Nucleotide signaling and cutaneous mechanisms of pain transduction.

Sensory neurons that innervate the skin provide critical information about physical contact between the organism and the environment, including information about potentially-damaging stimuli that give rise to the sensation of pain. These afferents also contribute to the maintenance of tissue homeostasis, inflammation and wound healing, while sensitization of sensory afferents after injury results in painful hypersensitivity and protective behavior. In contrast to the traditional view of primary afferent terminals as the sole site of sensory transduction, recent reports have lead to the intriguing idea that cells of the skin play an active role in the transduction of sensory stimuli. The search for molecules that transduce different types of sensory stimuli (mechanical, heat, chemical) at the axon terminal has yielded a wide range of potential effectors, many of which are expressed by keratinocytes as well as neurons. Emerging evidence underscores the importance of nucleotide signaling through P2X ionotropic and P2Y metabotropic receptors in pain processing, and implicates nucleotide signaling as a critical form of communication between cells of the skin, immune cells and sensory neurons. It is of great interest to determine whether pathological changes in these mechanisms contribute to chronic pain in human disease states such as complex regional pain syndrome (CRPS). This review discusses recent advances in our understanding of communication mechanisms between cells of the skin and sensory axons in the transduction of sensory input leading to pain.

Development of sensory neurons in the absence of NGF/TrkA signaling in vivo.

The neurotrophin survival dependence of peripheral neurons in vitro is regulated by the proapoptotic BCL-2 homolog BAX. To study peripheral neuron development in the absence of neurotrophin signaling, we have generated mice that are double null for BAX and nerve growth factor (NGF), and BAX and the NGF receptor TrkA. All dorsal root ganglion (DRG) neurons that normally die in the absence of NGF/TrkA signaling survive if BAX is also eliminated. These neurons extend axons through the dorsal roots and collateral branches into the dorsal horn. In contrast, superficial cutaneous innervation is absent. Furthermore, rescued sensory neurons fail to express biochemical markers characteristic of the nociceptive phenotype. These findings establish that NGF/TrkA signaling regulates peripheral target field innervation and is required for the full phenotypic differentiation of sensory neurons.

The Meissner corpuscle revised: a multiafferented mechanoreceptor with nociceptor immunochemical properties.

Meissner corpuscles (MCs) in the glabrous skin of monkey digits have at least three types of innervation as revealed by immunofluorescence. The previously well known Aalphabeta-fiber terminals are closely intertwined with endings from peptidergic C-fibers. These intertwined endings are segregated into zones that alternate with zones containing a third type of ending supplied by nonpeptidergic C-fibers. Although MCs are widely regarded as low-threshold mechanoreceptors, all three types of innervation express immunochemical properties associated with nociception. The peptidergic C-fiber endings have readily detectable levels of immunoreactivity (IR) for calcitonin gene-related peptide (CGRP) and substance P (SP). The Aalphabeta endings have relatively lower levels of IR for CGRP and SP as well as the SP neurokinin 1 receptor and vanilloid-like receptor 1. Both the Aalphabeta and peptidergic C-fiber endings were also labeled with antibodies for different combinations of adrenergic, opioid, and purinergic receptors. The nonpeptidergic C-fiber endings express IR for vanilloid receptor 1, which has also been implicated in nociception. Thus, MCs are multiafferented receptor organs that may have nociceptive capabilities in addition to being low-threshold mechanoreceptors.

Structure, vascularization, and innervation of the mystacial pad of the rat as revealed by the lectin Griffonia simplicifolia.

