Estimation of numbers of vomeronasal synapses in the glomerular layer of the accessory olfactory bulb of the mouse at different ages

The total number of vomeronasal nerve synapses was estimated in a series of mice from 1 to 12 months of age. The volume of the glomerular layer of the accessory olfactory bulb was derived from area measurements through a series of semithin sections. The specialized appositions constituting the vomeronasal nerve synapses were identified in electron micrographs of the glomerular layer by the characteristic electron dense matrix, vesicles, shape and arrangement of the presynaptic elements, and the pale, vesicle-containing dendritic postsynaptic elements.The volume of the glomerular layer showed an overall increase from 66 x 10³ cu. μm at 1 month of age to 82 x 10⁶ cu. μm at 12 months (although there was almost equally large intergroup variation probably not associated with age). The number of synapses per unit area was between 0.114 and 0.131 per sq. μm and unrelated to age. The average length of the synaptic appositions increased from0.32 ± 0.01 μm at 1 month to0.36 ± 0.02 μm at 12 months (the major increase occurring between 1 and 4 months). The calculated total numbers of synapses in the glomerular layer were between 20 and 21 millions at 1, 4 and 8 months and showed a statistically insignificant increase to slightly over 23 millions at 12 months. Since there are about 140 thousand vomeronasal axons, each axon makes on the average about 150 synapses.New vomeronasal neurosensory cells are continually being formed during life and the new cells develop axons which grow to the bulb. However, both the overall number of cells in the epithelium and the total number of synapses in the bulb are constant after about 8 months of age. This could be achieved without synaptic turnover if the newly formed cells die before making synapses. Alternatively, it could be achieved if the entire population of neurosensory cells and their synapses are undergoing continual replacement.     

Age-related changes in the neurosensory epithelium of the mouse vomeronasal organ: Extended period of post-natal growth in size and evidence for rapid cell turnover in the adult

The total number of neurosensory cell in the mouse vomeronasal organ was estimated during postnatal development by counting the cell density and measuring the total volume of the neurosensory cell layer. There is a 43% increase in neurosensory cell number between 1 and 4 months of age, followed by a 21% fall in cell number between 4 and 8 months. There is no further significance change in cell number between 8 and 18 months of age. Cell division was shown to be occuring in the vomeronasal organ of animals at 7 months of age by labelling dividing cells with [³H]thymidine continuously administered by means of implated ‘osmotic pumps’. At least 1 in 6 cells were labelled by 12 days of thymidine administration, indicating a turnover time of 2–3 months for the whole epithelium. This raises the general problem of how a fixed central nervous system accomodates a changing peripheral input.     

Cell division in the vomeronasal organ of the adult mouse

Using [³H]thymidine labelling we could demonstrate the presence of a population of dividing cells in the vomeronasal neurosensory epithelium of the adult mouse. These cells are localised in the regions of the epithelium adjacent to its boundaries with the ciliated respiratory epithelium. With increasing survival times after thymidine administration, the labelled cells become situated progressively further away from the boundary region. The cluster of cells with labelled nuclei forms a loose column, consisting of labelled receptor cells, but in addition the immediately overlying supporting cell nuclei are also labelled. By 56 days after thymidine administration the cluster of labelled cells is separated from the epithelial boundary by a distance equivalent to about one-fifth of the total width of the epithelial sheet. There is little further change in position at 102 days. It is not clear to what extent this represents a turnover process as opposed to a continuing growth of the epithelium by accretion at the edges.     

Vicia villosa agglutinin inhibits the fasciculation of vomeronasal axons in fetal rat vomeronasal organ culture

The vomeronasal organ of rat was cultured from embryonic 15-day littermates. During 4–8 days in vitro, vomeronasal axons originating from the explants of vomeronasal organ formed 2–3 large fascicles. WhenVicia villosa agglutinin (VVA) was added to the culture medium, fasciculation of the vomeronasal axons was inhibited. The timing of addition and the duration of the presence of VVA were related to the inhibition of fasciculation of vomeronasal axons. Glycocojugates that bind with the VVA may therefore play an important role in the fasciculation of developing vomeronasal axons.     

