Overexpression of nerve growth factor in epidermis disrupts the distribution and properties of sympathetic innervation in footpads

Sympathetic and sensory neurons form distinct axonal arborizations in several peripheral targets. The developmental mechanisms responsible for partitioning sympathetic and sensory axons between potential target tissues are poorly understood. We have used rodent footpads to study this process because three populations of peripheral axons innervate topographically segregated targets in the footpad; cholinergic sympathetic axons innervate sweat glands, noradrenergic sympathetic axons innervate blood vessels, and sensory axons form a plexus at the epidermal/dermal junction. To examine how nerve growth factor (NGF), a trophic and survival factor for sympathetic and some sensory neurons, may contribute to the generation of the patterned distribution of axons among targets, we studied transgenic mice (K14-NGF mice) in which NGF expression was significantly increased in the epidermis. Whereas the temporal sequence in which sensory and sympathetic fibers arrived in the footpad was not affected, the normal partitioning of axons between target tissues was disrupted. The two sympathetic targets in footpads, sweat glands, and blood vessels lacked substantial innervation and instead a dense plexus of catecholaminergic sympathetic fibers was found commingled with sensory fibers in the dermis. Those sympathetic fibers present in sweat glands expressed an abnormal dual catecholaminergic/cholinergic phenotype. Our findings indicate that overexpression of NGF in skin interferes with the segregation of sensory and sympathetic axonal arbors and suggests a role for target-derived NGF in the establishment of distinct axonal territories. Our data also suggest that by determining where axon arbors form, NGF can indirectly influence the phenotypic properties of sympathetic neurons. J. Comp. Neurol. 393:231–243, 1998. © 1998 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.     

Neonatal 6-hydroxydopamine treatment eliminates cholinergic sympathetic innervation and induces sensory sprouting in rat sweat glands

Previous studies of the development of cholinergic sympathetic innervation of sweat glands in rat footpads suggested that these terminals initially exhibit noradrenergic properties which are lost as the glands and their innervation mature. We have treated neonatal and adult rats with 6-hydroxydopamine (6-OHDA), a toxic congener of norepinephrine, and compared its effects on the cholinergic sympathetic innervation of sweat glands and the noradrenergic sympathetic innervation of the iris, salivary gland, and blood vessels. As reported by others, 6-OHDA treatment of neonates caused the destruction of noradrenergic fibers in the iris and salivary gland but did not affect other fibers projecting to these targets that stain for acetylcholinesterase (AChE). We found that 6-OHDA treatment of neonatal animals also caused the destruction of the sympathetic axons in immature sweat glands that possess catecholamine histofluorescence and tyrosine-hydroxylase-like immunoreactivity. Furthermore, when such animals were examined as adults, we found no AChE staining, vasoactive intestinal peptide (VIP)-like immunoreactivity, or characteristic sympathetic axonal varicosities. However, the denervated glands were invested by a plexus of sensory axons, some of which exhibited substance P-like immunoreactivity (SP-IR). An increase in the number of SP-IR fibers also occurred in the sympathetically denervated irides of these animals. Chronic treatment of neonates with guanethidine, another adrenergic sympathetic neurotoxin, resulted in similar loss of cholinergic sweat gland innervation. Treatment of adults rats with doses of 6-OHDA identical to those used to treat neonates caused the loss of noradrenergic fibers from the iris, salivary gland, and many blood vessels but did not noticeably affect AChE and VIP staining or axonal ultrastructure in the sweat glands. However, treatment with higher doses of 6-OHDA did cause significant axonal degeneration. The response of the sympathetic innervation of developing but not mature sweat glands to 6-OHDA provides evidence for a transition from noradrenergic to cholinergic phenotype during the development of sympathetic neurons in vivo similar to the transition observed in cell culture. The sprouting of sensory axons may be caused by NGF-like trophic influences present in some sympathetically denervated tissues.     