The mystacial pad of the rat is endowed with rows of vibrissal follicle-sinus complexes (F-SCs) that receive a dense and rich variety of innervation, much of which is C fibers. Each F-SC consists of a follicle at the core of a spindle-shaped, encapsulated vascular sinus. Previous studies have shown that the B subunit of the lectin Griffonia simplicifolia (GSA I-B4) binds selectively to a subset of small neurons in the trigeminal ganglion and to a subset of C fibers preferentially distributed to inner lamina II and outer lamina III of nucleus caudalis in the brainstem trigeminal complex in the rat. These laminae are also a major site of termination for afferents in superficial vibrissal nerves (SVNs) that innervate the upper portion of F-SCs. To determine the peripheral distribution of the afferents that bind GSA I-B4, mystacial pads from rats were prepared for fluorescence microscopy with GSA I-B4 conjugated to rhodamine. At the neck of each F-SC, numerous circumferentially oriented bundles of fine-caliber axonal profiles were labeled in the inner conical body, which receives nearly all of its innervation from the SVNs. A sparse, random distribution of fine-caliber profiles from deep vibrissal nerves was labeled at the level of the cavernous sinus in the deep half of the F-SCs. GSA I-B4 also labeled a variety of nonneural structures. By binding to vascular linings, GSA I-B4 revealed a dense, highly organized capillary system within the mesenchymal sheath that forms the inner lining of the vascular sinuses. Thus each F-SC appears to have a closed capillary system within the open vascular sinus. Trabeculae within the lumen of the cavernous sinus were also revealed to span between the sinus capsule and the mesenchymal sheath only about midway along the length of the follicle instead of the entire deeper half, as was previously believed. in addition, GSA I-B4 bound to the surface of follicular cells preferentially in the superficial half of the F-SCs. Sweat glands within the intervibrissal fur and some cells within sebaceous glands in F-SCs were also labeled as well as their ducts. The potential functional implications of these various features are discussed.

Gradual changes in the structure of the barrels during maturation of the primary somatosensory cortex in the rat.

The maturation of the barrel field in the primary somatosensory cortex was observed in Nissl-stained preparations from rats ranging in age from 12 days to 1.5 years postpartum. Prior to the 20th day, the barrels in the rat resemble those of the mouse and have distinct cell-sparse hollows that are surrounded by cell-dense sides. They span the full thickness of layer IV. Between the 20th and 34th days, the barrels in only the posteromedial part of the barrel field gradually change and the distinction between the hollows and sides is lost throughout all but the deepest part of layer IV. The resulting mature barrels are relatively indistinct and have a uniformly high cell density that extends well into the supragranular layers. In contrast, the barrels in the anterolateral part of the barrel field remain essentially unchanged. The remodeling apparently is not due passively to cortical growth because, by P20, the thicknesses of the cortical layers and the dimensions of the barrels are virtually the same as in the adult. Several mechanisms are considered that may account for the changes. These include a redistribution of the neurons that originally were in barrel sides; a reduction in the neuropil between the neurons that originally were within hollows; and differential growth of layer IV dendrites. The changes in the barrel structure may be related to the differentiation and quantity of innervation in the hairy skin between the vibrissae.

Central projections of primary sensory neurons innervating different parts of the vibrissae follicles and intervibrissal skin on the mystacial pad of the rat.

The cell bodies and central projections of neurons innervating the vibrissae follicles and adjacent skin in the rat were investigated by retrograde and transganglionic transport of HRP. The cell bodies of neurons innervating the vibrissa follicle via the deep vibrissa nerve (DVN) were the largest, followed by those innervating the follicle via the superficial vibrissa nerve (SVN). The smallest cell bodies were those innervating the intervibrissal skin. The DVN neurons terminated centrally as an almost uninterrupted column through the trigeminal sensory nuclear complex. The DVN projections to nucleus caudalis and C1 dorsal horn were entirely restricted to laminae III, IV, and V. Besides the projections to lamina V, the DVN projections were strictly localized somatotopically at all levels replicating the peripheral organization of the vibrissae. The SVNs projected sparsely to midlevels of the main sensory nucleus but not to nuclei oralis and interpolaris. The main SVN projections appeared in laminae I-III of nucleus caudalis. In addition, a small projection to lamina V was observed. The projections to laminae II and III were organized mediolaterally in a similar way as the DVN projections; those to laminae I and V were less restricted. The intervibrissal skin neurons projected sparsely to the caudal main sensory nucleus and to the border between nuclei oralis and interpolaris. The projections to nucleus caudalis were restricted to laminae I-III and V and were organized in a similar way as the SVN projections.

