Combination of TRP channel dietary agonists induces energy expending and glucose utilizing phenotype in HFD-fed mice.

BACKGROUND: Bioactive dietary constituents activating Transient receptor potential (TRP) channels have emerged as promising candidates for the prevention of metabolic disorders. OBJECTIVE: The present study is an attempt to evaluate anti-obesity potential of a dietary TRP-based tri-agonist, combination of sub-effective doses of capsaicin (TRPV1 agonist), menthol (TRPM8 agonist), and cinnamaldehyde (TRPA1 agonist) in high-fat diet (HFD)-fed mice. DESIGN: Male C57BL/6 J mice divided into three groups (n = 8), were fed on normal pellet diet (NPD), or high-fat diet (HFD) (60% energy by fat) and HFD + CB (combination of capsaicin 0.4 mg/Kg, menthol 20 mg/Kg, and cinnamaldehyde 2 mg/Kg; p.o) for 12 weeks. Effects on HFD-induced weight gain, biochemical, histological and genomic changes in the WAT, BAT, liver and hypothalamus tissues were studied. RESULTS: Administration of tri-agonist prevented HFD-induced increase in weight gain, improved altered morphometric parameters, glucose homeostasis, and adipose tissue hypertrophy. Tri-agonist supplementation was found to induce browning of white adipose tissue and promote brown adipose tissue activation. Enhanced glucose utilization and prevention of lipid accumulation and insulin resistance in the liver was observed in mice supplemented with a tri-agonist. CONCLUSION: The present work provides evidence that the new approach based on combination of sub-effective doses of TRP channel agonists (TRI-AGONIST) can be employed to develop concept-based functional food for therapeutic and preventive strategies against HFD-associated pathological complications.

Brown adipose tissue prevents glucose intolerance and cardiac remodeling in high-fat-fed mice after a mild myocardial infarction.

BACKGROUND: Obesity increases the risk of developing impaired glucose tolerance (IGT) and type 2 diabetes (T2D) after myocardial infarction (MI). Brown adipose tissue (BAT) is important to combat obesity and T2D, and increasing BAT mass by transplantation improves glucose metabolism and cardiac function. The objective of this study was to determine if BAT had a protective effect on glucose tolerance and cardiac function in high-fat diet (HFD) fed mice subjected to a mild MI. METHODS: Male C57BL/6 mice were fed a HFD for eight weeks and then divided into Sham (Sham-operated) and +BAT (mice receiving 0.1 g BAT into their visceral cavity). Sixteen weeks post-transplantation, mice were further subdivided into ±MI (Sham; Sham-MI; +BAT; +BAT-MI) and maintained on a HFD. Cardiac (echocardiography) and metabolic function (glucose and insulin tolerance tests, body composition and exercise tolerance) were assessed throughout 22 weeks post-MI. Quantitative PCR (qPCR) was performed to determine the expression of genes related to metabolic function of perigonadal adipose tissue (pgWAT), subcutaneous white adipose tissue (scWAT), liver, heart, tibialis anterior skeletal muscle (TA); and BAT. RESULTS: +BAT prevented the increase in left ventricle mass (LVM) and exercise intolerance in response to MI. Similar to what is observed in humans, Sham-MI mice developed IGT post-MI, but this was negated in +BAT-MI mice. IGT was independent of changes in body composition. Genes involved in inflammation, insulin resistance, and metabolism were significantly altered in pgWAT, scWAT, and liver in Sham-MI mice compared to all other groups. CONCLUSIONS: BAT transplantation prevents IGT, the increase in LVM, and exercise intolerance following MI. MI alters the expression of several metabolic-related genes in WAT and liver in Sham-MI mice, suggesting that these tissues may contribute to the impaired metabolic response. Increasing BAT may be an important intervention to prevent the development of IGT or T2D and cardiac remodeling in obese patients post-MI.

Developmental milestones in neonatal and juvenile C57Bl/6 mouse - Indications for the design of juvenile toxicity studies.

There is a growing demand for wild type mice and mouse models of disease that may be more representative of human conditions but there is little information on neonatal and juvenile mouse anatomy. This project produces sound and comprehensive histology background data on the developing neonatal mouse at different time points from Day 0 until Day 28. The work describes optimal methods for tissue harvesting, fixation and processing from the neonatal and juvenile mice which can be used in routine toxicology studies. A review of the available literature revealed inconsistencies in the developmental milestones reported in the mouse. Although it is true that the sequence of events during the development is virtually the same in mice and rats, important developmental milestones in the mouse often happen earlier than in the rat, and these species should not be used interchangeably.