Coexistence of calcitonin gene-related peptide and vasoactive intestinal peptide in cholinergic sympathetic innervation of rat sweat glands

Immunoreactivity for calcitonin gene-related peptide (CGRP) has been localized with indirect immunofluorescence techniques in the cholinergic sympathetic fibers that innervate eccrine sweat glands in the rat. This innervation also contains vasoactive intestinal peptide-like immunoreactivity (VIP-IR). A small proportion of principal neurons in stellate and lumbar sympathetic ganglia which provide innervation to the sweat glands contain detectable CGRP-immunoreactivity. The CGRP-IR neurons are immunoreactive for VIP; however, many VIP-IR neurons in these ganglia do not contain detectable levels of CGRP-IR.     

Immunohistochemical study of skin reinnervation by regenerative axons

The time sequence of sensory and sudomotor nerve regeneration to the mouse footpad was studied between one and seven weeks after crush or section of the sciatic nerve. Protein gene product 9.5, vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide were localized in thick sections by using indirect immunofluorescence techniques and imaged by confocal microscopy. Nerve regeneration was visually assessed in all nerves and quantified in sweat glands. After denervation, protein gene product 9.5 immunoreactivity remained as dim fluorescence within thick fibers of dermal nerve trunks, whereas thin nerve fibers to sweat glands and to epidermis disappeared. By 14 days postcrush and 35 days postsection, the first protein gene product 9.5 immunoreactive regenerating axons appeared in large nerve trunks, quickly extending to epidermis and sweat glands. Reinnervation of Meissner's corpuscles occurred nearly simultaneous with return of epidermal free nerve endings and sudomotor network. Calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P immunoreactivity disappeared completely one week after denervation, then reappeared at 17–18 days postcrush and 35 days postsection. Fewer nerve fibers were immunoreactive to these peptides than to protein gene product 9.5. The overall density of reinnervation, although reduced, more closely resembled normal in the sweat glands and Meissner's corpuscles than in the epidermis. Reinnervation was more successful after crush than after nerve section. The time course for functional return of sweating paralleled the return of protein gene product 9.5 immunoreactivity, whereas reappearance of vasoactive intestinal peptide was delayed by several days. J. Comp. Neurol. 380:164–174, 1997. © 1997 Wiley-Liss, Inc.     

Innervation of cutaneous structures in the mouse hind paw: A confocal microscopy immunohistochemical study

The normal innervation of structures in mouse foot pads was investigated with immunohistochemistry and confocal microscopy. Nerves were visualized by incubating Zamboni fixed, thick, frozen sections with antibodies to protein gene product 9.5 (PGP 9.5), vasoactive intestinal peptide, substance P, calcitonin gene-related peptide, and protein zero. The antibodies were localized using cyanine 3.18 labeled anti-rabbit gamma globulin. PGP 9.5 immunolocalization showed dense nerve bundles at the base of the foot pad with branches to larger blood vessels, sweat glands and epidermis. Sweat gland tubules were surrounded by numerous sudomotor axons; single fibers accompanied the sweat duct toward the skin's surface. Nerve bundles containing myelinated and unmyelinated axons ran through and around the centrally located sweat gland cluster to end in free nerve endings and Meissner's-like corpuscles at the apex of the foot pad. Other bundles running parallel to the epidermis gave arcuate branches that supplied epidermis on the sides of the pads with a rich nerve network, principally with free nerve endings that often reached the most superficial cell layers of epidermis. Calcitonin gene-related peptide-immunoreactive (-ir) nerves were distributed to dermis and epidermis in lower density than PGP 9.5-ir fibers. Substance P-ir fibers were less numerous; most terminated as free endings in deeper layers of epidermis. Vasoactive intestinal peptide-ir nerves almost exclusively innervated sweat glands, ducts and blood vessels, but not epidermis. The mouse hind paw has potential to serve as a model system for investigations of functional and morphological changes that affect peripheral and au tonomic nerves under diverse experimental conditions. © 1995 Wiley-Liss, Inc.     

