Lesions of the vomeronasal organ disrupt mating-induced pair bonding in female prairie voles (Microtus ochrogaster)

The prairie vole (Microtus ochrogaster) is a highly social, monogamous species and displays pair bonding that can be assessed by the presence of selective affiliation with the familiar partner versus a conspecific stranger. In female prairie voles, exposure to a male or to male sensory cues is essential for estrus induction, and the subsequent mating facilitates pair bond formation. In the present study, we examined the role of the vomeronasal organ (VNO) in estrus induction and pair bonding in female prairie voles. VNO lesions did not alter olfaction mediated by the main olfactory system, but did prevent male-induced estrus induction. We by-passed the necessity of the VNO for estrus induction by estrogen priming the females. Despite the fact that all subjects displayed similar levels of mating, social contact and locomotor activities, VNO lesioned females failed to show mating-induced pair bonding whereas intact and sham-lesioned females displayed a robust preference for the familiar partner. Our data not only support previous findings that the VNO is important for estrus induction but also indicate that this structure is crucial for mating-induced pair bonding, suggesting an important role for the VNO in reproductive success in prairie voles.     

Molecular heterogeneity of Vicia villosa-recognized perineuronal nets surrounding pyramidal and nonpyramidal neurons in the guinea pig cerebral cortex

Of three monoclonal antibodies (mAbs Cat 301, 1B5 and 473) which recognize epitopes on perineuronal nets (PnNs), only mAb 473 displayed considerably varied degrees of staining intensity on Vicia villosa-labeled pyramidal (P) neurons (5%, intensely; 54%, very weakly; and 41%, unstained). These results indicate the heterogeneity on molecular structure or composition of the terminal N-acetylgalactosamine-containing PnNs within P class as well as between P and nonpyramidal classes.     

Innervation of blood vessels in the vomeronasal complex of the rat

We have made an immunohistochemical study of the vomeronasal (VN) complex of 12-day-old rats to characterize the innervation of its blood vessels. The VN complex can be subdivided into rostral, middle and caudal segments, each one with a particular vascularization pattern. Several small vessels were associated with the rostral segment, whereas a large venous sinus ran along the middle and caudal segments. Immunostaining for α-smooth muscle actin demonstrated that the muscular sheath was asymmetric, with more cells layers in its lateral than in its medial walls. Nerves were demonstrated with antisera against protein gene product 9.5 (PGP), and against several molecules associated with specific classes of nerve fibers: the C-terminal peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (NOS). The latter, was also studied with NADPH-diaphorase. Vascular associated fibers exhibited NOS-, CPON-, GAL-, CGRP-, SP- and VIP-immunoreactivity. Only the vessels of the rostral segment showed VIP-immunoreactive fibers. Each wall of the venous sinus exhibited different types of nerve fibers. CPON-, GAL-, CGRP- and SP-immunoreactive fibers concentrated in the medial wall, whereas NOS-immunoreactive ones concentrated in the lateral wall. This distribution of vascular fibers, plus the presence of sensory fibers exhibiting CGRP-, SP- and GAL-immunoreactivity within the pseudostratified epithelium of the VN tube, would be relevant to understand the operation of the pumping mechanism regulating influx and efflux from the VN tube.     

Calbindin-like immunoreactivity in epithelial cells of the newborn and adult human vomeronasal organ

The vomeronasal organs (VNOs) of two humans, a male neonate and a female adult, were examined for immunolocalization of calbindin-D28k (calbindin) which has been immunolocalized to VNO receptor cells in other mammals. The present study demonstrates that epithelial cells within the VNOs of both subjects expressed calbindin-like immunoreactivity. These results suggest that human VNO epithelial cells of both genders express calbindin during development and in the adult.     

Histochemical and scanning electron microscopic studies of supernumerary hair cells in embryonic rat cochlea in vitro

In the embryonic organ of Corti supernumerary hair cells were observed when developed in organotypic cultures. Hair cells ranging in up to two rows of inner hair cells (IHCs) and up to nine rows of outer hair cells (OHCs), were observed by phalloidin histochemistry. The total number of hair cells may double in some explanted cochlea compared to control ones. Cuticular plates of hair cells displayed an actin-free zone corresponding to the kinocilium location, differently located and indicating different degrees of differentiation and maturation. Moreover, some hair cells had a small apical surface area and a centrally located kinocilium, revealing immaturity. Under scanning electron microscopy, stereocilia appeared to differentiate normally, as compared to the in vivo development. The staircase pattern of the stereociliary bundles was reached on most of the hair cells with a ‘V’ shape on the OHCs and hemispherical one on the IHCs. Hair cell polarity was not homogeneous along the length of the tissue. Organs of Corti explanted at birth developed a weaker number of supernumerary hair cells showing a decrease of supernumerary hair cells with the developmental stage of the explant. These results provide evidence for supernumerary hair cells in the mammalian cochlea in culture, without loss or injury to preexisting hair cells.     