A comparative light microscopic analysis of the sensory innervation of the mystacial pad. II. The common fur between the vibrissae.

The innervation to the common fur between the vibrissae was examined in the hamster, mouse, rat, gerbil, rabbit, guinea pig, and cat. Samples were taken from central locations among the more caudal vibrissae in the mystacial pad and processed with Richardson's variant of the Bielschowsky silver technique or with Winkelmann's silver technique to selectively stain peripheral axons and terminals. Additional samples were taken among the rostral vibrissae in the rat. We found major unpredictable species-related variations in the distribution of receptor types, innervation density, and the quantity of innervation in the skin between neighboring vibrissae. The common fur is composed of numerous larger guard hairs and even more numerous smaller vellus hairs. The guard hairs usually are richly innervated with fully developed piloneural complexes composed primarily of a pallisade of lanceolate endings and a circumferential array of Ruffini and free nerve endings. The vellus hairs are usually innervated by individual or shared free nerve endings. The piloneural complexes in the cat, rat, and mouse are usually complete, whereas those in the other species were usually incomplete and lacked Ruffini endings. There is considerable interspecies variation in the relative quantity of innervation between homologous neighboring vibrissae. The quantity of innervation is related to a combination of receptor completeness, innervation density, and distance between vibrissae. The quantity of intervibrissal fur innervation is by far highest in the cat, relatively high in the rabbit, relatively low in the hamster and caudal mystacial pad of the rat, and lowest in the mouse, gerbil, guinea pig, and rostral mystacial pad of the rat. The differences in the innervation between the cat and the rabbit correlate well with published physiologic data on types of receptor units. Also, barrels are most prominent in species having relatively low quantities of intervibrissal innervation and are less prominent or absent in species having high quantities of intervibrissal innervation.

Successive waves of differentiation of cutaneous afferents in rat mystacial skin.

The present study has traced the sequence of maturation of sensory receptors in the mystacial pad of postnatal rats. At birth the follicle-sinus complexes (F-SC) are well innervated by deep vibrissal nerves although the number of axons entering the sinus is less than that in the adult. The innervation of the F-SC by the conus or superficial vibrissal nerves derived from skin nerves that form the superficial dermal nerve plexus is limited to the Merkel rete ridge collar at birth, and the innervation to the inner conical body is conspicuously absent. The inner conical body innervation begins to appear 3-4 days after birth and rapidly matures over the week. By 3 weeks of age the F-SCs have a mature sensory innervation. At birth small guard hairs are present in the intervibrissal pelage and are associated with scant axons of the superficial dermal nerve plexus, but no mature sensory terminals are present. The sensory innervation of the intervening pelage begins to differentiate during the second week and mature piloneural complexes can be recognized by 3 weeks of age. Innervation to vellus hairs is still developing at 3-4 weeks of age. These maturational changes in peripheral sensory innervation correlate with gradual changes in the structure of barrels in the first somatosensory cortex (SI). Sequential waves of differentiation of sensory receptors appear to be a general feature of neural development.

Sequential differentiation of sensory innervation in the mystacial pad of the ferret.

The mystacial pad of the ferret has an elaborate sensory innervation provided by three types of terminal nerves that arise from the infraorbital branch of the trigeminal nerve. Deep and superficial vibrissal nerves innervate nearly exclusive targets in the large follicle-sinus complexes (F-SCs) at the base of each tactile vibrissa. Dermal plexus nerves innervate the fur between the vibrissae. Each type of nerve provides a similar variety of sensory endings, albeit to different targets. In this study, Winkelmann and Sevier-Munger reduced silver techniques revealed that most of the endings differentiate postnatally in an overlapping sequence like that observed previously in the rat. Afferents from the deep vibrissal nerves begin to differentiate first, followed successively by those from superficial vibrissal nerves and the dermal plexus. Within each type of nerve, Merkel endings begin to differentiate first, followed successively by lanceolate endings and circumferential endings. In the ferret, the differentiation of the intervibrissal fur and its innervation is slightly delayed but substantially overlaps the development of the vibrissal innervation, whereas in the rat it occurs almost entirely later. There was no evidence of a transient exuberant or misplaced innervation or other secondary remodeling. Differentiating afferents and endings are located only in the sites normally seen in the adult, suggesting a high degree of afferent-target specificity. In the ferret, innervation is virtually lacking in one target--the inner conical body of the F-SCs, which is densely innervated in the rat. This lack was due to a failure of innervation to develop rather than to a secondary elimination of a transient innervation.

