have suggested that calcitonin gene-related peptide (CGRP) can be used as a marker for a subpopulation of nociceptive primary afferents. Consequently, CGRP-immunoreactive (CGRP-IR) primary afferents have been reported to project many segments rostral to their segment of entry and to send collaterals into the superficial and deep laminae of the dorsal horn. This study reports that some CGRP-IR primary afferents of sacral origin project rostral through the ipsilateral lumbar enlargement in the cat. The ultrastructure of these multisegmentally projecting primary afferent axons and terminals identified in a partially denervated cat was examined and compared to the ultrastructure of CGRP-IR afferents from an intact cat. Retrograde transport of wheatgerm agglutinin-colloidal gold injected into the cat L4 spinal cord resulted in labeling of primary afferent cell bodies in the ipsilateral L4-S1 dorsal root ganglia (DRG). Analysis of every fourth section through the ipsilateral S1 DRG revealed as many as 1,000 retrogradely labeled neuronal cell bodies. One third of these cell bodies were double labeled for CGRP-like immunoreactivity. The number of single- and double-labeled cells increased in ganglia closer to the injection site (L4-L7). At the ultrastructural level, in the lumbosacral dorsal spinal cord of a normal cat, most CGRP-IR axons were unmyelinated, while the rest were small myelinated axons. In both the superficial dorsal horn and lamina V, CGRP-IR varicosities were dome shaped, scallop shaped, or elongated. The CGRP-IR varicosities contained small agranular vesicles and frequently a few dense core vesicles. These labeled varicosities formed asymmetric synapses on unlabeled dendritic spines, shafts, or neuronal somata. One cat received multiple unilateral dorsal rhizotomies (S1-L4) and an ipsilateral hemisection (mid L4). CGRP-IR axons and terminals were found within each of the rhizotomized segments, although their density was greatly reduced compared to that in the intact animals. In Lissauer's tract the majority (greater than 90%) of CGRP-IR fibers were unmyelinated. In laminae I and V, the remaining CGRP-IR varicosities were mainly the dome-shaped varicosities morphologically similar to those observed in the normal spinal cords. They contained small agranular vesicles and a few dense core vesicles and formed asymmetric synapses on unlabeled dendritic shafts and spines. These data demonstrate that unmyelinated, presumably C-fiber nociceptive primary afferents and some small myelinated A-delta nociceptive primary afferents of sacral origin project rostral through the cat lumbar enlargement and make synaptic connections in both the superficial and deep laminae of the cat dorsal spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of CaMKIIalpha in rat primary sensory neurons: increase in inflammation

This study investigates Ca(2+)/calmodulin kinase IIalpha (CaMKIIalpha) in primary sensory neurons. Immunohistochemical staining with a CaMKIIalpha antibody demonstrates 28% of dorsal root ganglion (DRG) cells are positively stained and have a diameter of 27 +/- 2.4 microm (mean +/- S.D.). Placement of tight ligatures around the sciatic nerve demonstrates a build up of immunoreaction product proximal to the ligatures indicating that CaMKIIalpha is transported into the peripheral processes of DRG cells. Immunostaining of lumbar dorsal roots at the electron microscopic level demonstrates reaction product in 15.4 +/- 2.1% of unmyelinated and 2.4 +/- 1.0% of myelinated axons, indicating that CaMKIIalpha is transported into the central processes of DRG cells. Electron microscopic analysis of normal digital nerves demonstrates CaMKIIalpha labeling in 3.3 +/- 0.3% of unmyelinated and 2.0 +/- 1.1% of myelinated cutaneous axons. These percentages increase significantly to 14.1 +/- 2.3% for unmyelinated and 5.1 +/- 1.4% for myelinated axons 48 h after complete Freund's adjuvant-induced inflammation of the hindpaw. The data indicate that CaMKIIalpha is present in small diameter primary sensory neurons, that it is transported into the peripheral and central processes of these cells and may play a role in processing noxious input, particularly in the inflamed state.