Calbindin D28K immunoreactive neurons in vomeronasal organ and their projections to the accessory olfactory bulb in the rat

The vomeronasal system is a nasal chemosensory system involved in pheromone detection. The chemosensory receptor neurons are located in the sensory epithelium of the vomeronasal organ (VNO). Their axons terminate in the glomeruli of the accessory olfactory bulb (AOB). In this study, we examined the expression of calbindin D28k (CB) in the rat VNO and AOB. In the VNO, a subpopulation of receptor neurons in the middle layer of the sensory epithelium was immunostained with antibodies to CB. Their axons could be traced to terminate in a group of glomeruli in the anterior half of the AOB glomerular layer. This group of CB-immunostained glomeruli in the anterior half of the AOB included a few large glomeruli close to the boundary between the anterior and posterior halves of the AOB, and several small glomeruli scattered in the anterior region of the AOB glomerular layer. The positions of the CB-immunostained glomeruli in the AOB, especially those close to the anterior-posterior boundary, were similar in the two bulbs and in different rats. No sex difference was found. A developmental study showed that the CB-immunoreactive receptor neurons in the middle layer of the VNO sensory epithelium and CB-immunoreactive glomeruli in the anterior AOB were present on the 14th postnatal day and older. The distribution pattern of the CB-immunostained receptor neurons and their localized projection suggest the possibility that these neurons may express the same or functionally related pheromone receptor genes.     

Development of serotonin, substance P and thyrotrophin-releasing hormone in mouse medullary raphe grown in organotypic tissue culture: developmental regulation by serotonin

Substance P (SP) and thyrotrophin-releasing hormone (TRH) are co-localized with serotonin (5-HT) in cells of the medullary raphe nuclei. In order to examine the factors that control development of multiple neurotransmitters within individual brain nuclei, we have grown presumptive raphe nuclei in organotypic tissue culture, an environment in which mammalian embryonic brain is easily accessible and manipulable. Tissue was obtained from E13 mice. A discrete midline segment of the rhombencephalon was dissected intact or was separated into 'rostral' (RR) and 'medullary' (MR) fragments. Tissue was explanted onto collagen coverslips and grown for up to two weeks in Maximow depression chambers. Tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT biosynthesis, was barely detectable at explantation. During the first week in culture, however, TPH activity increased 7-fold. After two weeks, TPH activity increased almost 2.5-fold above the one-week level. Immunocytochemical analysis of the cultures confirmed a widespread distribution of 5-HT-positive cells and fibers throughout the explant. SP, monitored by radioimmunoassay, was detected after two days in culture, and attained a level of 111.7 +/- 9.8 pg/culture after two weeks. TRH activity was similarly elevated after two weeks in vitro. Therefore, developmental increases in TPH, SP, and TRH occurred in culture, mimicking the condition in vivo. RR and MR fragments, when grown apart on separate coverslips, developed 1.57-2.26 times the TPH activity that developed in the undivided piece. Inclusion of 1 microM pargyline in the fragments restored TPH to control levels. The effect of pargyline was blocked by methiothepin, suggesting autoreceptor-mediated regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proliferation in the vomeronasal organ of the rat during postnatal development

We investigated proliferation of sensory cell precursors in the rat vomeronasal organ (VNO) at various postnatal ages from birth (P1) to P666. In the rat, which continues to grow during most of its adult life, proliferation might be related to growth and/or replacement. Proliferating cells were labelled by BrdU injection, and histological sections of the VNO were evaluated after immunohistochemical detection of BrdU. Proliferation density (number of proliferating cells/section) decreased dramatically from 115 at P1 to 27.2 at P21, although the area increased. Adult values were reached at P66-P333 (10.3 cells/section); at P400-P666 the value was 8.6 cells/section. Distribution of labelled cells changed considerably with age: in neonates the cells were nearly equally distributed throughout the sensory epithelium, whereas from P21 onwards most proliferating cells were concentrated in clusters near the boundaries with non-sensory epithelium. Labelled cells in the sensory neuronal layer were adjacent to the undulating basement membrane-bordering capillaries that intrude into the sensory epithelium, indicating that they were true basal cells. The volume of the sensory epithelium increased between P1 and P66, and remained constant thereafter, although the length still increased. Length and volume of the sensory epithelium were related to body size, not to sex; males and females of the same body size had the same VNO size. The complex changes in proliferation pattern during postnatal development indicate differential growth and replacement. We suggest that in adults the labelled cell clusters near the boundaries are a pool for growth, whereas proliferation in the central parts represents a replacement pool.     