Innervation of the digit on the forepaw of the raccoon.

The innervation of the digits on the raccoon forepaw was examined by using immunochemistry for protein gene product 9.5, calcitonin-gene related peptide, substance P, neuropeptide-Y, tyrosine hydroxylase, and neurofilament protein. The larger-caliber axons in the ventral glabrous skin terminate as Pacinian corpuscles deep in the dermis, small corpuscles and Merkel endings around the base of dermal papillae, and Merkel endings on rete pegs in dermal papillae. Extensive fine-caliber innervation terminates in the epidermis and on the microvasculature. The innervation is more dense in the distal than in the proximal volar pads. Pacinian endings are also concentrated in the transverse crease separating the distal and proximal pads. In the dorsal hairy skin, hair follicles are well innervated with piloneural complexes. Merkel innervation is located under slight epidermal elevations and in some large Merkel rete pegs located at the apex of transverse skin folds just proximal to the claw. No cutaneous Ruffini corpuscles were found anywhere on the digit. The claw is affiliated with dense medial and lateral beds of Pacinian endings, bouquets of highly branched Ruffini-like endings at the transition from the distal phalanx and unmyelinated innervation in the skin around the perimeter. Encapsulated endings are located at the lateral edge of the articular surface of the distal phalanx. Extensive fine-caliber innervation is affiliated with sweat glands and with the vasculature and is especially dense at presumptive arteriovenous sphincters. Virtually all of the sweat gland and vascular innervation is peptidergic, whereas most of the unmyelinated epidermal innervation is nonpeptidergic.

Innervation of nonmystacial vibrissae in the adult rat.

Vibrissal follicle-sinus complexes (F-SCs) in the mystacial pad of rodents are heavily innervated by different types of sensory nerve endings. One site in mystacial F-SCs, the inner conical body (ICB), is uniquely well innervated only in those species, such as the rat, that rhythmically whisk their mystacial vibrissae. In this study, we examined the innervation of rat nonmystacial F-SCs, which are not whisked. Supraorbital, posteroorbital, lateral cervical, median cervical, submental, and carpal forelimb F-SCs were cut on a cryostat and were either prepared for anti-human protein gene product (PGP 9.5) immunofluorescence or stained using the Winkelmann silver technique. Much of the innervation of the nonmystacial F-SCs is similar to that of mystacial F-SCs. All are innervated by a large deep vibrissal nerve (DVN) and several smaller superficial vibrissal nerves (SVNs). As in the mystacial pad, the SVNs show a distribution of Merkel and free nerve endings qualitatively similar to the rete ridge collar of all the nonmystacial F-SCs as well as provide circumferentially oriented endings to the ICBs to all but median-cervical and carpal F-SCs. Not only was the ICB innervation relatively sparse in median-cervical and carpal F-SCs, but a large portion of the carpal ICB innervation also ascended from the DVNs, which make only a small ICB contribution in other locations. Similar to mystacial pad F-SCs, the DVNs provided Merkel and lanceolate endings to the level of the ring sinus as well as reticular and irregular lanceolate-like endings to the level of the cavernous sinus. However, all but the posteroorbital F-SCs have relatively few lanceolate endings. Carpal F-SCs also have relatively few ring-sinus Merkel endings, which are diffusely distributed, are limited to the superficial portion of the outer root sheath. They also lack reticular and irregular lanceolate-like endings in the cavernous sinus. However, carpal F-SCs have a unique set of corpuscular endings in the ICB, ring sinus, and cavernous sinus that are rarely seen in other F-SCs. PGP 9.5 immunofluorescence also revealed two sets of fine-caliber profiles at the level of the ICB and ring sinus that were not previously seen in mystacial F-SCs. Although there was no correlation between ICB innervation and whisking, the regional variations in F-SC innervation suggest that functional differences may exist between vibrissae at different locations in the body.