Developmental expression of the high affinity choline transporter in cholinergic sympathetic neurons

Choline uptake by the high affinity choline transporter (CHT) is the rate-limiting step in acetylcholine synthesis. Induction of CHT is therefore a critical step in cholinergic differentiation, and we examined the developmental expression of CHT in cholinergic sympathetic neurons that innervate rodent sweat glands. During postnatal development the earliest sympathetic axons in the rear footpads are noradrenergic, containing intense tyrosine hydroxylase immunoreactivity and lacking CHT-immunoreactivity (CHT-IR). By postnatal day 7 (P7) in mouse, and P10 in rat, weak CHT-IR appeared in axons associated with the sweat gland anlagen. CHT staining intensity increased during the following weeks in conjunction with plexus arborization and gland maturation. The pattern of CHT-immunoreactivity (CHT-IR) in the sweat gland innervation was similar to staining for the vesicular acetylcholine transporter and vasoactive intestinal peptide. Immunoblots of tissue from sympathectomized rats confirmed that most of the CHT in footpad was contained in sympathetic neurons. Although CHT expression has been reported in noradrenergic sympathetic neurons of the superior cervical ganglion, these data indicate that in the sympathetic neurons projecting to sweat glands CHT is present at detectable levels only after association with the glands.     

Sympathetic cholinergic target innervation requires GDNF family receptor GFR alpha 2

Many cholinergic parasympathetic and enteric neurons require neurturin signaling through GDNF family receptor GFRalpha2 for target innervation. Since a distinct minority of sympathetic neurons are cholinergic, we examined whether GFRalpha2 is important for their development. We detected GFRalpha2 in neonatal sympathetic cholinergic neurons and neurturin mRNA in their target tissues, sweat glands in footpads, and periosteum. Lack of GFRalpha2 in mice did not affect the number of sympathetic cholinergic neurons, but their soma size was decreased in comparison to wild types. In adult and in 3-week-old GFRalpha2 knockout mice, the density of sympathetic cholinergic innervation was reduced by 50-70% in the sweat glands, and was completely absent in the periosteum. Sympathetic noradrenergic innervation of blood vessels in the footpads was unchanged. The density of sympathetic axons in sweat glands was unaffected at postnatal day P4 reflecting successful growth into the target area. Our results indicate that the cholinergic subpopulation of sympathetic neurons requires GFRalpha2 signaling for soma size and for growth or maintenance of target innervation. Thus, neurturin may be a general target-derived innervation factor for postganglionic cholinergic neurons in all parts of the autonomic nervous system.     

Developmental changes in the transmitter properties of sympathetic neurons that innervate the periosteum.

During the development of sweat gland innervation, interactions with the target tissue induce a change from noradrenergic to cholinergic and peptidergic properties. To determine whether the change in neurotransmitter properties that occurs in the sweat gland innervation occurs more generally in sympathetic neurons, we identified a new target of cholinergic sympathetic neurons in rat, the periosteum, which is the connective tissue covering of bone, and characterized the development of periosteal innervation of the sternum. During development, sympathetic axons grow from thoracic sympathetic ganglia along rib periosteum to reach the sternum. All sympathetic axons displayed catecholaminergic properties when they reached the sternum, but these properties subsequently disappeared. Many axons lacked detectable immunoreactivities for vesicular acetylcholine transporter and vasoactive intestinal peptide when they reached the sternum and acquired them after arrival. To determine whether periosteum could direct changes in the neurotransmitter properties of sympathetic neurons that innervate it, we transplanted periosteum to the hairy skin, a noradrenergic sympathetic target. We found that the sympathetic innervation of the transplant underwent a noradrenergic to cholinergic and peptidergic change. These results suggest that periosteum, in addition to sweat glands, regulates the neurotransmitter properties of the sympathetic neurons that innervate it.     