Assessment of impulsivity in adolescent mice: A new training procedure for a 3-choice serial reaction time task.

Immaturity in impulse control among adolescents could result in substance abuse, criminal involvement, and suicide. The brains of adolescents and adults are anatomically, neurophysiologically, and pharmacologically different. Therefore, preclinical models of adolescent impulsivity are required to screen drugs for adolescents and elucidate the neural mechanisms underlying age-related differences in impulsivity. The conventional 3- or 5-choice serial reaction time task, which is a widely used task to assess impulsivity in adult rodents, cannot be used for young mice because of two technical problems: impaired growth caused by food restriction and the very long training duration. To overcome these problems, we altered the conventional training process, optimizing the degree of food restriction for young animals and shortening the training duration. We found that almost all basal performance levels were similar between the novel and conventional procedures. We also confirmed the pharmacological validity of our results: the 5-hydroxytryptamine 2C (5-HT2C) receptor agonist Ro60-0175 (0.6 mg/kg, subcutaneous) reduced the occurrence of premature responses, whereas the 5-HT2C receptor antagonist SB242084 (0.5 mg/kg intraperitoneal) increased their occurrence, consistent with results of previous studies using conventional procedures. Furthermore, we detected age-related differences in impulsivity using the novel procedure: adolescent mice were found to be more impulsive than adult mice, congruent with the results of human studies. Thus, the new procedure enables the assessment of impulsivity in adolescent mice and facilitates a better understanding of the neurophysiological/pharmacological properties of adolescents.

Subtype-Specific Genes that Characterize Subpopulations of Callosal Projection Neurons in Mouse Identify Molecularly Homologous Populations in Macaque Cortex.

Callosal projection neurons (CPN) interconnect the neocortical hemispheres via the corpus callosum and are implicated in associative integration of multimodal information. CPN have undergone differential evolutionary elaboration, leading to increased diversity of cortical neurons-and more extensive and varied connections in neocortical gray and white matter-in primates compared with rodents. In mouse, distinct sets of genes are enriched in discrete subpopulations of CPN, indicating the molecular diversity of rodent CPN. Elements of rodent CPN functional and organizational diversity might thus be present in the further elaborated primate cortex. We address the hypothesis that genes controlling mouse CPN subtype diversity might reflect molecular patterns shared among mammals that arose prior to the divergence of rodents and primates. We find that, while early expression of the examined CPN-enriched genes, and postmigratory expression of these CPN-enriched genes in deep layers are highly conserved (e.g., Ptn, Nnmt, Cited2, Dkk3), in contrast, the examined genes expressed by superficial layer CPN show more variable levels of conservation (e.g., EphA3, Chn2). These results suggest that there has been evolutionarily differential retraction and elaboration of superficial layer CPN subpopulations between mouse and macaque, with independent derivation of novel populations in primates. Together, these data inform future studies regarding CPN subpopulations that are unique to primates and rodents, and indicate putative evolutionary relationships.

Postnatal Development of the Craniofacial Skeleton in Male C57BL/6J Mice.

C57BL/6J is one of the most commonly used inbred mouse strains in biomedical research, including studies of craniofacial development and teratogenic studies of craniofacial malformation. The current study quantitatively assessed the development of the skull in male C57BL/6J mice by using high-resolution 3D imaging of 55 landmarks from 48 male mice over 10 developmental time points from postnatal day 0 to 90. The growth of the skull plateaued at approximately postnatal day 60, and the shape of the skull did not change markedly thereafter. The amount of asymmetry in the craniofacial skeleton seemed to peak at birth, but considerable variation persisted in all age groups. For C57BL/6J male mice, postnatal day 60 is the earliest time point at which the skull achieves its adult shape and proportions. In addition, C57BL/6J male mice appear to have an inherent susceptibility to craniofacial malformation.

Littermate influence on infant growth in mice: comparison of SJL/J and ICR as cotransferred carrier embryos.

In mice, a minimum number of healthy embryos is required to trigger and maintain pregnancy. Therefore, when recovering mouse embryos from a limited litter, one useful technique is to transfer carrier ICR embryos along with the embryos of interest, a technique referred to as cotransfer. In this study, we examined suitable mouse strains for cotransfer with C57BL/6J (B6) embryos in regards to the maintenance of pregnancy, number of pups born, intrauterine growth, and postnatal growth. Because the coat color of B6 is black, we compared two white coat-colored strains, SJL/J and ICR. Cotransfer of SJL/J and ICR embryos had similar effects on maintenance of pregnancy, number of pups born, and intrauterine growth. However, the postnatal growth of B6 mouse pups cotransferred and grown with SJL/J pups was better than for B6 mouse pups cotransferred and grown with ICR pups, suggesting competition among littermates. These results demonstrate that cotransfer of SJL/J embryos will be useful not only as carrier embryos with B6-background embryos but also as a model system to examine littermate competition.