A comparative light microscopic analysis of the sensory innervation of the mystacial pad. I. Innervation of vibrissal follicle-sinus complexes.

This comparative study was conducted to provide a detailed, comprehensive description of the innervation to the follicle-sinus complex (F-SC) of mystacial vibrissae and to determine if interspecies variability in the innervation of the F-SCs may be related to differences in the structure or existence of barrels in the primary somatosensory (SI) cortex. Two silver techniques (Winkelmann on 100 micron-thick-frozen sections and Sevier-Munger on 8 micron-thick paraffin sections) were applied to comparable mystacial skin samples from adult hamsters, mice, rats, gerbils, rabbits, guinea pigs and cats. The basic structure and innervation of the F-SCs is the same in all species. Six distinct populations of sensory receptors are identified at consistent locations: Merkel endings in the epidermal rete ridge collar at the mouth of the follicle; circularly disposed presumptive lanceolate, Ruffini, and free nerve endings (FNE) in the inner conical body; longitudinal lanceolate endings in a dense palisade in the mesenchymal sheath at the level of the ring sinus; Merkel endings in the external root sheath at the level of the ring sinus; scattered corpuscular and FNEs (possibly lanceolate or Ruffini endings) in the cavernous sinus; and a few FNEs in the dermal papilla. In each F-SC, the first two locations are supplied by several superficial vibrissal nerves that arise from several small nerves that also innervate the skin between the vibrissae. These superficial nerves may innervate more than one F-SC. The next three locations are supplied by a single large deep vibrissal nerve that is derived directly from a row fascicle of the infraorbital nerve. Each deep nerve innervates a single F-SC. The source of the papilla innervation was not found. The ring sinus locations are consistently the most heavily innervated in all species. The number of axons in comparable deep vibrissal nerves is similar among the rodents, higher in the cat, and lower in the rabbit. Innervation of the inner conical body varies considerably, being dense in species that vigorously whisk their vibrissae (hamster, mouse, rat, and gerbil) and sparse or absent in species that minimally or never whisk (guinea pig, rabbit, and cat). Innervation to the cavernous sinus is sparse particularly in hamsters and gerbils. The innervation to the rete ridge is uniquely absent in the rabbit.(ABSTRACT TRUNCATED AT 400 WORDS)

Different distributions of the sensory and autonomic innervation among the microvasculature of the rat mystacial pad.

The regulation of the vasculature in the skin is a complex process involving both perivascular nerves and local endothelial-mediated control. In this study, the perivascular innervation in the mystacial pad of the rat was characterized based upon immunochemical and lectin binding characteristics and distribution. All of the innervation labeled with anti-protein gene product 9.5 (PGP 9.5), which was used in double- and triple-labeling combinations with the Griffonia simplicifolia lectin (GSA) and antibodies against a variety of neuropeptides, enzymes, and structural proteins. GSA histofluorescence revealed an intricate microvasculature within the rows of tactile vibrissae, which form a natural grid to standardize analyses. Specific features of the vascular organization were confirmed by scanning electron microscopy. Each interval between adjacent vibrissae contained a predictably organized microvascular module composed of separate arterial channels and capillary networks for each of several different structures: papillary muscles, facial muscles, the interior of vibrissal follicle-sinus complexes, vibrissal papillae, and the upper dermis of the intervibrissal fur. Each module was innervated by at least two sets of sensory, at least two sets of sympathetic, and at least one possible set of parasympathetic. These sets not only differed in their biochemical characteristics, but also in their relative position within the arterial walls and their distribution among the microvasculature to the various structures. As such, the microvasculature to each type of structure had a particular combination of innervation, suggesting that separate neuronal mechanisms may be involved in regulating the blood flow to different types of targets even within the confines of a small territory of tissue.

Development of the barrels and barrel field in the somatosensory cortex of the mouse.

