Innervation of the canine mammary gland: an immunohistochemical study

The distribution of peptidergic nerves in canine mammary tissues was studied by immunohistochemical techniques. In addition, the general and the noradrenergic innervations were demonstrated using protein gene product 9.5 and tyrosine hydroxylase immunoreactivities as markers, respectively. Tissue specimens from the caudal mammary glands were obtained from adult, non-lactating, female dogs. The overall innervation of the mammary gland tissue was sparse and primarily associated with the arterial vasculature. Nerve fibres positive for protein gene product 9.5 were rarely found in the secretory parenchyma. The nipple was not richly innervated, although it displayed a greater amount of nerve fibres than the mammary parenchyma. Nerve fibres supplying nonvascular structures of the nipple expressed immunoreactivity for the sensory neuropeptides calcitonin gene-related peptide, substance P and neuropeptide K, but not for vasoactive intestinal peptide, peptide histidine isoleucine and C-flanking peptide of neuropeptide Y. Somatostatin immunoreactivity was not detected in mammary gland tissue. Our results indicate that the innervation of the canine mammary gland is mainly affiliated with the vasculature and comprises peptidergic nerves which may be involved in the regulation of local blood flow. The presence of sensory neuropeptides in nerves supplying the mammary nipple suggest that these peptides may play a role in the afferent pathway of the milk ejection reflex.     

TH-, NPY-, SP-, and CGRP-immunoreactive nerves in interscapular brown adipose tissue of adult rats acclimated at different temperatures: an immunohistochemical study

Interscapular brown adipose tissue (IBAT), a site of nonshivering thermogenesis in mammals, is neurally controlled. The co-existence of sympathetic and peptidergic innervation has been demonstrated in different brown adipose depots. We studied the morphological profile of IBAT innervation and tested by immunohistochemical methods whether cold and warm stimulation are accompanied by modifications in the density of parenchymal noradrenergic nerve fibers. We also studied the immunoreactivity of afferent fibers—which contain calcitonin gene-related peptide (CGRP) and substance P (SP)<197>in different functional conditions. IBAT was obtained from adult rats (6 weeks old) acclimated at different temperatures (4°, 20°, and 28°C). Tissue activity was evaluated by studying the immunolocalization of uncoupling protein (UCP-1), a specific marker of brown adipose tissue. Noradrenergic and peptidergic innervation were seen to arise from morphologically different nerves. Fibers staining for tyrosine hydroxylase (TH) were thin, unmyelinated hilar nerves, and CGRP- and SP-positive fibers were in thick nerves containing both myelinated and unmyelinated fibers. Under cold stimulation, noradrenergic neurons produce greater amounts of TH, and their axons branch, resulting in increased parenchymal nerve fibers density. Neuropeptide Y (NPY) probably co-localizes with TH in noradrenergic neurons, but only in the perivascular nerve fiber network. The parenchymal distribution of NPY to interlobular arterioles and capillaries suggests that this peptide must have other functions besides that of innervating arteriovenous anastomoses, as hypothesized by other researchers. The different distribution of CGRP and SP suggests the existence of different sensory neuronal populations. The detection of CGRP at the parenchymal level is in line with the hypothesis of a trophic action of this peptide.     

Postnatal development of sympathetic and sensory innervation of the rhesus monkey ovary.

We have used immunofluorescence to study the postnatal development of the sympathetic and sensory innervation to the rhesus monkey (Macaca mulatta) ovary. Sympathetic nerves were identified as adrenergic by their content of tyrosine hydroxylase (TH)-like immunoreactivity and as peptidergic by the presence of neuropeptide Y (NPY). Fibers containing substance P (SP) or calcitonin gene-related peptide (CGRP)-like immunoreactivity were considered as sensory, whereas vasoactive intestinal peptide (VIP)-positive fibers were only defined as peptidergic because VIP may be present in both sympathetic and sensory nerves. Ovaries from neonatal (2-mo-old), juvenile (9-18-mo-old), peripubertal (3-3.5-yr-old), adult (9-14-yr-old), and senescent (20-27-yr-old) monkeys were studied. At all ages, with the exception of senescence, TH-, NPY-, and VIP-containing fibers were associated with follicles in different developmental stages. In peripubertal and adult animals, some primordial follicles were found to be selectively innervated by VIPergic fibers that almost completely encircled each follicle. Both sympathetic and VIP fibers were also detected in the interstitial tissue and associated with the ovarian vasculature at all ages. The number of sympathetic and VIP fibers increased significantly (p < 0.01) between 2 mo and 9-18 mo of age, and again increased (p < 0.01) around the age of puberty (approximately 3 yr of age). After this time, the number of NPY and TH fibers remained constant. Conversely, the number of VIP fibers decreased (p < 0.05) by 9-14 yr of age, but remained constant thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Innervation of radicular and extraparenchymal arteries of spinal cord