Enhancing the value of psychiatric mouse models; differential expression of developmental behavioral and cognitive profiles in four inbred strains of mice.

The behavioral characterization of animal models of psychiatric disorders is often based upon independent traits measured at adult age. To model the neurodevelopmental aspects of psychiatric pathogenesis, we introduce a novel approach for a developmental behavioral analysis in mice. C57BL/6J (C57) mice were used as a reference strain and compared with 129S1/SvImJ (129Sv), BTBR T+tf/J (BTBR) and A/J (AJ) strains as marker strains for aberrant development. Mice were assessed at pre-adolescence (4 weeks), adolescence (6 weeks), early adulthood (8 weeks) and in adulthood (10-12 weeks) on a series of behavioral tasks measuring general health, neurological reflexes, locomotor activity, anxiety, short- and long-term memory and cognitive flexibility. Developmental delays in short-term object memory were associated with either a hypo-reactive profile in 129Sv mice or a hyper-reactive profile in BTBR mice. Furthermore, BTBR mice showed persistent high levels of repetitive grooming behavior during all developmental stages that was associated with the adult expression of cognitive rigidity. In addition, strain differences in development were observed in puberty onset, touch escape, and body position. These data showed that this longitudinal testing battery provides sufficient behavioral and cognitive resolution during different development stages and offers the opportunity to address the behavioral developmental trajectory in genetic mouse models for neurodevelopmental disorders. Furthermore, the data revealed that the assessment of multiple behavioral and cognitive domains at different developmental stages is critical to determine confounding factors (e.g., impaired motor behavior) that may interfere with the behavioral testing performance in mouse models for brain disorders.

Development, structure, and keratin expression in C57BL/6J mouse eccrine glands.

Eccrine sweat glands in the mouse are found only on the footpads and, when mature, resemble human eccrine glands. Eccrine gland anlagen were first apparent at 16.5 days postconception (DPC) in mouse embryos as small accumulations of cells in the mesenchymal tissue beneath the developing epidermis resembling hair follicle placodes. These cells extended into the dermis where significant cell organization, duct development, and evidence of the acrosyringium were observed in 6- to 7-postpartum day (PPD) mice. Mouse-specific keratin 1 (K1) and 10 (K10) expression was confined to the strata spinosum and granulosum. In 16.5 and 18.5 DPC embryos, K14 and K17 were both expressed in the stratum basale and diffusely in the gland anlagen. K5 expression closely mimicked K17 throughout gland development. K6 expression was not observed in the developing glands of the embryo but was apparent in the luminal cell layer of the duct by 6 to 7 PPD. By 21 PPD, the gland apertures appeared as depressions in the surface surrounded by cornified squames, and the footpad surface lacked the organized ridge and crease system seen in human fingers. These data serve as a valuable reference for investigators who use genetically engineered mice for skin research.

Sociability and brain development in BALB/cJ and C57BL/6J mice.

Sociability--the tendency to seek social interaction--propels the development of social cognition and social skills, but is disrupted in autism spectrum disorders (ASD). BALB/cJ and C57BL/6J inbred mouse strains are useful models of low and high levels of juvenile sociability, respectively, but the neurobiological and developmental factors that account for the strains' contrasting sociability levels are largely unknown. We hypothesized that BALB/cJ mice would show increasing sociability with age but that C57BL/6J mice would show high sociability throughout development. We also hypothesized that littermates would resemble one another in sociability more than non-littermates. Finally, we hypothesized that low sociability would be associated with low corpus callosum size and increased brain size in BALB/cJ mice. Separate cohorts of C57BL/6J and BALB/cJ mice were tested for sociability at 19-, 23-, 31-, 42-, or 70-days-of-age, and brain weights and mid-sagittal corpus callosum area were measured. BALB/cJ sociability increased with age, and a strain by age interaction in sociability between 31 and 42 days of age suggested strong effects of puberty on sociability development. Sociability scores clustered according to litter membership in both strains, and perinatal litter size and sex ratio were identified as factors that contributed to this clustering in C57BL/6J, but not BALB/cJ, litters. There was no association between corpus callosum size and sociability, but smaller brains were associated with lower sociability in BALB/cJ mice. The associations reported here will provide directions for future mechanistic studies of sociability development.