Inhibition by puromycin of transneuronal transport in the mouse accessory olfactory bulb

Application of [³H]proline to the vomeronasal organ (VNO) in mice results in the transport of labelled material along the vomeronasal axons to their terminals in the glomerular layer of the accessory olfactory bulb (AOB). In addition labelled material leaves the vomeronasal nerve terminals and is found over the external plexiform layer (EPL), where a previous electron microscopic autoradiographic study showed that it is preferentially accumulated in mitral cells. Grain densities over the glomerular layer and the EPL were counted in light micrographs. After subtracting background, the overall density of grains in the EPL is about 10% of that over the glomerular layer at 6 h after administration of [³H]proline to the VNO (5 mice). In a further 7 mice, puromycin (or saline) was applied directly to the AOB at hourly intervals during the 6 h after [³H]proline administration. Under these circumtances the labelling in the EPL is only 2–4% of that in the glomerular layer (9% for the 2 saline controls). These observations are evidence that a major part of the transsynaptic transfer mechanism is dependent on protein synthesis, and also favour the view that free amino acids are an important component of the material transferred.     

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.     

β3GnT2 null mice exhibit defective accessory olfactory bulb innervation

Vomeronasal sensory neurons (VSNs) extend axons to the accessory olfactory bulb (AOB) where they form synaptic connections that relay pheromone signals to the brain. The projections of apical and basal VSNs segregate in the AOB into anterior (aAOB) and posterior (pAOB) compartments. Although some aspects of this organization exhibit fundamental similarities with the main olfactory system, the mechanisms that regulate mammalian vomeronasal targeting are not as well understood. In the olfactory epithelium (OE), the glycosyltransferase β3GnT2 maintains expression of axon guidance cues required for proper glomerular positioning and neuronal survival. We show here that β3GnT2 also regulates guidance and adhesion molecule expression in the vomeronasal system in ways that are partially distinct from the OE. In wildtype mice, ephrinA5⁺ axons project to stereotypic subdomains in both the aAOB and pAOB compartments. This pattern is dramatically altered in β3GnT2^−/− mice, where ephrinA5 is upregulated exclusively on aAOB axons. Despite this, apical and basal VSN projections remain strictly segregated in the null AOB, although some V2r1b axons that normally project to the pAOB inappropriately innervate the anterior compartment. These fibers appear to arise from ectopic expression of V2r1b receptors in a subset of apical VSNs. The homotypic adhesion molecules Kirrel2 and OCAM that facilitate axon segregation and glomerular compartmentalization in the main olfactory bulb are ablated in the β3GnT2^−/− aAOB. This loss is accompanied by a two-fold increase in the total number of V2r1b glomeruli and a failure to form morphologically distinct glomeruli in the anterior compartment. These results identify a novel function for β3GnT2 glycosylation in maintaining expression of layer-specific vomeronasal receptors, as well as adhesion molecules required for proper AOB glomerular formation.     

Loss of short dystrophin isoform Dp71 in olfactory ensheathing cells causes vomeronasal nerve defasciculation in mouse olfactory system

The Duchenne muscular dystrophy (DMD) gene encodes dystrophin, which is a protein defective in DMD patients, as well as a number of shorter isoforms, which have been shown to be expressed in various non-muscle, primarily neural, tissues. As of yet, the physiological function of the various dystrophin isoforms is not fully understood. In the present study, we investigated the neurological phenotype that arises in the DMD-null mice, where expression of all dystrophin isoforms had been disrupted. We demonstrate that vomeronasal axons in the DMD-null mice are defasciculated, and some of the defasciculated vomeronasal axons aberrantly entered into the main olfactory bulb, which indicates that the product(s) of the DMD gene plays an important role in vomeronasal nerve organization. Through western blot and immunofluorescence analyses, we determined that the dystrophin isoform Dp71 was exclusively expressed in the mouse olfactory system: mainly in the olfactory ensheathing cells (OECs), an olfactory system-specific glia cell that ensheaths fascicles of the olfactory nerve. In the OECs, Dp71 was co-localized with β-dystroglycan, utrophin, laminin, and perlecan. Since β-dystroglycan and perlecan expression was decreased in the OECs of DMD-null mice, we hypothesize that Dp71 expressed in the OECs participates in fasciculation of the vomeronasal nerve, most likely through interactions with extracellular matrix.     