A comparative immunocytochemical study of development and regeneration of chemosensory neurons in the rat vomeronasal system

Vomeronasal neurons undergo continuous neurogenesis during development and after neuronal injury. We used immunocytochemical methods to compare different stages of the vomeronasal organ development to those of regeneration following vomeronasal nerve transection. At E15 and at 6 to 10 days after injury, nestin-positive cells were observed throughout the sensory epithelium. We did not find nestin immunoreactivity to be localized to the boundary region of the epithelium. The early appearance and wide distribution of nestin-positive cells suggests that they represent chemosensory precursor cells that develop and migrate vertically in the epithelium. Vomeronasal receptor cells degenerated 6 to 8 days after nerve transection, but axon terminals in the accessory olfactory bulb (AOB) continued to show the presence of the chemosensory specific marker (OMP) for up to ten days, a significant finding observed in this study. It is likely that the distance from the site of nerve transection may contribute to differences in the time course of anterograde and retrograde axon degradation. OMP-positive neurons were observed in the normal adult epithelium and to a much lesser extent 10-60 days after recovery from nerve transection. Axons from regenerated receptor cells did not reach the AOB during this time period. This failure to reestablish connections with target cells in the AOB could explain why OMP-positive cells were rarely observed among the regenerated cells in the vomeronasal epithelium.     

A model of organotypic rat spinal slice culture and biolistic transfection to elucidate factors that drive the preprotachykinin-A promoter

The tachykinin substance P (SP) is a neuropeptide that is expressed in some nociceptive primary sensory afferents and in discrete populations of spinal cord neurons. Expression of spinal SP and the preprotachykinin-A (PPT-A) gene that encodes SP exhibits plasticity in response to conditions such as peripheral inflammation but the mechanisms that regulate expression are poorly understood. We have developed a spinal cord organotypic culture system that is suitable for the analysis of PPT-A gene promoter activity following biolistic transfection of recombinant DNA constructs. Spinal cord organotypic slices showed good viability over a 7-day culture period. Immunostaining for phenotypic markers such as NeuN and β-III tubulin demonstrated preservation of neurons and their structure, although there was evidence of axotomy-induced down-regulation of NeuN in certain neuronal populations. Neurokinin-1 receptor (NK-1R) immunostaining in laminae I and III was similar to that seen in acute slices. Biolistic transfection was used to introduce DNA constructs into neurons of these organotypic cultures. Following transfection with a construct in which expression of enhanced green fluorescent protein (EGFP) is controlled by the PPT-A promoter, we showed that induction of neuronal activity by administration of a forskolin analogue/high K⁺ (10 μM/10 mM) for 24 h resulted in a fourfold increase in the number of EGFP-positive cells. Similarly, a twofold increase was obtained after treatment with the NK-1R-specific agonist [Sar⁹,Met (O₂)¹¹]-substance P (10 μM). These data demonstrate the usefulness of this model to study physiological and pharmacological factors relevant to nociceptive processing that can modulate PPT-A promoter activity.     

Morphological complexity of the glomerulus in the rat accessory olfactory bulb-a Golgi study

Using the rapid Golgi method, the accessory olfactory bulb (AOB) of the adult rat was examined. The principal neurons of the AOB were very variable in terms of their dendritic branching pattern and morphology of the glomerular arbors. Some glomerular arbors occupied small portions, other large portions of a single glomerulus. These morphological patterns suggest that complex integrative events occur in the AOB.     

Monoclonal antibodies (2C5 and 4C9) against lactoseries carbohydrates identify subsets of olfactory and vomeronasal receptor cells and their axons in the rabbit

Monoclonal antibodies (MAbs) against lactoseries carbohydrates were used to study immunohistochemically the olfactory and vomeronasal receptor cells and their axons in the rabbit. MAb 2C5, which recognizes Gal alpha 1----3Gal beta 1----4G1cNAc----R structure, selectively labeled a subset of olfactory receptor cells and the majority of vomeronasal receptor cells. MAb 4C9, which reacts with fucosyl poly-N-acetyllactosamine, identified a subset of vomeronasal receptor cells. The above two MAbs also labeled the axons of these chemosensory receptor cells and thus revealed their axonal projection sites in the main and accessory olfactory bulbs.     