Barrels of the PMBSF of the mouse somatosensory cortex become apparent in Nissl-stained tangential sections simultaneously, on the fourth postnatal day. At this time they are miniatures of those in the adult and are situated in the deepest sublamina of the trilaminar cortical plate. An early barrel appears as a patch of decreased cell density: the prospective hollow of the barrel. Septa become noticeable during the sixth postnatal day. From that period to adulthood, the relative contribution of the PMBSF to the total cortical surface area increases -- an increase that goes against one's expectation: the barrel related periphery matures very early and so does the central, lateral region of the cortex. Barrel growth parallel to the pial surface is greater along the major axes than along the minor axes. By using the barrels to identify prospective layer IV in immature cortex, we could determine that layers V and VI attain their adult height during the sixth postnatal day -- an age when prospective layers I-IV are only half their adult height. The onset of barrel formation coincides with the moment after which injury to the pertinent somatosensory periphery (the vibrissal papillae) no longer causes profound alterations in barrel morphology.

The innervation of the mystacial pad of the rat as revealed by PGP 9.5 immunofluorescence.

The innervation of the mystacial pad in the rat was investigated with the aid of antihuman protein gene product (PGP) 9.5 immunofluorescence. PGP 9.5 is ubiquitin carboxyl-terminal hydrolase, which is distributed throughout neuronal cytoplasm. This technique revealed all previously known innervation as well as a wide variety of small-caliber axons and some endings of large-caliber afferents that had not been observed before. Newly revealed innervation affiliated with vibrissal-follicle sinus complexes included 1) fine-caliber, radially oriented processes in the epidermal rete ridge collar; 2) a loose network of fine-caliber, circumferentially arrayed processes in the centrifugal part of the mesenchymal sheath at the level of the ring sinus; 3) a loose haphazard network of fine-caliber and medium-caliber processes in the mesenchymal sheath and among the trabeculae of the cavernous sinus; 4) a loose network of circumferentially arrayed processes within the mesenchymal sheath of the cavernous sinus and in close proximity to the basement membrane; 5) a dense network of reticular-like endings provided by large-caliber afferents to the mesenchymal sheath in the upper part of the cavernous sinus; and 6) fine-caliber innervation to the dermal papilla at the base of all vibrissal shafts. In the intervibrissal skin, a dense distribution of fine-caliber individual and clustered profiles was detected in the epidermis. In addition to previously known innervation, Merkel endings were consistently observed in the epidermis at the mouths of guard hairs, loose networks of fine-caliber axons were found around the necks of occasional guard hairs, and fine-caliber profiles were frequently affiliated with vellus hairs. Vascular profiles were heavily innervated throughout the dermis. Axons and motor end plates of the facial nerve innervation to papillary muscles also were labeled. Transection of the infraorbital nerve eliminated all but the facial nerve innervation. Unilateral removal of the superior cervical ganglion eliminated the innervation to the dermal papillae but caused no other noticeable reduction. PGP 9.5-like immunofluorescence was also moderately expressed in apparent Schwann cells, in Merkel cells only in the external root sheath of vibrissal follicles, and in apparent dendritic and/or Langerhans cells usually located in the epidermis and occasionally in the follicles. PGP 9.5-like immunofluorescence persisted in highly vacuolated profiles along the usual courses of medium to large-caliber axons 2 weeks after nerve transection. The possible functional role of the newly discovered innervation is considered along with that of previously identified afferents.

Sensory innervation in the inner conical body of the vibrissal follicle-sinus complex of the rat.

The innervation of the inner conical body of the vibrissal follicle-sinus complex of the rat was examined by high-voltage and conventional transmission electron microscopy of serial and semi-serial sections. The inner conical body is innervated by axons supplied almost exclusively by several superficial vibrissal nerves that arise from the infraorbital branch of the trigeminal nerve and converge upon the neck of the follicle-sinus complex. Each superficial vibrissal nerve contains a few A delta myelinated axons and several bundles of 20-30 unmyelinated axons. These axons enter the inner conical body and distribute circumferentially within 7-10 ring-like arrays that encircle the vibrissal follicle and are stacked through the superficial-to-deep extent of the inner conical body. Each ring consists of 1 or 2 myelinated axons and several small bundles of 2-15 unmyelinated axons enclosed in sheaves of parallel collagen fibrils. Myelinated axons provide exclusively lanceolate endings that may arise at the termination of the axon or at nodes of Ranvier. Within the small bundles, unmyelinated axons individually terminate in succession as abrupt cytoplasmic swellings referred to as cytoplasmic blebs, which contain mitochondria or clusters of clear or dense-core vesicles. Because of their affiliation with collagen fibrils and the proximity of myelinated axons, the blebbed endings may have been misinterpreted as Ruffini endings in previous studies. Their structure, distribution, and origin from unmyelinated axons suggest that the blebbed endings may constitute a unique array of low-threshold C-mechanoreceptors.