Parasympathetic nerve fibers invade the upper dermis following sensory denervation of the rat lower lip skin

The sympathetic division of the autonomic nervous system is known to play a role in the genesis of neuropathic pain. In the skin of the rat lower lip (hairy skin), sympathetic and parasympathetic fibers normally innervate the same blood vessels in the lower dermis but do not occur in the upper dermis. However, we have shown that sympathetic fiber migration into the upper dermis occurs following mental nerve lesions (Ruocco et al. [2000] J. Comp. Neurol. 422:287-296). As sensory denervation has a dramatic effect on sympathetic fiber innervation patterns in the rat lower lip skin, we decided to investigate the possible changes in the other autonomic fiber type in the skin-the parasympathetic fiber. Sensory denervation of the rat lower lip was achieved by bilateral transection of the mental nerve, and animals were allowed to recover for 1-8 weeks. Lower lip tissue was processed for double-labeling light microscopic immunocytochemistry (ICC), using antibodies against substance P (SP), which labels a subpopulation of peptidergic sensory fibers, and against the vesicular acetycholine transporter (VAChT), as a marker for parasympathetic fibers. In sham-operated rats, SP-immunoreactive (IR) sensory fibers were found in the epidermis and upper and lower dermal regions, whereas VAChT-IR fibers were confined to the lower dermis. Mental nerve lesions induced the gradual disappearance of SP-IR fibers from all skin layers accompanied by the progressive migration of VAChT-IR fibers into the upper dermis. Cholinergic fiber migration was evident by the second week post surgery, and the ectopic innervation of the upper dermis by these fibers persisted even at the last time point studied (8 weeks) when SP-IR fibers have completely regrown. VAChT-IR fibers were observed in the upper dermis, well above the opening of the sebaceous glands into the hair follicles. These results show that considerable changes occur in the innervation patterns of parasympathetic fibers following mental nerve lesions.     

Development of choline acetyltransferase (CAT) in the sympathetic innervation of rat sweat glands

It has been postulated that the developing sympathetic innervation of rat eccrine sweat glands changes from adrenergic to cholinergic under the influence of its target. In agreement with previous evidence that the sympathetic innervation of adult rat sweat glands is cholinergic, we found that choline acetyltransferase (CAT)-immunoreactive nerve fibers are present in adult glands, and that gland-rich chunks of adult footpads contain CAT enzyme activity. We were therefore interested in determining when CAT activity is first expressed in the developing gland innervation. Low levels of acetylating activity were observed in rat footpads as early as postnatal day 4, when sympathetic fibers first contact the glands. A greater than fourfold increase in CAT specific activity occurred between postnatal days 11 and 21. Neonatal treatment of rats with the adrenergic neurotoxin 6-hydroxydopamine (6-OHDA) eliminated most of the CAT activity in 14 and 19 d footpads. In contrast, the acetylating activity observed prior to day 11 was unaffected by neonatal 6-OHDA treatment, and only slightly reduced by the selective CAT inhibitor, naphthylvinylpyridine. These results indicate that the sympathetic fibers that innervate rat sweat glands do not acquire detectable levels of CAT activity until a full week after they contact the glands.     

Peripheral patterns of calcitonin-gene-related peptide general somatic sensory innervation: cutaneous and deep terminations

