Excess target-derived neurotrophin-3 alters the segmental innervation of the skin.

It is thought that dermatomes are established during development as a result of competition between afferents of neighbouring segments. Mice that overexpress neurotrophins in the skin provide an interesting model to test this hypothesis, as they possess increased numbers of sensory neurons, and display hyperinnervation of the skin. When dermatomal boundaries were mapped in adult mice, it was found that those in nerve growth factor and brain-derived neurotrophic factor overexpressers were indistinguishable from wild-type animals but that overlap between adjacent segments was greatly reduced in neurotrophin-3 (NT-3) overexpressers. However, dermatomes in heterozygous NT-3 knockout mice displayed no more overlap than wild-types. In order to quantify differences across strains, innervation territories of thoracic dorsal cutaneous nerves were mapped and measured in adult mice. Overlap between adjacent dorsal cutaneous nerves was normal in nerve growth factor overexpressing mice, but much reduced in NT-3 overexpressers. However, this restriction was not reflected in the central projection of the dorsal cutaneous nerve, creating a mismatch between peripheral and central projections. Dorsal cutaneous nerve territories were also mapped in neonatal mice aged postnatal day 7-8. In neonates, nerve territories of NT-3 overexpressers overlapped less than wild-types, but in neonates of both strains the amount of overlap was much greater than in the adult. These results indicate that substantial separation of dermatomes occurs postnatally, and that excess NT-3 enhances this process, resulting in more restricted dermatomes. It may exert its effects either by enhancing competition, or by direct effects on the stability and formation of sensory endings in the skin.

Central anatomy of individual rapidly adapting low-threshold mechanoreceptors innervating the "hairy" skin of newborn mice: early maturation of hair follicle afferents.

Adult skin sensory neurons exhibit characteristic projection patterns in the dorsal horn of the spinal gray matter that are tightly correlated with modality. However, little is known about how these patterns come about during the ontogeny of the distinct subclasses of skin sensory neurons. To this end, we have developed an intact ex vivo somatosensory system preparation in neonatal mice, allowing single, physiologically identified cutaneous afferents to be iontophoretically injected with Neurobiotin for subsequent histological analyses. The present report, centered on rapidly adapting mechanoreceptors, represents the first study of the central projections of identified skin sensory neurons in neonatal animals. Cutaneous afferents exhibiting rapidly adapting responses to sustained natural stimuli were encountered as early as recordings were made. Well-stained representatives of coarse (tylotrich and guard) and fine-diameter (down) hair follicle afferents, along with a putative Pacinian corpuscle afferent, were recovered from 2-7-day-old neonates. All were characterized by narrow, uninflected somal action potentials and generally low mechanical thresholds, and many could be activated via deflection of recently erupted hairs. The central collaterals of hair follicle afferents formed recurrent, flame-shaped arbors that were essentially miniaturized replicas of their adult counterparts, with identical laminar terminations. The terminal arbors of down hair afferents, previously undescribed in rodents, were distinct and consistently occupied a more superficial position than tylotrich and guard hair afferents. Nevertheless, the former extended no higher than the middle of the incipient substantia gelatinosa, leaving a clear gap more dorsally. In all major respects, therefore, hair follicle afferents display the same laminar specificity in neonates as they do in adults. The widely held misperception that their collaterals extend exuberant projections into pain-specific regions of the dorsal horn during early postnatal life is shown to have multiple, deep-rooted underpinnings.

Maturation of cutaneous sensory neurons from normal and NGF-overexpressing mice.