A comparative analysis of the development of the primary somatosensory cortex: interspecies similarities during barrel and laminar development.

The development of the barrels and layers II-V was examined in Nissl-stained preparations of the primary somatosensory cortex in six species--hamster, mouse, rat, gerbil, rabbit, and guinea pig--that have increasingly longer gestation periods. The barrels and layers II-V begin to differentiate postnatally during the first week postpartum in the hamster, mouse, rat and gerbil; perinatally in the rabbit; and approximately 4 weeks prenatally in the guinea pig. The structure of the barrels and layers II-V is similar at the onset of their differentiation in each species, even though there are interspecies differences in the mature structure of the barrels and layer V. The rate of the initial differentiation of the barrels and layers II-V is also similar in each species, even though there are considerable interspecies differences in the duration of the preceding period of development. In each species, layer V begins to differentiate first from the cortical plate and, within 1 or 2 days, contains sublayers that eventually disappear in the rabbit and guinea pig. About 3 days after the initial differentiation of layer V, layers II-IV begin to differentiate, seemingly simultaneously, causing the cortical plate to have a trilaminar appearance. Barrels are first evident just before the appearance of the trilaminar plate in hamsters; concomitant with the trilaminar plate in mice, rats, and guinea pigs; and just after the trilaminar plate in gerbils and rabbits. Septa appear 1 or 2 days after the barrels except in rabbits, which never have septa. Barrel maturation proceeds rapidly after the initial appearance in all species except the hamster, in which continued maturation seems to be delayed until the appearance of the trilaminar plate. The barrels in immature rats and rabbits become more prominent than they will eventually be in the adults. Our results indicate a close and rapid developmental affiliation between layers II-V, especially layers II-IV, that seems quite separate from the development of layers I and VI. However, barrel development and differentiation of layers II-IV seem to be closely, but independently initiated. Secondary remodeling occurs in layer V and the barrels of some species.

Comprehensive immunofluorescence and lectin binding analysis of vibrissal follicle sinus complex innervation in the mystacial pad of the rat.

The innervation of the vibrissal follicle sinus complexes (FSCs) in the mystacial pad of the rat was examined by lectin binding histofluorescence with the B subunit of Griffonia simplicifolia (GSA) and by immunofluorescence with a wide variety of antibodies for neuronal related structural proteins, enzymes, and peptides. Only anti-protein gene product 9.5 labeled all sets of innervation. Several types of mechanoreceptors were distributed to specific different targets by medium to large caliber myelinated axons. All were positive for 200 kDa neurofilament subunit, peripherin, and carbonic anhydrase. Their endings expressed synaptophysin. Labeling for the 160 kDa neurofilament subunit, calbindin, and parvalbumin varied. Anti-Schwann cell protein S100 was completely co-extensive with the axons, terminal arbors, and endings of the mechanoreceptor afferents including Merkel innervation. At least 15 different sets of unmyelinated innervation were evident based upon distribution and labeling characteristics. They consisted of four basic types: 1) peptidergic; 2) GSA binding; 3) peptidergic and GSA binding; and 4) nonpeptidergic and GSA negative (peptide-/GSA-). Previous studies had not revealed that several major sets of unmyelinated innervation were peptide-/GSA-. The unmyelinated innervation had detectable peripherin but not 160 kDa or 200 kDa neurofilament subunits. GSA-positive axons uniquely lacked anti-S100 immunoreactivity. The dense circumferentially oriented unmyelinated innervation of the inner conical body contained major sets of peptide-/GSA- and GSA innervation as well as a smaller peptidergic GSA component. A small contingent of sympathetic and possibly parasympathetic innervation was affiliated with microvasculature in the FSCs. This study confirms and refutes some previous hypotheses about biochemical and morphological relationships between peripheral innervation and sensory ganglion cells.