The distribution of calcitonin-gene-related peptide (CGRP) immunoreactivity (IR) was studied in peripheral tissues of rats. The ganglionic origin, somatosensory nature, and anatomic relations of this thin-axon population were evaluated with particular emphasis on possible nociceptive roles. In animals untreated with colchicine, CGRP-IR is found in a vast proportion of small- and medium-diameter sensory ganglion cells that give rise to numerous thinly myelinated and unmyelinated axons that display CGRP-IR throughout the body. The integumentary innervation consists, in part, of an extensive subpapillary network largely traced to dermal blood vessels, sweat glands, and "free" nerve endings, some of which are found within regions containing only mast cells, fibroblasts, and collagen. Dermal papillae contain CGRP-IR axons surrounding each vascular loop; other papillary axons end freely or occasionally surround Meissner corpuscles. Intraepithelial axons enter glabrous epidermal pegs, branching and exhibiting terminals throughout the stratum spinosum. A similar pattern is found in hairy skin with additional innervation entering the base and surrounding the lower third of each hair follicle, but apparently not supplying sebaceous glands and arrector pili muscle. Axons innervating nonkeratinized oral epithelium are similar or greater in number and distribution compared to epidermis, often with more extensive branching. The high density of intraepithelial CGRP-IR innervation does not appear to correlate with the sensitive mechanoreceptor-based increase in spatial sensory discriminative capacities in the distal portions of the limb. In deep somatic tissues, CGRP-IR is principally related to vasculature and motor end plates of striated muscle, but there is an extensive network of thin axons within bone, principally in the periosteum, and focally in joint capsules, but not in relation to muscle spindles or tendon organs. These findings, together with the distribution in cranial tissues described in an accompanying paper (Silverman and Kruger: J. Comp. Neurol. 280:303-330, '89), are considered in the context of a "noceffector" concept incorporating the efferent role of these sensory axons in various tissues. It is suggested that involvement in tissue maintenance and renewal during normal function, as well as following injury, may predominate over the relatively infrequent nociceptive role of this peptidergic sensory system.     

Absent innervation of skin and sweat glands in congenital insensitivity to pain with anhidrosis.

OBJECTIVES: A case of a 10-year-old girl with congenital insensitivity to pain with anhidrosis (CIPA) is reported. METHODS AND RESULTS: Parents referred several hyperpyretic episodes without sweating occurring since birth, and insensitivity to pain, noticed when the child was 2 years old. Her body had many bruises and scars, bone fractures and signs of self-mutilation. Neurological examination was normal except for insensitivity to pain. Her IQ was 52. Electrical and tactile sensory nerve conduction velocities were normal. The patient was unable to detect thermal stimuli. Histamine injection evoked a wheal but not a flare; pilocarpine by iontophoresis did not induce sweat. Microneurography showed neural activity from A-beta sensory fibers while nociceptive and skin sympathetic C fiber nerve activity was absent. No small myelinated fibers and very rare unmyelinated fibers were found in the sural nerve. Immunohistochemistry showed a lack of nerve fibers in the epidermis and only few hypotrophic and uninnervated sweat glands in the dermis. CONCLUSIONS: The lack of innervation of the skin (C and A-delta fibers) appears to be the morphological basis of insensitivity to pain and anhidrosis, and is consistent with the loss of unmyelinated and small myelinated fibers in the sural nerve biopsy.     

Encapsulated nerve end-organs in the rabbit, mouse, sheep and man

There is a system of highly specialized blood vessels around each sensory hair follicle in the rabbit. The system includes two arteriovenous shunts and a cylinder of dilative blood vessels enclosed in a large bulbous capsule of connective tissue. The shunts, dilative blood vessels and capsule surround two cylinders of nerve fibers; together these structures constitute the largest neurovascular end-organ in skin. The end-organ is similar to the glomus-Pacinian corpuscle complex in the hairless digital skin of man. The blood vessel cylinder and arteriovenous shunts in rabbit skin connect with a system of dilative blood vessels that are enclosed in another capsule of connective tissue under a small thick pad of sensory epidermis, beihnd the sensory hair follicle. The pad of epidermis contains expanded nerve fibers (Merkel's discs) that end near a cluster of Merkel (tactile) cells. In some regions of skin, each area of sensory epidermis is associated with the orifice of a sweat gland duct. In hairy skin of the rabbit, mouse and sheep, the areas of sensory epidermis, Merkel cells and Merkel's discs are distributed in parallel chains or in whorls and loops. Similarly, in the hairless skin of man the sensory epidermis, clusters of Merkel's discs and sweat duct orifices are distributed together in rows, whorls and loops that form the finger and palmer prints. In some regions of hairy human skin the same components occur together in small circular whorls called the Haarscheiben or Rosetten.     

