Cutaneous overexpression of NT-3 increases sensory and sympathetic neuron number and enhances touch dome and hair follicle innervation.

Target-derived influences of nerve growth factor on neuronal survival and differentiation are well documented, though effects of other neurotrophins are less clear. To examine the influence of NT-3 neurotrophin overexpression in a target tissue of sensory and sympathetic neurons, transgenic mice were isolated that overexpress NT-3 in the epidermis. Overexpression of NT-3 led to a 42% increase in the number of dorsal root ganglia sensory neurons, a 70% increase in the number of trigeminal sensory neurons, and a 32% increase in sympathetic neurons. Elevated NT-3 also caused enlargement of touch dome mechanoreceptor units, sensory end organs innervated by slowly adapting type 1 (SA1) neurons. The enlarged touch dome units of the transgenics had an increased number of associated Merkel cells, cells at which SA1s terminate. An additional alteration of skin innervation in NT-3 transgenics was an increased density of myelinated circular endings associated with the piloneural complex. The enhancement of innervation to the skin was accompanied by a doubling in the number of sensory neurons expressing trkC. In addition, measures of nerve fibers in cross-sectional profiles of cutaneous saphenous nerves of transgenics showed a 60% increase in myelinated fibers. These results indicate that in vivo overexpression of NT-3 by the epidermis enhances the number of sensory and sympathetic neurons and the development of selected sensory endings of the skin.

Overexpression of nerve growth factor in skin causes preferential increases among innervation to specific sensory targets.

The impact of increased levels of skin-derived nerve growth factor (NGF) neurotrophin on sensory and sympathetic innervation to the mouse mystacial pad and postero-orbital vibrissae was determined. Consistent with an approximate doubling of neuron number in trigeminal and superior cervical ganglia, many components of the sensory and sympathetic innervation were substantially enhanced. Although the increased number of neurons raised the possibility that all types of innervation were increased, immunohistochemical analysis indicated that enhanced NGF production had a differential effect upon sensory innervation, primarily increasing unmyelinated innervation. This increased innervation occurred in specific locations known to be innervated by small, unmyelinated fibers, suggesting that NGF modulated sensory innervation density, but not targeting. In contrast, sympathetic innervation was not only increased but also was distributed to some aberrant locations. In the intervibrissal fur of the mystacial pad, both the number of sensory axons and branches appeared increased, whereas in vibrissal follicle sinus complexes, only branching increased. In some areas, sensory ending density was lower than expected based upon the size of the source nerve bundles suggesting that many axons and branches were surviving but failing to form functional endings. Furthermore, the immunochemical profile of innervation was altered in some sensory populations as demonstrated by the coexistence of RT-97 neurofilament labeling in calcitonin gene-related peptide (CGRP) positive axons, by the loss of substance P colocalization in some CGRP axons, and by an absence of neuropeptide Y labeling in tyrosine hydroxylase positive sympathetic axons. Collectively, these results indicate that the NGF mediated increase in neuron number may be selective for particular sets of innervation and that increases among some populations may result from phenotypic switching.

Over-expression of NGF in skin causes formation of novel sympathetic projections to trkA-positive sensory neurons.

Previous studies have shown that transgenic mice over-expressing NGF in the skin have novel sympathetic 'basket-like' projections to sensory neurons similar to that seen in models of chronic pain. Since only a subset of sensory neurons in the NGF-transgenic mice received the sympathetic projections, we hypothesized that sympathetic sprouting was targeted to those neurons affected by increased levels of NGF. To test this, double-label immunohistochemistry for the NGF receptor (trkA) and sympathetic baskets was performed. Thirty-nine percent of all neurons in transgenic trigeminal ganglia were trkA-positive. Moreover, of the population of sensory neurons that received sympathetic input, 84% were trkA-positive. These results indicate that retrogradely transported NGF can induce and direct growth of sympathetic axons in vivo.

Effects of NGF overexpression on anatomical and physiological properties of sympathetic postganglionic neurons.

To examine the effects of increased target derived nerve growth factor (NGF) on the sympathetic nervous system, the superior cervical ganglion was characterized in transgenic mice overexpressing NGF in keratinized epithelium (e.g. skin, tongue and oral cavity). In these mice NGF overexpression was achieved via expression of an NGF transgene driven by the K14 keratin promoter. This promoter is expressed at approximately embryonic day 11 and thereafter expressed constitutively in the adult. This expression results in supranormal levels of NGF in targets of sympathetic postganglionic neurons prior to the period of programmed cell death. Examination of postnatal day 6 (PN6) and adult transgenic mice shows ca. 2.5-fold increase in neuron number in the superior cervical ganglion (SCG). Analysis of SCG neuronal size revealed a dramatic hypertrophy in the transgenic mice that is present by PN6 and is maintained in the adult. Intracellular physiological measurements of control superior cervical ganglia identified two distinct types of neurons identified on the basis of their response to depolarizing current; 'phasic' neurons fire a single action potential while 'tonic' neurons fire continuously. In adult transgenic mice the phasic neurons were 102% larger than control phasic neurons while the tonic neurons only increased 44% relative to controls. The hypertrophy of sympathetic ganglia in the transgenic mice was correlated with an increased innervation of skin and dorsal root ganglia, structures that either express the transgene or concentrate NGF produced by the skin.

Overexpression of nerve growth factor in epidermis of transgenic mice preserves excess sensory neurons but does not alter the somatotopic organization of cutaneous nerve projections.

To determine how target-derived nerve growth factor (NGF) affects sensory neuronal survival and the development of topographic nerve projections in the spinal cord, anatomical studies were performed on transgenic mice that overexpress NGF in skin and other keratinized epithelial structures. Transgenic animals showed a 100% increase in the number of sensory neurons in specific dorsal root ganglia and exhibited significantly more fibers immunoreactive for calcitonin gene-related peptide in the dorsal horn compared to control animals. This confirms earlier studies which suggested that naturally occurring sensory neuronal death is decreased, or eliminated, in the transgenic mice. Nerve labeling studies showed that the somatotopic organization of cutaneous nerve projections was not altered in the transgenic animals. These data suggest that neuronal death does not act to remove sensory neurons that project to inappropriate regions of the spinal cord.

Overexpression of nerve growth factor in skin increases sensory neuron size and modulates Trk receptor expression.

Sensory neuron development and differentiation is dependent on a family of growth factors known as neurotrophins. Neurotrophins modulate neuron development via trk tyrosine kinase receptor proteins trkA, trkB and trkC. To determine how elevated levels of a target-derived neurotrophin might affect neuronal differentiation, we analysed trk expression in the trigeminal ganglion of transgenic mice that overexpressed nerve growth factor (NGF) in the skin. Increased levels of NGF caused a five-fold increase in neurons expressing trkA mRNA and a two-fold increase in neurons expressing trkC. In control mice, cell size distributions of neuronal subpopulations expressing each trk mRNA showed the three subpopulations distributed over a narrow, overlapping range. In contrast, cell size distribution in NGF-transgenic mice was significantly divergent due in large part to hypertrophy of trkA neurons and, to a lesser extent, trkC neurons. In addition, we examined neurons that bound the isolectin B4 from Bandeiraea simplicifolia (BS-IB4) because most of these neurons do not express any trk receptor in the adult. There was a significant increase in the size of BS-IB4-positive neurons in transgenic mice; however, there was no increase in their number. These studies indicate that an increased level of target-derived NGF affects the development of sensory neurons that in the adult express trkA or trkC, as well as neurons that do not express trk receptors.