Maturation of newly born vomeronasal neurons in the adult mice

The olfactory and vomeronasal epithelia detect chemical stimuli in most tetrapods. Both epithelia undergo neural replacement during adulthood. In the central regions of vomeronasal epithelium, similar rates of neurogenesis and apoptosis evidence balanced replacement mechanisms. In the margins, the rate of neurogenesis is several times higher as compared with the rate of apoptosis suggesting net addition of neural receptor cells during adulthood. Herein, the fate of these marginal neuroblasts has been investigated in adult mice. Newly born and mature receptor neurons have been labeled. In the margins, more than 60% of new-born cells send axons to the accessory olfactory bulb. These results evidence that new neural elements from the vomeronasal epithelium are added to the accessory olfactory bulb preexisting circuitry.     

Olfactory ensheathing cells form the microenvironment of migrating GnRH‐1 neurons during mouse development

During development, GnRH‐1 neurons differentiate extracerebraly from the nasal placode and migrate from the vomeronasal organ to the forebrain along vomeronasal and terminal nerves. Numerous studies have described the influence of different molecules on the migration of GnRH‐1 neurons, however, the role of microenvironment cells remains poorly understood. This study used GFAP‐GFP transgenic mice to detect glial cells at early developmental stages. Using nasal explant cultures, the comigration of glial cells with GnRH‐1 neurons was clearly demonstrated. This in vitro approach showed that glial cells began migrating from the explants before GnRH‐1 neurons. They remained ahead of the GnRH‐1 migratory front and stopped migrating after the GnRH‐1 neurons. The association of these glial cells with the axons combined with gene expression analysis of GFAP‐GFP sorted cells enabled them to be identified as olfactory ensheathing cells (OEC). Immunohistochemical analysis revealed the presence of multiple glial cell‐type markers showing several OEC subpopulations surrounding GnRH‐1 neurons. Moreover, these OEC expressed genes whose products are involved in the migration of GnRH‐1 neurons, such as Nelf and Semaphorin 4. In situ data confirmed that the majority of the GnRH‐1 neurons were associated with glial cells along the vomeronasal axons in nasal septum and terminal nerves in the nasal forebrain junction as early as E12.5. Overall, these data demonstrate an OEC microenvironment for migrating GnRH‐1 neurons during mouse development. The fact that this glial cell type precedes GnRH‐1 neurons and encodes for molecules involved in their nasal migration suggests that it participates in the GnRH‐1 system ontogenesis. © 2013 Wiley Periodicals, Inc. © 2013 Wiley Periodicals, Inc.     

Expression of pheromone receptor gene families during olfactory development in the mouse: expression of a V1 receptor in the main olfactory epithelium

In the mouse, two large gene families, V1R and V2R, encoding putative pheromone receptors have been described. Studies have suggested a homotypic recognition role for V1Rs and V2Rs during development in the targeting of vomeronasal axons to specific sets of glomeruli in the accessory olfactory bulb (AOB). Analysis of the onset of expression of the V1R and V2R gene families in developing vomeronasal neurons using polymerase chain reaction and in situ hybridization now suggests that a role for these receptors in the organization of axon projections is only likely at the final stages of targeting within the AOB. Surprisingly, our studies reveal expression of a V1Rd receptor in scattered cells within the main olfactory epithelium, suggesting that limited pheromone detection may also take place in this structure. The pheromone sensory neurons of the vomeronasal system and the neuroendocrine gonadotrophin-releasing hormone (GnRH) neurons that regulate fertility both arise from progenitor cells of the nasal placode. The development of these two cell types is intimately linked, and the GnRH neuron population migrates into the forebrain during embryogenesis in close association with a subset of vomeronasal sensory axons; how GnRH neurons recognize this axon subset is unknown. We report selective expression of a V1Ra gene in the clonal NLT GnRH cell line, raising the possibility of a similar role for V1Rs or V2Rs in the directed migration of GnRH neurons. However, no expression of this gene or of other V1Rs and V2Rs is detectable at the cellular level in migrating GnRH neurons in the mouse.     