Summary The perivascular innervation of extraparenchymal arteries of spinal cord and the radicular arteries was examined using histochemical and immunohistochemical technics in monkey. The radicular and the extraparenchymal arteries of spinal cord were found to be invested with adrenergic, neuropeptide Y, vasoactive intestinal peptide, substance P and calcitonin gene-related peptide containing nerve fibres. The pattern of arrangement of fibres differed among the various fibre types. SP-and CGRP-containing fibres were less in density as compared to other nerve plexus. There was no difference in density of an individual type of nerve fibre in arteries of different cord segments or between the radicular arteries from different levels. The study reveals the existence of a comprehensive perivascular adrenergic and peptidergic innervation of spinal cord arterial system, with a possible role in neurogenic regulation of spinal cord circulation.     

Innervation of radicular and extraparenchymal arteries of spinal cord. Histochemical and immunohistochemical study in primate

The perivascular innervation of extraparenchymal arteries of spinal cord and the radicular arteries was examined using histochemical and immunohistochemical technics in monkey. The radicular and the extraparenchymal arteries of spinal cord were found to be invested with adrenergic, neuropeptide Y, vasoactive intestinal peptide, substance P and calcitonin gene-related peptide containing nerve fibres. The pattern of arrangement of fibres differed among the various fibre types. SP- and CGRP-containing fibres were less in density as compared to other nerve plexus. There was no difference in density of an individual type of nerve fibre in arteries of different cord segments or between the radicular arteries from different levels. The study reveals the existence of a comprehensive perivascular adrenergic and peptidergic innervation of spinal cord arterial system, with a possible role in neurogenic regulation of spinal cord circulation.     

Topographically distinct epidermal nociceptive circuits revealed by axonal tracers targeted to Mrgprd

The brain receives sensory input from diverse peripheral tissues, including the skin, the body's largest sensory organ. Using genetically encoded axonal tracers expressed from the Mrgprd locus, we identify a subpopulation of nonpeptidergic, nociceptive neurons that project exclusively to the skin, and to no other peripheral tissue examined. Surprisingly, Mrgprd(+) innervation is restricted to the epidermis and absent from specialized sensory structures. Furthermore, Mrgprd(+) fibers terminate in a specific layer of the epidermis, the stratum granulosum. This termination zone is distinct from that innervated by most CGRP(+) neurons, revealing that peptidergic and nonpeptidergic epidermal innervation is spatially segregated. The central projections deriving from these distinct epidermal innervation zones terminate in adjacent laminae in the dorsal spinal cord. Thus, afferent input from different layers of the epidermis is conveyed by topographically segregated sensory circuits, suggesting that at least some aspects of sensory information processing may be organized along labeled lines.     

Anatomy of the renal innervation: intrarenal aspects and ganglia of origin.

The intrinsic innervation of the kidney is described based on studies using ultrastructural, fluorescent, immunocytochemical, and autoradiographic techniques. The efferent sympathetic innervation reaches all the segments of the renal vasculature and to a much lesser extent the tubular nephron. The afferent renal nerves are localized predominantly in the pelvic region, the major vessels, and the corticomedullary connective tissue. The pathways of the renal innervation to the corresponding ganglia, as reported from observations resulting from the combination of axonal transport labeling and immunocytochemical methods, are presented. In the rat the ganglia of origin of the sympathetic efferent innervation include T13-L1 ipsilateral and contralateral paravertebral ganglia and the prevertebral superior mesenteric and celiac ganglia. The sensory afferent innervation presents a different segmental distribution of the dorsal root ganglia for the right and left kidney. For the left kidney, the corresponding ganglia extend from T8 to L2 with the greatest numbers in T12 and T13. For the right kidney, ganglia as high as T6 and as low as L2 harbor neurons innervating the kidney. Current knowledge of the anatomical bases of the function of the renal nerves is discussed.     