Effects of the physical form of the diet on food intake, growth, and body composition changes in mice.

The present study investigated effects of the physical form of the diet on food intake, growth, and body composition in male C57BL/6 mice. Three-week-old mice were fed isocaloric diets (AIN93G or a modification containing 25% wheat) in powdered or pelleted form. In experiment 1, mice were assigned into 4 groups offered the AIN93G or the wheat-modified diet in powdered or pelleted form. In experiment 2, mice were pair-fed the powdered diets to the ad libitum level of food intake of those fed the pelleted form of the respective diets. Body weight, food intake, and fecal excretion were recorded, and body composition was assessed on mice 1 wk before termination of the experiment. Mice fed the powdered diets showed greater increases in body weight in 2 wk of feeding than did mice fed the pelleted diets. Compared with the pelleted diets, the powdered diets supported an approximately 85% increase in the fat-mass:body-mass ratio and a 2-fold increase in the abdominal-fat-weight:carcass-weight ratio. In addition, mice fed the powdered diet showed significantly greater plasma concentrations of insulin and leptin and significantly lower plasma adiponectin, compared with their pellet-fed counterparts. Food intake of mice fed the powdered diet was 11% greater for the AIN93G and 16% greater for the wheat diet compared with that of the respective pelleted diet. These results demonstrate that C57BL/6 mice responded to the physical form of these diets in terms of food intake, which affected their growth, body composition, and plasma concentrations of insulin and adipocytokines.

The water delivery system affects the rate of weight gain in C57BL/6J mice during the first week after weaning.

Facility planners, IACUCs, veterinary staff, and researchers make choices on water delivery systems for rodents on the basis of cost effectiveness, water quality, risk of malfunction, and potential effect on animal health and welfare. Here we compare biometrics, including weight trends, of newly arrived mice unfamiliar with automated watering; weight trends of weanlings; fecundity of mice; and risk of malfunction among 3 water delivery techniques: water bottle only, combination of automated delivery and water bottle, and automated system only. There was no statistically significant difference among the 3 experimental groups with respect to fecundity, mortality, and delivery malfunction. On the basis of body weight trends, the health and wellbeing of the mice used in these studies were not affected by the water delivery system or housing density after the first week; however, there was a significant difference in the growth rate at 21 to 28 d of age among the 3 groups of pups. The mice receiving both automated delivery and water bottles experienced higher growth rates from 21 to 28 d of age than did the other experimental groups. However, after 35 d of age, weight trends did not differ among the groups. Our results suggest that mice weaned into the same method of water delivery as their respective dams thrive equally well among the 3 tested water delivery systems.

Identification methods in newborn C57BL/6 mice: a developmental and behavioural evaluation.

The use of group-housed rodents in many fields of biomedical research imposes a need to identify individuals in a cage. Few studies have been designed to assess possible negative effects of identification methods of newborn mice on their development and wellbeing. In the present study, three different identification methods were applied to newborn C57BL/6J mice on postnatal day (pnd) 5 (toe clipping, toe tattoo ink puncture and subcutaneous implantation of a small transponder). All identification methods used proved to be effective for long-term marking of individual animals. Newborn mice showed the least reaction to toe clipping followed by toe tattoo ink puncture and transponder implantation was the most distressful individual identification procedure in newborn mice. Importantly, clipped toe tissue proved to be enough for genotyping purposes. No overall consistent differences in somatic and neurological reflex development during the postnatal period were shown as a result of the newborn individual identification procedures used. Further, none of the methods interfered significantly with the adult animals' general normal behaviour (e.g. ability to move, grasp, climb) and sensory-motor functions as assessed with a simplified SHIRPA battery of tests, as well as Rotarod and Elevated Plus Maze tests. Postmortem thymus and adrenal gland weights gave no indication of chronic stress as a consequence of the identification method. We conclude that toe clipping might even be advisable in newborn mice at a very young age, when genotyping is needed. Toe tattoo ink puncture is also a good identification method for newborn mice and transponder implantation should only be used in older newborns or applied at weaning.

The impact of tail tip amputation and ink tattoo on C57BL/6JBomTac mice.