Analysis of wheat germ agglutinin (WGA),Phaseolus vulgaris leucoagglutinin (PHA-L), andLens culinaris agglutinin (LCA) binding to isolated rat neocortical membrane glycoproteins and to brain tissue sections

Different lectin transport directions — either anterograde or retrograde in tracing neuronal pathways — have been attributed to different sugar specificities of the lectins. To test this hypothesis, transmembrane neocortical glycoproteins were isolated to analyze their lectin-binding properties. The lectins tested exhibited a broad binding pattern to these glycoproteins. Arguing against the above-mentioned hypothesis, sugar residues of transmembranal glycoproteins probably are not exclusively responsible for the different transport directions observed in lectin tracing.     

Regrowth of retinal axons after lesions of the brachium and pretectal region in the rat

Suction lesions of the left rostral superior colliculus (SC) and pretectal area were made in young rats. One to 50 days later, the right eye of each animal was injected with horseradish peroxidase (HRP). Animals were subsequently perfused and the brains processed with tetramethylbenzidine. In many older animals, lesioned areas were covered with thin membranes composed of glial and connective tissue cells. HRP-labelled optic axons were traced running through these membranes for up to 3 mm. On occasion, regrowing retinal fibres reached the rostral border of the remaining SC but did not grow into the tectal neuropil.     

Proportion of unmyelinated axons in rat molar and incisor tooth pulps following neonatal capsaicin treatment and/or sympathectomy

The occurrence of unmyelinated axons was examined ultrastructurally in rat molar and incisor root pulps of normal rats, of neonatally capsaicin-treated rats, of rats subjected to neonatal capsaicin treatment followed by resection of the superior cervical ganglion and of sympathectomized but otherwise normal rats. Following capsaicin treatment the occurrence of unmyelinated pulpal axons was slightly subnormal. Sympathectomy was largely without effect on the population of unmyelinated axons in tooth pulps of capsaicin-treated rats. In normal rats the proportion of unmyelinated axons in molar pulps was not altered by sympathectomy but it caused a slight decrease in the number of unmyelinated incisor pulpal axons. These findings support the view that most of the unmyelinated axons in rat molar and incisor pulps are sensory, that the parent neurons of these axons differ from nociceptive neurons at other sites by being largely resistant to neonatal capsaicin treatment and that very few unmyelinated tooth pulp axons represent postganglionic efferents.     

Inverse expression of olfactory cell adhesion molecule in a subset of olfactory axons and a subset of mitral/tufted cells in the developing rat main olfactory bulb

The projection of olfactory sensory neuron (OSN) axons from the olfactory epithelium (OE) to the olfactory bulb (OB) is highly organized but topographically complex. Evidence suggests that odorant receptor expression zones in the OE map to the OB about orthogonal axes. One candidate molecule for the formation of zone-specific targeting of OSN axon synapses onto the OB is the olfactory cell adhesion molecule (OCAM). OCAM(+) OSNs are restricted to three of the four zones in the OE and project their axons to the ventral OB where they form synapses with mitral/tufted (M/T) cells. To determine when this zonal connection is established, we have examined OCAM expression in rat olfactory system, during seminal periods of glomerular formation. OCAM(+) axons sort out in the ventral olfactory nerve layer of the OB before glomerular formation. Surprisingly, OCAM was also expressed transiently by subsets of M/T cell dendrites located in the dorsal OB. The expression of OCAM by OSN axons and M/T dendrites was asymmetrical; in the dorsal OB, OCAM(-) OSN axons synapsed on OCAM(+) M/T dendrites, whereas in the ventral OB, OCAM(+) OSN axons synapsed on OCAM(-) M/T dendrites. The restricted spatial map of OCAM(+) M/T cells appeared earlier in development than the zonal segregation of OCAM(+) OSN axons. Thus, OCAM on M/T cell dendrites may act in a spatiotemporal window to specify regions of the developing rat OB, thereby establishing a foundation for mapping of the OE zonal organization onto the OB.     