Adrenergic innervation of the gills of brown and rainbow trout,Salmo trutta and S. gairdneri

The adrenergic innervation of structures in the gills of brown and rainbow trout was studied with catecholamine fluorescence histochemistry. In the arterio-arterial vascular pathway, there was an innervation of the afferent and efferent lamellar arterioles, but the afferent and efferent filamental arteries and the secondary lamellae were devoid of any fluorescent nerve fibres. In S. trutta only, there was an additional innervation of the afferent and efferent branchial arteries and the base of the efferent filamental artery. The innervation of the arterio-venous vascular pathway was similar in both trout species. Many fluorescent nerve fibres were found on nutritive arterioles in the gill arch and interbranchial septum, and in the core of each filament between the surface epithelium and the wall of the filament venous sinus. No fluorescent nerve fibres were observed at the origins of the capillaries arising from the efferent filamental artery. The sympathetic nerve supply is provided to the gills mainly through the posttrematic nerve, with an occasional small contribution through the pretrematic nerve. The presence of adrenergic nerves in the gills is discussed in relation to the regulation of blood flow through the arterio-arterial and arterio-venous pathways.     

The autonomic innervation of Rana ridibunda intestine

1. The innervation of Rana ridibunda intestine has been studied by the following methods: (a) demonstration of cholinesterase activity; (b) FIF method for catecholamines; (c) immunohistochemistry for VIP, SP and SOM, and (d) conventional electron microscopy. 2. The intrinsic intestinal innervation is represented by cholinergic-, VIPergic-, SP- and SOM-like plexuses. The intestinal adrenergic component is of extrinsic origin. 3. The intestinal peptidergic innervation is the most developed, the large intestine being the portion where the studied peptidergic plexuses are more widely distributed. 4. Against a poorly developed cholinergic/adrenergic innervation, it seems that there is a predominant peptidergic innervation in the amphibians intestine wall. 5. Taking into account that amphibians sacral parasympathetic as well as sympathetic innervation development are limited, it could be considered that in vertebrates the intestinal peptidergic innervation is phylogenetically earlier and hence better developed.     

The bovine tubouterine junction: general innervation pattern and distribution of adrenergic,cholinergic, and peptidergic nerve fibers

The innervation of the bovine tubouterine junction was studied in sexually mature heifers using antisera against various neuronal markers and a modified acetylcholinesterase method. The vast majority of the nerve fibres in the bovine tubouterine junction belongs to the sympathetic nervous system; peptidergic and cholinergic fibers are restricted to characteristic locations. The endosalpinx in the adovarian portion of the terminal tubal segment is poorly innervated. The mucosa of the aduterine portion and of the tubouterine transitinal region proper receives a strikingly dense innervation, which is observed mainly in combination with a strong vascularisation of specialised mucosal structures. In the endometrium, perivascular nerves accompany the ascending spiral arteries but sporadic contacts between nerve fibres and uterine glands are also observed. From the muscular coat the inner longitudinal layer of the terminal tubal segment is more richly supplied by nerve fibres than the intermediate circular and outer longitudinal layers of the tubouterine junction. No changes in the innervation pattern were seen during the different stages of the sexual cycle.     

Endometriosis as a neurovascular condition: estrous variations in innervation, vascularization, and growth factor content of ectopic endometrial cysts in the rat

Endometriosis is a poorly understood, estradiol-dependent condition associated with severe pelvic pains and defined by vascularized endometrial growths outside the uterus. Endometriosis is produced in cycling rats by autotransplanting pieces of uterus onto abdominal arteries where they develop into cysts. The surgery induces vaginal and abdominal muscle hyperalgesia, whose severity is greatest in proestrus and nearly absent in estrus. The cysts contain growth factors and cytokines and develop their own sympathetic and sensory C- and Adelta-fiber innervation. Here, we used quantitative immunostaining and protein array analyses to test the hypothesis that the innervation and growth factor/cytokine content of the cysts, but not uterine horn, contribute to proestrous-to-estrous changes in hyperalgesic severity. If so, these characteristics in the cysts, but not the uterine horn, should change with estrous stage. In cysts, the density of sympathetic (but not sensory) neurites and amounts of NGF and VEGF proteins (but not cytokines IL-1, IL-6, IL-10, or TNF-alpha) were greater in proestrus than estrus. These changes were accompanied by vascular changes. Both sympathetic and sensory fibers in both stages colabeled with TrkA, indicating that changes in NGF could act on both afferent and efferent fibers. In contrast with the cysts, no changes occurred in the uterine horn between proestrus and estrus. Together, these results suggest that coordinated proestrous-to-estrous changes in innervation and vascularization of the cysts contribute to similar changes in hyperalgesic severity. The findings also encourage consideration of endometriosis as a neurovascular condition.     