In the rodent, cutaneous sensory neurons mature over the first two postnatal weeks, both in terms of their electrical properties and their responses to mechanical stimulation of the skin. To examine the coincidence of these events, intracellular recordings were made from neurons in the dorsal root ganglion (DRG) in an in vitro spinal cord, DRG, and skin preparation from mice between the ages of postnatal day 0 and 5 (P0-P5). We also examined mice in which nerve growth factor (NGF) is overexpressed in the skin. NGF has been shown to be involved in a number of aspects of sensory neuron development and function. Therefore we ask here whether excess target-derived NGF will alter the normal course of development, either of somal membrane properties, physiological response properties, or neuropeptide content. In wild-type mice, somal action potentials (APs) were heterogeneous, with some having simple, uninflected falling phases and some displaying an inflection or break on the falling limb. The proportion of neurons lacking an inflection increased with increasing age, as did mean conduction velocity. A variety of rapidly and slowly adapting responses could be obtained by gently probing the skin; however, due to relatively low thresholds and firing frequencies, as well as lack of mature peripheral receptors such as hairs, it was not possible to place afferents into the same categories as in the adult. No correlation was seen between the presence or absence of an inflection on the somal AP (a marker for high-threshold mechanoreceptors in adult animals) and either peripheral threshold or calcitonin-gene related peptide (CGRP) content. Small differences in the duration and amplitude of the somal AP were seen in the NGF-overexpressing mice that disappeared by P3-P5. Excess target-derived NGF did not alter physiological response properties or the types of neurons containing CGRP. The changes that did occur, including a loss of the normal relationship between AP duration and conduction velocity, and a decrease in mean conduction velocity in the inflected population, might best be explained by an increase in the relative proportions of myelinated nociceptors. Of greatest interest was the finding that in both NGF overexpressers and wild-type mice, the correlation between mechanical threshold and presence or absence of an inflection on the somal spike is not apparent by P5.

On the problem of lamination in the superficial dorsal horn of mammals: a reappraisal of the substantia gelatinosa in postnatal life.

Although it is one of the most distinctive and earliest recognized features in the spinal cord, the substantia gelatinosa (SG) remains among the most enigmatic of central nervous system regions. The present neuroanatomical studies employed transganglionic transport of horseradish peroxidase conjugates of choleragenoid (B-HRP) and the B4 isolectin of Bandeiraea simplicifolia (IB4-HRP) on opposite sides to compare the projection patterns of myelinated and unmyelinated cutaneous primary afferents, respectively, within the superficial dorsal horn of the spinal cord in postnatal mice, from shortly after birth to adulthood. Putative unmyelinated afferents labeled with IB4-HRP gave rise to a dense sheet of terminal-like labeling restricted to the outer half of the SG. In contrast, myelinated inputs labeled with B-HRP gave rise to a similarly dense sheet of terminal-like labeling that occupied the inner half of the SG. This adult organization, with two dense sheets of terminal labeling in the superficial dorsal horn, was clearly evident shortly after birth using these markers, prior to the emergence of the SG. Furthermore, the location of the SG proper varied considerably within the dorsoventral plane of the dorsal horn according to mediolateral and segmental locations, a finding that was also seen in comparative studies in rat and cat. These findings caution against equating the SG in particular, and the superficial dorsal horn in general, with nociceptive processing; at minimum, the SG subserves a clear duality of function, with only a thin portion of its outermost aspect devoted to pain.

Cutaneous and muscle afferents: interactions with potential targets in vitro.

Previous studies suggest that cutaneous and muscle afferents use different environmental cues in growing to their peripheral targets. As a first approach to learning whether these earlier observations reflect differences in the behavior of individual growth cones, trigeminal cutaneous and muscle afferents of embryonic day 10 chicks were cocultured with explants of epidermis or dermis or with myotubes, and interactions of their growth cones with these potential targets were followed with time lapse video microscopy. Cutaneous and muscle afferents differed in their response to all three targets. In birds, few cutaneous afferents innervate epidermis. Accordingly, most cutaneous neurites retracted within minutes of touching an epidermal cell. In contrast, most muscle afferents stopped growing but remained in contact with epidermis for as long as they were observed (> 1 hr). Further, most cutaneous afferents grew readily across explants of dermis, their normal target, at rates comparable to their growth on the substrate. In contrast, most muscle afferents advanced only poorly on dermis. Finally, most cutaneous afferents grew readily across myotubes, the normal targets of muscle afferents. In contrast, few muscle afferents grew across myotubes; most either retracted or changed course and grew along the myotube. Overall, muscle afferents stayed in contact with myotubes longer than cutaneous afferents. These cell-type-specific responses reflect in large part the patterns of cutaneous and muscle afferent growth in vivo. Further studies are required to determine whether these observed differences between the behavior of regenerating cutaneous and muscle afferent growth cones could potentially play a role in the selection of targets or pathways during embryonic development.