Sympathetic sprouting and changes in nociceptive sensory innervation in the glabrous skin of the rat hind paw following partial peripheral nerve injury

Previous studies have suggested that sympathetic sprouting in the periphery may contribute to the development and persistence of sympathetically maintained pain in animal models of neuropathic pain. In the present study, we examined changes in the cutaneous innervation in rats with a chronic constriction injury to the sciatic nerve. At several periods postinjury, hind paw skin was harvested and processed by using a monoclonal antibody against dopamine-beta-hydroxylase to detect sympathetic fibers and a polyclonal antibody against calcitonin gene-related peptide to identify peptidergic sensory fibers. We observed migration and branching of sympathetic fibers into the upper dermis of the hind paw skin, where they were normally absent. This migration was first detected at 2 weeks, peaked at 4-6 weeks, and lasted for at least 20 weeks postlesion. At 8 weeks postlesion, there was a dramatic increase in the density of peptidergic fibers in the upper dermis. Quantification revealed that densities of peptidergic fibers 8 weeks postlesion were significantly above levels in sham animals. The ectopic sympathetic fibers did not innervate blood vessels but formed a novel association and wrapped around sprouted peptidergic nociceptive fibers. Our data show a long-term sympathetic and sensory innervation change in the rat hind paw skin after the chronic constriction injury. This novel fiber arrangement after nerve lesion may play an important role in the development and persistence of sympathetically maintained neuropathic pain after partial nerve lesions.     

Neurophysiologic and histopathologic studies in a case of congenital sensory neuropathy with anhidrosis]

A three year-old boy with congenital sensory neuropathy with anhidrosis (CSNA) was described. Sural nerve biopsy specimens revealed an almost complete absence of unmyelinated fibers and a marked decrease of the density of small myelinated fibers with preservation of the density of large myelinated fibers. No evidence of active degeneration of unmyelinated or myelinated fibers was found. Skin biopsy specimens revealed the absence of nerve terminals and fibers innervating sweat glands, although sweat glands seemed to be apparently normal in their morphological findings. Therefore, it was concluded that the absence of pain and temperature sensations with preservation of touch sensation in our patient was compatible with the morphometric findings of nerve fibers of the sural nerve described. Similarly anhidrosis was concluded to be well explained by the absence of the innervation of sweat glands and the vessels around them. On the other hand, electrophysiologic studies, such as motor and sensory nerve conduction, short latency somatosensory evoked potential and auditory brainstem response, in which the function of the large myelinated fibers is presumably tested, were all normal. Therefore, the structure and function of such large myelinated fibers were spared in this case. From clinical viewpoints, electrophysiologic studies described above are useful to differentiate CSNA from other types of congenital sensory neuropathies, in which large myelinated fibers are affected.     

A case of hereditary sensory autonomic neuropathy type II with late onset]

We reported a 22-year-old man with hereditary sensory autonomic neuropathy (HSAN) type II. His initial neurological symptom at the age of 18 years was hypoesthesia on the feet and legs. In spite of late onset and absence of multilating acropathy, we diagnosed this case as HSAN type II because of an absence of sensory nerve action potentials with normal motor nerve conduction velocities and of a total loss of myelinated fibers with a decrease of unmyelinated fibers in the sural nerve. The sweating induced by iontophoretic pilocarpine stimulation was decreased on the dorsum of the foot. In addition, the morphometric analysis of sudomotor nerves around sweat glands showed a decrease of nerve terminals and unmyelinated axons. Because decrease or loss of sweating is one of the cardinal signs in HSAN type II, the quantitative sweating test and morphometric evaluation of the innervation of sweat glands are important for the proof of the autonomic signs.     