Adrenergic terminal structures in the mesentery of mammals

This work is devoted to the study of adrenergic terminal structures in the mesentery of mammals (cat, dog). The investigation was performed with the Falck-Hillarp method of catecholamine fluorescence microscopy on total stretch mesentery preparations. The investigation showed that richly developed perivascular plexus constitute the basis of the adrenergic innervation system of the mesentery. In numerous points of these plexuses, single adrenergic fibers or polyaxonal structures are observed to issue into nonvascular areas of the mesentery where after repeated dichotomic division they pass into the preterminal and terminal parts. Being constructed on the principle of extended or restrained arborizations, these innervating structures have a morphological similarity with free sensory nerve endings. In this connection, the question of the possible existence of the sensory (afferent) links in the catecholamine-containing vegetative nerve plexuses is discussed.     

Norepinephrine reduces ω-conotoxin-sensitive Ca2+ currents in renal afferent neurons in rats

Sympathetic efferent and peptidergic afferent renal nerves likely influence hypertensive and inflammatory kidney disease. Our recent investigation with confocal microscopy revealed that in the kidney sympathetic nerve endings are colocalized with afferent nerve fibers (Ditting T, Tiegs G, Rodionova K, Reeh PW, Neuhuber W, Freisinger W, Veelken R. Am J Physiol Renal Physiol 297: F1427-F1434, 2009; Veelken R, Vogel EM, Hilgers K, Amman K, Hartner A, Sass G, Neuhuber W, Tiegs G. J Am Soc Nephrol 19: 1371-1378, 2008). However, it is not known whether renal afferent nerves are influenced by sympathetic nerve activity. We tested the hypothesis that norepinephrine (NE) influences voltage-gated Ca(2+) channel currents in cultured renal dorsal root ganglion (DRG) neurons, i.e., the first-order neuron of the renal afferent pathway. DRG neurons (T11-L2) retrogradely labeled from the kidney and subsequently cultured, were investigated by whole-cell patch clamp. Voltage-gated calcium channels (VGCC) were investigated by voltage ramps (-100 to +80 mV, 300 ms, every 20 s). NE and appropriate adrenergic receptor antagonists were administered by microperfusion. NE (20 μM) reduced VGCC-mediated currents by 10.4 ± 3.0% (P < 0.01). This reduction was abolished by the α-adrenoreceptor inhibitor phentolamine and the α(2)-adrenoceptor antagonist yohimbine. The β-adrenoreceptor antagonist propranolol and the α(1)-adrenoceptor antagonist prazosin had no effect. The inhibitory effect of NE was abolished when N-type currents were blocked by ω-conotoxin GVIA, but was unaffected by other specific Ca(2+) channel inhibitors (ω-agatoxin IVA; nimodipine). Confocal microscopy revealed sympathetic innervation of DRGs and confirmed colocalization of afferent and efferent fibers within in the kidney. Hence NE released from intrarenal sympathetic nerve endings, or sympathetic fibers within the DRGs, or even circulating catecholamines, may influence the activity of peptidergic afferent nerve fibers through N-type Ca(2+) channels via an α(2)-adrenoceptor-dependent mechanism. However, the exact site and the functional role of this interaction remains to be elucidated.     

Functional morphology of the neural apparatus of the hen ovary in postnatal ontogeny

When comparing the data of neurohistochemical and electron microscopic investigations in the hen and chick ovaries, adrenergic, cholinergic and, possibly, peptidergic nerve fibers have been identified. Previously described cells in the follicular internal theca are mainly SIF-cells and AChE-positive neurocytes of afferent and efferent nature. Axonal terminals make synaptic (or synaptic-like) contacts with chromaffin cells, thecocytes, pericytes of capillaries, AChE-positive motor neurons. Integral estimation, taking into account informative parameters, demonstrates that the degree of the neuromediator differentiation and age resistivity of nervous structures correlates the gland steroidogenic activity. The vascular adrenergic apparatus and chromaffin cells can be considered as potential sources of innervation and catecholamines, able to perform a compensatory function at ageing and other conditions, that are accompanied with a local deficit of sympathetic mediation.     