Coexistence of nitric oxide synthase and neuropeptides in the mouse vomeronasal organ demonstrated by a combination of double immunofluorescence labeling and a multiple dye filter

Nitric oxide synthase (NOS)-immunofluorescence techniques were applied to the mouse vomeronasal organ. Immunoreactivity for NOS was found in the nerve fibers distributed in the receptor-free epithelium, and around the blood vessels and glands in the cavernous tissue. No NOS fibers were seen in the receptor area. A combination of double immunofluorescence labeling and multiple dye filter revealed that a part of the substance P (SP)-immunoreactive nerve fibers in the cavernous tissue contained NOS and that all the vasoactive intestinal polypeptide (VIP)-immunoreactive nerve fibers around the blood vessels and glands in the cavernous tissue contained NOS. A few SP-immunoreactive cell bodies in the trigeminal ganglion showed coexistence with NOS, and almost all VIP-immunoreactive cell bodies in the sphenopalatine ganglion showed coexistence with NOS. Immunoreactivity for NOS without VIP in the cell bodies in the sphenopalatine ganglion was also found. These results suggest that NOS-immunoreactive nerve fibers in the mouse vomeronasal organ originate from the trigeminal and the sphenopalatine ganglia, and may modulate the vascular tone and the glandular secretion. In addition, these functions may be controled in part by the interaction of nitric oxide and neuropeptides.     

Influence of neurotrophins on the synaptogenesis of inner hair cells in the deaf Bronx waltzer (bv) mouse organ of Corti in culture

The Bronx waltzer (bv) deaf mouse is characterized by massive degeneration of the primary auditory receptors, the inner hair cells, which occurs during the time of expected afferent synaptogenesis. The process is associated with degeneration and protracted division of the normally postmitotic afferent spiral ganglion neurons. To investigate the potential role of neurotrophins in the afferent synaptogenesis of inner hair cells, we exposed bv newborn cochleas in organotypic culture to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF), and also to gamma aminobutyric acid (GABA), for up to 8 days. The study was done using light and electron microscopy. Only about 20% of the inner hair cells survived in culture, regardless of the treatment, similar to the number in the intact mutant in our colony. Depending on the exogenous treatment, this population consisted of either innervated ultrastructurally normal cells or denervated dedifferentiated cells wrapped-in lieu of nerve endings-by the supporting inner phalangeal and border cells. In the control and GABA cultures, inner hair cells were mostly denervated. BDNF and NT-3 alone or combined increased synaptogenesis and hair cell survival only during the first 3 days (by about 10%); however, the cells became denervated by 8 postnatal (PN). Only NGF induced stable innervation and differentiation of neurosensory relationships, including supernumerary innervation characteristic of the intact bv. Denervation among the remaining 20% of inner hair cells induced a reactive wrapping by inner phalangeal and border cells which evidently extended inner hair cell survival. Immunocytochemical studies of these reactive supporting cells were done in the intact (8 PN) mutant cochlea. The supporting cells that provide sustenance to the denervated inner hair cells displayed strong BDNF (and possibly NT-3) immunoreactivity. Subsequently, we revealed the presence of all three neurotrophins in the inner hair cell region of the developing (1-8 PN) cochlea of the normal ICR mouse. The inner hair cells expressed all three neurotrophins; BDNF prevailed in the inner phalangeal cells, NT-3 in the pillar cells and inner phalangeal cells, and NGF in the pillar cells. In conclusion: initially, the 80% loss of inner hair cells is apparently caused by their failed afferent synaptogenesis. Exogenous neurotrophins influence synaptogenesis in the bv in culture, but NGF alone is successful in promoting stable neurosensory relationships. The presence of neurotrophins in supporting cells in the normal and degenerating cochlea indicates their role in the sustenance of inner hair cells.     

Ascending myelinated axons of primary sensory neurons of the sciatic nerve in the posterior column of the rat spinal cord

The present study was undertaken to obtain morphologic data about the posterior column of the spinal cord to characterize ascending myelinated axons of primary sensory neurons of the sciatic nerve. By applying doxorubicin to the right sciatic nerve in eight male Wistar rats, selective degeneration of centrally directed axons of these neurons in the posterior column was produced. Epon-embedded transverse sections of the posterior column at spinal cord segments C1, C3, C8, T6, L3 and L5 showed a circumscribed area (R) that contained a cluster of degenerated myelinated fibers. To characterize area R, its size and distances between various defined points on transverse sections of the posterior column were measured and compared at several spinal segments. The location of area R was illustrated in representative rats. The posterior intermediate septum corresponded to the lateral border of area R at C8 and T6. To characterize the putatively degenerating and degenerated myelinated fibers, area L in the left posterior column, corresponding to area R, was defined, and subsequently the number and size distribution of normal-appearing myelinated fibers in areas R and L were evaluated at C3, T6 and L3 in four rats. After comparative evaluation of these data, it was concluded that large myelinated fibers degenerated preferentially in area R. The number of putatively degenerating and degenerated myelinated fibers in area R at segments C3 and T6 was estimated to be 38.6% and 50.1%, respectively, of that at segment L3. Received: 25 August 1995 / Revised, accepted: 25 January 1996