Genetic material for polymerase chain reaction (PCR) and Southern blot analysis on transgenic mice is normally obtained by tail biopsy. Additionally, it may be necessary to tattoo the mice, as it is essential to have a good and permanent identification. The aim of this study was to evaluate the effects of amputating the tip of the tail to obtain a biopsy for genetic analysis and of ink tattooing on welfare in C57BL/6J mice, a strain often used as genetic background for transgenes. The behaviour of the animals, fluctuating asymmetry (FA, a measure of developmental instability) and the level of restitution in the remaining part of the tail were evaluated and used for an assessment of the impact of these procedures on the welfare of the animals. One group of mice was marked by tail tattooing at various ages. Another group of mice were tail amputated at 12 or 20 days of age. Body weight and FA were followed, and at the end of the experiment, the level of fear/anxiety was assessed using a light-dark box. In the group of tail-amputated animals observation of climbing behaviour and a beam walking test for balance was performed. Seven weeks after tail amputation, the animals were euthanized. The remaining part of the tail was evaluated histopathologically. Body weight, behaviour in the light-dark box and balance test results were not influenced by tail amputation or tattooing. FA was only transiently increased by tattooing. Climbing behaviour was reduced just after tail amputation at 20 days of age. No signs of neuromas were found in the amputated tails, but seven weeks after amputation a significant number of mice did not have fully regenerated glandular tissue and hair follicles in the tail. It is concluded that both tail amputation and tail tattooing seem to have minor short-term negative effects on welfare and that the tissues on the tail probably do not regenerate fully after amputation.

Adult but not aged C57BL/6 male mice are capable of using geometry for orientation.

Geometry, e.g., the shape of the environment, can be used by numerous animal species to orientate, but data concerning the mouse are lacking. We addressed the question of whether mice are capable of using geometry for navigating. To test whether aging could affect searching strategies, we compared adult (3- to 5-mo old) and aged (20- to 21-mo old) C57BL/6 male mice. We established a water maze task in which spatial information is provided by one landmark proximal to the target (featural information) and by the rectangular shape of the maze (geometric information). By means of probe trials in which we manipulated the presence of these two kinds of information, we show that adult mice can use both geometry and landmark to orientate. By contrast, aged mice do not use geometry and rely exclusively on the landmark to locate the platform. This study provides the first evidence that mice are capable of using geometric information for orientation and that this ability declines in aged animals.

Further mapping of quantitative trait loci for postnatal growth in an inter-sub-specific backcross of wild Mus musculus castaneus and C57BL/6J mice.

We performed a quantitative trait locus (QTL) analysis of eight body weights recorded weekly from 3 weeks to 10 weeks after birth and two weight gains recorded between 3 weeks and 6 weeks, and between 6 weeks and 10 weeks in an inter-sub-specific backcross population of wild Mus musculus castaneus mice captured in the Philippines and the common inbred strain C57BL/6J ( M. musculus domesticus ), to elucidate the complex genetic architecture of body weight and growth. Interval mapping identified 17 significant QTLs with main effects on 11 chromosomes. In particular, the main effect of the most potent QTL on proximal chromosome 2 increased linearly with age, whereas other QTLs exerted effects on either the early or late growth period. Surprisingly, although wild mice displayed 60% of the body size of their C57BL/6J counterparts, the wild-derived allele enhanced growth at two QTLs. Interestingly, five of the 17 main-effect QTLs identified had significant epistatic interaction effects. Five new epistatic QTLs with no main effects were identified on different chromosomes or regions. For one pair of epistatic QTLs, mice that were heterozygous for the wild-derived allele at one QTL and homozygous for that allele at another QTL exhibited the most rapid growth in all four possible genotypic combinations. Out of the identified QTLs, several showed significant sex-specific effects.

Abnormality in the cerebellar folial pattern of C57BL/6J mice.

The characteristic folial pattern of the mouse cerebellum is formed during postnatal development. We observed this process in C57BL/6J (B6) mice in detail, and found an abnormal folial pattern in a specific region (lobules VIII and IX of the vermis) in a substantial number of B6 mice. The frequency of this abnormality increased during postnatal development and reached 55% in the adult. Thus, the present study showed an abnormality in the cerebellar folial pattern of B6 mice, a mouse widely used in knockout studies, and called for caution in the phenotypic analysis of knockout mice of the B6 genetic background.

Biotelemetry transmitter implantation in rodents: impact on growth and circadian rhythms.