Monoaminergic Modulation of Spinal Viscero-Sympathetic Function in the Neonatal Mouse Thoracic Spinal Cord

Descending serotonergic, noradrenergic, and dopaminergic systems project diffusely to sensory, motor and autonomic spinal cord regions. Using neonatal mice, this study examined monoaminergic modulation of visceral sensory input and sympathetic preganglionic output. Whole-cell recordings from sympathetic preganglionic neurons (SPNs) in spinal cord slice demonstrated that serotonin, noradrenaline, and dopamine modulated SPN excitability. Serotonin depolarized all, while noradrenaline and dopamine depolarized most SPNs. Serotonin and noradrenaline also increased SPN current-evoked firing frequency, while both increases and decreases were seen with dopamine. In an in vitro thoracolumbar spinal cord/sympathetic chain preparation, stimulation of splanchnic nerve visceral afferents evoked reflexes and subthreshold population synaptic potentials in thoracic ventral roots that were dose-dependently depressed by the monoamines. Visceral afferent stimulation also evoked bicuculline-sensitive dorsal root potentials thought to reflect presynaptic inhibition via primary afferent depolarization. These dorsal root potentials were likewise dose-dependently depressed by the monoamines. Concomitant monoaminergic depression of population afferent synaptic transmission recorded as dorsal horn field potentials was also seen. Collectively, serotonin, norepinephrine and dopamine were shown to exert broad and comparable modulatory regulation of viscero-sympathetic function. The general facilitation of SPN efferent excitability with simultaneous depression of visceral afferent-evoked motor output suggests that descending monoaminergic systems reconfigure spinal cord autonomic function away from visceral sensory influence. Coincident monoaminergic reductions in dorsal horn responses support a multifaceted modulatory shift in the encoding of spinal visceral afferent activity. Similar monoamine-induced changes have been observed for somatic sensorimotor function, suggesting an integrative modulatory response on spinal autonomic and somatic function.     

Peptidergic innervation of rat lymphoid tissue and lung: Relation to mast cells and sensitivity to capsaicin and immunization

Summary The peptidergic innervation of lymphoid tissue and the lung in relation to mast cells was studied in rat. The sensitivity of neuropeptide-containing nerves to capsaicin treatment and immunization was also examined. Measurements of the content of neurokinin A and calcitonin gene-related peptide revealed that the lung contained the highest content of both neuropeptides; lymph nodes had intermediate levels, whereas the spleen had the lowest content. Immuhohistochemistry showed that the calcitonin gene-related peptide- and neurokinin A-immunoreactive nerves in lymph nodes were mainly found around blood vessels, whereas in the lung the nerves were present within the lining respiratory epithelium, bronchial smooth muscle, around blood vessels and close to lymphoid aggregates. Combined immunohistochemistry for serotonin (5-hydroxytryptamine), as a marker for mast cells, and tachykinins or calcitonin gene-related peptide revealed that a close association was often present between the nerves and 5-hydroxytryptamine-positive cells in the bronchi of the lung, while 5-hydroxytryptamine-positive cells were not observed in lymph nodes. The neurokinin A and calcitonin gene-related peptide content in lymph nodes, spleen and lung, but not the content of neuropeptide Y, was markedly decreased by capsaicin treatment, suggesting a sensory origin for the two former peptides. Aerosol immunization increased the levels of calcitonin gene-related peptide in the lung, whereas the content in mediastinal lymph nodes was not affected. These data demonstrate a peptidergic innervation mainly of blood vessels in lymphoid tissue and a close relation between sensory nerves and mast cells as well as lymphoid aggregates in the bronchi of the lung. This further suggests that the sensory innervation of lymph nodes is mainly related to regulation of vascular tone and lymph flow. Furthermore, at the site of immunization, i.e., in the airway mucosa, sensory nerve mediators may interact both with mast cells and lymphoid cells.     