Sympathetic co-activation of skin blood vessels and sweat glands

Abstract. Skin blood vessels and sweat glands are both innervated by sympathetic C fibers. We investigated whether during diverse respiratory maneuvers the vasomotor responses (VRs) and the sympathetic skin responses (SSRs) were frequently or occasionally co-activated. We simultaneously recorded the amplitude of the vasomotor responses and the sympathetic skin responses, the ECG and the respiratory movements in 30 healthy subjects during natural breathing at rest, rhythmic respirations at 6 per minute, sudden deep inspiration and Valsalva maneuver. We found: 1) The SSR habituates with all respiratory maneuvers whereas the VRs do not habituate. 2) There was slight co-activation between the SSRs and VRs during natural default breathing (56 percent). 3) During rhythmic breathing at 6 per minute the VRs and the SSRs were frequently co-activated (97 percent). The SSR appeared at the end of the inspiration coinciding with the end of the decreased blood flow. However the SSR habituated after few rhythmic respirations. 4) During sudden deep inspiration one hundred percent of co-activations were between the initial phase of the VRs and the SSR. The SSR is large in amplitude and longer in duration than during rhythmic breathing. 5) During the Valsalva maneuver there was a strong co-activation (100 percent) particularly during the phases II and III that are characterized by vaso-constriction but also during phase IV. The SSR is the longest of duration in all of the maneuvers. The sympathetic innervation to the sweat glands of the palm of the hand and to the skin blood vessels of the fingertips is differentiated. Under normothermic conditions sudden deep inspiration and Valsalva maneuver induced a large sympathetic simultaneous outflow to the skin blood vessels and sweat glands. The simultaneous recording of skin blood flow and the SSRs provides a more complete assessment of the sympathetic outflow to the skin than either one alone.     

Cutaneous and sympathetic denervation in neonatal rats with a mutation in the delta subunit of the cytosolic chaperonin-containing t-complex peptide-1 gene

The mutilated-foot rat (mf rat) is an autosomal recessive mutant with characteristic digit deformities in adult animals, and this phenotype mimics many aspects of human sensory neuropathy. The genetics of mf rats was recently elucidated. To understand whether the genotype is responsible for cutaneous denervation before clinically overt mutilation in adult mf rats, we investigated skin innervation in postnatal day 7 (P7) mf rats and compared the patterns with P7 wild-type rats. The mf rat carries a G-->A mutation in the gene encoding the delta subunit of the cytosolic chaperonin-containing t-complex peptide-1 (Cct4). In the footpad skin of P7 mf rats, there was a >90% loss of epidermal nerves (0.7-7.9% of P7 wild-type rats) as indicated by neuronal markers including protein gene product 9.5 (PGP 9.5), growth-associated protein 43 (GAP43), calcitonin gene-related peptide (CGRP), and substance P (SP). The epidermis of hairy skin in hind feet was completely denervated in mf rats as well. Compared with an approximately 80% reduction in the size of dermal nerve fascicles and a parallel loss of nerve fibers, the nearly complete absence of epidermal innervation suggests further sensory nerve degeneration at the level of nerve terminals in the epidermis. In contrast, the loss of epidermal nerves in the abdominal skin of mf rats was less extensive than that in the footpad skin of mf rats; CGRP (+) and SP (+) fibers were moderately reduced (28.3-56.4% of levels of wild-type rats) with normal amounts of PGP 9.5 (+) and GAP43 (+) nerves. Sympathetic innervation as assessed by tyrosine hydroxylase immunoreactivity was absent from the footpad and abdominal skin of mf rats. In conclusion, there is regional skin denervation with diffuse sympathetic denervation in P7 mf rats. These results suggest that the mutation in Cct4 underlies cutaneous nerve degeneration in mf rats.