The implantation of a biotelemetry transmitter for core body temperature (T(c)) and motor activity (MA) measurements is hypothesized to have effects on growth and circadian rhythmicity depending on animal body-to-transmitter (B:T) size ratio. This study examined the impact of transmitter implantation (TM) on body weight, food intake (FI), water intake (WI), and circadian T(c) and MA rhythms in mice (23.8 +/- 0.04 g) and rats (311.5 +/- 5.1 g) receiving no treatment (NT), anesthesia, laparotomy (LAP), and TM. The B:T size ratio was 6:1 and 84:1 for mice and rats, respectively. In mice, body weight required 14 days to recover to presurgical levels and never attained the level of the other groups. FI recovered in 3 days, whereas WI never reached presurgical levels. Rat body weight did not decrease below presurgical levels. FI and WI recovered to presurgical levels in rats by day 2 postsurgery. Anesthesia decreased mouse body weight for 1 wk, but was without effect in rats. LAP significantly decreased body weight for 5 days in mice and 1 day in rats, showing a significant effect of the surgical procedure in the absence of TM in both species. Circadian T(c) and MA rhythms were evident within the first week in both species, indicating dissociation between circadian rhythmicity and recovery of growth variables. Cosinor analysis showed a TM effect on T(c) min, T(c) max, mesor, amplitude, and period of mice, whereas only the amplitude of the rhythm was affected in rats. These data indicate that a large B:T size ratio is associated with minimization of the adverse effects of surgical implantation. We recommend that B:T size ratio, recovery of presurgical body weight, and display of a robust circadian T(c) and MA rhythm be established before collection of biotelemetry data collection under an experimental paradigm.

Marked differences in olfactory sensitivity and apparent speed of forebrain neuroblast migration in three inbred strains of mice.

In the adult forebrain, new neuroblasts constantly migrate from the subventricular zone along the rostral migratory stream to the olfactory bulb, where many become neurons. It is unclear whether this process is different in commonly used mouse strains and whether it is related to olfactory function. Adult male BALB/c, C57BL/6, and 129/S1 (formerly 129SV) mice were tested for olfactory sensitivity plus discrimination, using male mouse urine from the two other strains. BALB/c mice had the greatest olfactory sensitivity, followed by 129/S1, and C57BL/6 mice, by an order of magnitude each. Newly formed cells were pulse-labeled for 3 h with i.p. 5-bromo-2'-deoxyuridine (BrdU) injections and the animals analyzed 24 h later. In 129/S1 mice, a greater proportion of neuroblasts were present closer to the olfactory bulb than in BALB/c mice, followed by C57BL/6 mice. The total number of BrdU-labeled cells did not differ, suggesting differences in migration and not proliferation. The impaired olfactory function in C57BL/6 mice might be caused by the reduced number of neuroblasts that reach the olfactory bulbs. However, olfactory function in BALB/c and 129/S1 mice did not correlate with their putative migration speed, suggesting a more complex nature of cellular processes that contribute to olfactory function. These results caution against comparing studies of olfactory function or neural precursors that use different strains of mice, and question the use of C57BL/6 mice as a "normal" strain or as transgenic background. Perhaps more importantly, the results point to an opportunity to identify genes that regulate olfactory function and neuroblast behavior.

Developmental timing of hair follicle and dorsal skin innervation in mice.

The innervation of hair follicles offers an intriguing, yet hardly studied model for the dissection of the stepwise innervation during cutaneous morphogenesis. We have used immunofluorescence and a panel of neuronal markers to characterize the developmental choreography of C57BL/6 mouse backskin innervation. The development of murine skin innervation occurs in successive waves. The first cutaneous nerve fibers appeared before any morphological evidence of hair follicle development at embryonic day 15 (E15). Stage 1 and 2 developing hair follicles were already associated with nerve fibers at E16. These fibers approached a location where later in development the follicular (neural) network A (FNA) is located on fully developed pelage hair follicles. Prior to birth (E18), some nerve fibers had penetrated the epidermis, and an additional set of perifollicular nerve fibers arranged itself around the isthmus and bulge region of stage 5 hair follicles, to develop into the follicular (neural) network B (FNB). By the day of birth (P1), the neuropeptides substance P and calcitonin gene-related peptide became detectable in subcutaneous and dermal nerve fibers first. Newly formed hair follicles on E18 and P1 displayed the same innervation pattern seen in the first wave of hair follicle development. Just prior to epidermal penetration of hair shafts (P5), peptide histidine methionine-IR nerve fibers became detectable and epidermal innervation peaked; such innervation decreased after penetration (P7- P17). Last, tyrosine hydroxylase-IR and neuropeptide Y-IR became readily detectable. This sequence of developing innervation consistently correlates with hair follicle development, indicating a close interdependence of neuronal and epithelial morphogenesis.