大鼠直肠肽能神经的支配与起源——HRP与免疫细胞化学法联合研究

用HRP追踪法与免疫细胞化学法观察了大鼠直肠内P物质（SP）、降钙素基因相关肽（CGRP）和血管活性肠肽（VIP）三种肽能神经的支配与来源。结果显示：（1）直肠GCRP和VIP肽能神经起源于盆丛副交感神经节（PSG）。（2）直肠感觉神经纤维来自骶2－4节段双侧背根神经节（S2－4－DRG）SP能或CGRP能神经元。（3）感觉神经元的中枢突进入骶髓2－3节段后角并形成较粗大的外侧束,其中大部分传入纤维经后角外侧缘走行,终止于侧角区中间外侧核交感神经元胞体周围。其余部分传入纤维延伸到骶髓2－3节段灰质第Ⅱ、Ⅲ层和灰质后连合核（中央自主神经核）,进入中间外侧核的传入纤维与后连合核也有联系。上述结果提示,支配直肠的VIP能神经元参与了直肠肌运动的调节;SP和CGRP能神经元可能与直肠的运动、感觉调节有关。     

The use of sensory-sympathetic coupling indices for diagnosing of sympathetically maintained pain with laser Doppler flowmetry

For the first time the objective diagnosis of sympathetically maintained pain was created with laser Doppler flowmetry (LDF), directed specially to discovery of skin sensory-sympathetic coupling at 49 patients with posttraumatic complex regional pain syndrome. Sensory-sympathetic coupling was diagnosed as combination of sympathetic vasomotor activity with the existence of sensory peptidergic blood flow oscillations in frequency range of 0.047-0.069 Hz in LDF wavelet-spectrum. The results of LDF diagnosis were compared with clinical evaluation of sympathetically maintained pain carried out after desympathization surgery (thoracoscopic clipping above and below the Th3 ganglion of sympathetic chain at 33 patients and perivascular sympathectomy at the level of brachial artery and veins at 16 patients). Sensitivity of preoperative LDF-diagnosis was 90.2%, specificity--87.5%, positive predictive value--97.3%, negative predictive value--63.6%, diagnostic effectiveness--89.8%.     

Neuropeptide Y and vasoactive intestinal polypeptide in cerebral arteries of the rat: relationships between innervation pattern and mechanical response

Possible relationships between the density of peptide innervation and the contractile response of rat cerebral arteries to exogenously applied neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) were examined. The effects of NPY on membrane potential and reactivity of cerebral arteries to exogenous norepinephrine also were studied. In normally innervated arteries there was no apparent correlation between degree of innervation and response to NPY. Marked, prolonged tachyphylaxis to NPY and VIP was observed following brief exposure to these peptides. Surgical removal of the superior cervical ganglia or the sphenopalatine ganglia greatly reduced and, in some cases, eliminated NPY- or VIP-immunoreactive perivascular nerves from cerebral arteries. However, responses of denervated middle cerebral arteries to exogenous NPY or VIP were not different from responses of innervated arteries. Doses of NPY that induced maximal contraction caused no change in membrane potential of the middle cerebral artery. NPY also did not alter the response of cerebral arteries to exogenous norepinephrine. Finally, electrical stimulation of normal or denervated arteries caused only minor constrictor or dilator responses. These results do not support a substantial role for peptidergic perivascular nerves in regulation of pial arterial contractility in the rat.     

Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea

The adult mammalian cochlea receives dual afferent innervation: the inner sensory hair cells are innervated exclusively by type I spiral ganglion neurons (SGN), whereas the sensory outer hair cells are innervated by type II SGN. We have characterized the spatiotemporal reorganization of the dual afferent innervation pattern as it is established in the developing mouse cochlea. This reorganization occurs during the first postnatal week just before the onset of hearing. Our data reveal three distinct phases in the development of the afferent innervation of the organ of Corti: (1) neurite growth and extension of both classes of afferents to all hair cells (E18-P0); (2) neurite refinement, with formation of the outer spiral bundles innervating outer hair cells (P0-P3); (3) neurite retraction and synaptic pruning to eliminate type I SGN innervation of outer hair cells, while retaining their innervation of inner hair cells (P3-P6). The characterization of this developmental innervation pattern was made possible by the finding that tetramethylrhodamine-conjugated dextran (TMRD) specifically labeled type I SGN. Peripherin and choline-acetyltransferase immunofluorescence confirmed the type II and efferent innervation patterns, respectively, and verified the specificity of the type I SGN neurites labeled by TMRD. These findings define the precise spatiotemporal neurite reorganization of the two afferent nerve fiber populations in the cochlea, which is crucial for auditory neurotransmission. This reorganization also establishes the cochlea as a model system for studying CNS synapse development, plasticity and elimination.