Comprehensive immunofluorescence and lectin binding analysis of intervibrissal fur innervation in the mystacial pad of the rat.

The innervation of the intervibrissal fur in the mystacial pad of the rat and mouse was examined by immunofluorescence with a wide variety of antibodies for neuronal related structural proteins, enzymes, and peptides as well as for lectin binding histofluorescence with Griffonia simplicifolia (GSA). Anti-protein gene product 9.5 (PGP) immunofluorescence labeled all sets of axons and endings. The innervation in the upper dermis and epidermis was distributed through a four tiered dermal plexus. From deep to superficial, the second tier was the source of all apparent myelinated mechanoreceptors, the third tier of nearly all the peptidergic and GSA binding innervation, and the fourth tier of nonpeptidergic GSA negative innervation (peptide-/GSA-). Three types of mechanoreceptors-Merkel, transverse lanceolate, and longitudinal lanceolate endings-innervated guard hair follicles. All had similar labeling characteristics for 160 kDa and 200 kDa neurofilament subunits, peripherin, carbonic anhydrase, synaptophysin, and S100. Palisades of longitudinal lanceolate endings were part of piloneural complexes along circumferentially oriented sets of transverse lanceolate endings, peptidergic free nerve endings (FNEs), and peptide-/GSA- FNEs. The longitudinal lanceolate endings were the only mechanoreceptors in the mystacial pad that had detectable calcitonin gene-related peptide. The epidermis contained four types of unmyelinated endings: simple free nerve endings (FNEs), penicillate endings, cluster endings and bush endings. Only the simple FNEs were clearly peptidergic. Virtually all others were peptide-/ GSA-. Each bush ending was actually an intermingled cluster of endings formed by several unmyelinated axons and occasionally an Adelta axon. In contrast to the other unmyelinated innervation to the epidermis, bush endings labeled with an antibody against the Schwann cell protein S100. The necks and mouths of follicles, as well as superficial vasculature, were innervated by a mixture of unmyelinated peptidergic and/or GSA labeled sensory and sympathetic axons. Small presumptive sweat glands were innervated by three sets of peptidergic axons of which one was immunoreactive for somatostatin. Potential functions of the various sets of innervation are discussed.

Axonal remodeling during postnatal maturation of CA3 hippocampal pyramidal neurons.

Anatomical substrates were investigated for local circuit hyperexcitability that occurs in the CA3 subfield of the rat hippocampus during postnatal week 2. A transient excess of excitatory local circuit connectivity was hypothesized to underlie this hyperexcitability. To test this hypothesis, recurrent excitatory axon arbors from single biocytin-filled CA3 pyramidal cells were reconstructed. Arbors were analyzed in segments of area CA3 comparable in size to in vitro minislice preparations, which were shown to reproduce the developmental hyperexcitability seen in intact slices during postnatal week 2. Segments were then adjusted for hippocampal growth, based on age-dependent changes in neuron density in stratum pyramidale. Axon arbors were found to be short and possessed very few branches during the first postnatal week. By the second postnatal week, arbors had undergone dramatic growth and were much longer and more complex in their branching patterns. By adulthood, a significant decrease in all measures of arbor length and complexity was observed. Following growth adjustment, measures of axon length and varicosity number during week 2 were not significantly different from that of adulthood. However, the number of axon branches decreased by 50%. These results suggest that, during early postnatal life, there is exuberant outgrowth of local CA3 recurrent axons, and with maturation these recurrent collaterals are remodeled. Short-ranging, profusely branched axons appear to be replaced by longer-ranging arbors that possess fewer branches. Maturational changes in the dendritic location rather than the number of early-formed recurrent excitatory synapses may explain developmental hyperexcitability of the hippocampal CA3 subfield.