Physiological studies of cutaneous inputs to dorsal horn laminae I-IV of adult chickens.

1. The dorsal horn (DH) of chickens exhibits a novel pattern of cytoarchitectonic lamination among vertebrates, whereby lamina III lies medial, rather than ventral, to lamina II. Indeed, cutaneous nerves labeled with horseradish peroxidase (HRP) form two separate projections across the mediolateral axis of the superficial DH; each projection is somatotopically organized, such that two non-overlapping somatotopic maps are formed: the medial map within lamina III and the lateral map within lamina II. Interestingly, these two projections of cutaneous nerves are differentially labeled by HRP ligands. The present experiments were designed to address whether the separate subpopulations of cutaneous afferents, as demonstrated neuroanatomically, also differ physiologically on the basis of myelination, fiber diameter, and/or sensory modality. 2. Extracellular multi- and single-unit recordings were obtained in the DH at the ninth synsacral level of spinal adult chickens anesthetized with alpha-chloralose. Activity in lateral (laminae I/II) and medial DH (laminae III/IV) was studied after both electrical stimulation of the caudal femoral cutaneous nerve (CFC) and natural stimulation of the skin. Single units were characterized in terms of the conduction velocity (CV) and sensory modality of their cutaneous afferent inputs. 3. In multiunit recordings, electrical stimulation of the CFC above C-fiber intensities elicited a robust, long-latency response in lateral (laminae I/II) but not medial DH (laminae III/V). The afferents responsible for this late lateral response were C-fibers, as evidenced by a CV of approximately 1 m/s; birds were spinalized to rule out long spinal loops. In contrast, only a single, short-latency (and low-threshold) multiunit response was seen in medial DH, even after activation of C-fibers. 4. Natural stimulation of skin revealed a clear segregation of sensory modalities between medial (laminae III/IV) and lateral DH (laminae I/II). Innocuous mechanical stimuli were extremely effective at eliciting multiunit activity in medial DH, but ineffective in lateral DH. In contrast, noxious mechanical and thermal stimuli were extremely effective at eliciting multiunit activity in lateral DH, but were ineffective in medial DH. 5. In single-unit studies, primary afferent inputs to units in medial DH (laminae III/IV) had an average CV close to 43 m/s; no medial units received exclusive inputs from afferents with CVs less than 5.5 m/s. In contrast, primary afferent inputs to units in lateral DH (laminae I/II) had an average CV close to 10 m/s; 20% of the lateral units received exclusive inputs from C-fibers (CVs less than 1.7 m/s; N = 1.2 m/s).(ABSTRACT TRUNCATED AT 250 WORDS)

Somatotopic organization of hindlimb skin sensory inputs to the dorsal horn of hatchling chicks (Gallus g. domesticus).

The somatotopic organization of skin sensory nerve projections to the lumbosacral dorsal horn of hatchling chickens was determined with the aid of transganglionic transport of horseradish peroxidase (HRP) processed with tetramethylbenzidine histochemistry. A total of eight hindlimb nerves were studied, five of which were purely cutaneous. When combined, the innervation fields of these nerves covered most of the hindlimb surface, allowing a nearly complete somatotopic map of the hindlimb to be generated. This report describes a novel pattern of cutaneous nerve projections to the dorsal horn. Unlike other vertebrates, cutaneous nerves of chickens formed two separate, somatotopically organized projections across the mediolateral axis of the dorsal horn; when serially reconstructed and superimposed, these projections produced two nonoverlapping somatotopic maps of the skin surface lying side by side. Each of these separate maps was nearly identical to the other in overall topology. These two separate maps appear to represent distinct modalities of sensory information, as projections composing the medial map were preferentially labeled by choleragenoid-HRP, whereas those composing the lateral map were preferentially labeled by wheat germ agglutinin-HRP. In mammals, these HRP ligands selectively label the central projections of myelinated and unmyelinated cutaneous afferents, respectively. The present study, therefore, strongly supports the cytoarchitectonic findings of Brinkman and Martin (Brain Res. 56:43-62, '73) that lamina III lies medial, rather than ventral, to lamina II in the chicken dorsal horn. Further, the present studies also suggest that laminae II and III of chickens are homologous to the homonymous laminae in the dorsal horn of mammals.