Reelin is expressed in the accessory olfactory system,but is not a guidance cue for vomeronasal axons

Reelin is an extracellular matrix protein that regulates neuronal migration in the developing cerebral cortex, and axon outgrowth in the hippocampus. In the developing vomeronasal system, Reelin mRNA is expressed in perineural cells near the vomeronasal nerve, as well as in the vomeronasal organ, olfactory epithelium and olfactory and accessory olfactory bulbs, suggesting that it might regulate axon guidance or fasiculation. We tested that hypothesis by crossing reeler mice with VN12-IRES-tau-lacZ mice to investigate the role of reelin. The vomeronasal nerves are indistinguishable in normal and reeler mutant mice, strongly suggesting that Reelin does not provide a guidance cue for vomeronasal axons.     

Replacement of receptor neurones after section of the vomeronasal nerves in the adult mouse

Eight days after vomeronasal nerve section or removal of the accessory olfactory bulb, the majority of receptor cells of the vomeronasal neuroepithelium degenerate and disappear, leaving a regular framework consisting of supporting cells and their radial processes. The cell clusters at the boundaries of the epithelial sheet (which have been shown to be actively dividing in the normal, unoperated adult mouse) are also spared. The epithelium is subsequently repopulated by receptor cells appearing first in the basal part of the receptor cell layer and later occupying the full width of the receptor layer. These cells are anatomically fully differentiated receptor cells with normal sensory dendrites. Their axons form conspicuous intraepithelial neuromatous masses. Administration of [3H]thymidine on days 10-20 postoperatively labels some clusters of supporting cells and virtually all of the receptor cells, indicating that the repopulation of the epithelium is due to new formation of receptor cells.     

Immunocytochemical demonstration of neural cell adhesion molecule (NCAM) along the migration route of luteinizing hormone-releasing hormone (LHRH) neurons in mice.

Contact between the developing forebrain and the ingrowing central processes of the olfactory, vomeronasal and terminal nerves is preceded by a migration of neural cell adhesion molecule (NCAM)-immunoreactive cells from the epithelium of the olfactory pit and the formation of an NCAM-immunoreactive cellular aggregate in the mesenchyme between the olfactory pit and the forebrain. The axons of the olfactory, vomeronasal, and terminal nerves, also NCAM-immunoreactive, grow into the cellular aggregate, which as development proceeds, becomes continuous with the rostral tip of the forebrain. The lateral and more rostral part of the cellular aggregate receives the ingrowing axons of the olfactory nerves and becomes the olfactory nerve layer of the olfactory bulb. The medial, more caudal part receives the central processes of the vomeronasal and terminal nerves. The vomeronasal nerve ends in the accessory olfactory bulb. The central processes of the terminal nerve end in the medial forebrain. Luteinizing hormone-releasing hormone (LHRH)-immunoreactive neurons, like the vomeronasal and terminal nerves, originate from the medial part of the olfactory pit. These LHRH cells migrate into the brain along and within a scaffolding formed by the NCAM-immunoreactive axons of the vomeronasal and terminal nerves, and they are never seen independent of this NCAM scaffold as they cross the nasal lamina propria. The results suggest that: (1) NCAM is likely to be necessary for scaffold formation, and (2) the scaffold may be essential for the subsequent migration of LHRH neurons into the brain. Because they aggregate, migrating LHRH-immunoreactive neurons, on which we did not detect NCAM immunoreactivity, may interact via other cell adhesion molecules (CAM). Inasmuch as the interaction between the LHRH-immunoreactive neurons and the NCAM-immunoreactive scaffold is heterotypic, the possibility of a heterophilic (NCAM to other CAM) interaction is not ruled out. These findings focus our attention on the functional role of NCAM in this migratory system.     

Topographic analysis of the retinal ganglion cell layer and optic nerve in the sandlance Limnichthyes fasciatus (Creeiidae,Perciformes)

The sandlance or tommy fish Limnichthyes fasciatus (Creeiidae, Perciformes) is a tiny species that lives beneath the sand with only its eyes protruding and is found throughout the Indopacific region. The retina of the sandlance possesses a deep convexiclivate fovea in the central fundus of its minute eye (1.04 mm in diameter). A Nissl-stained retinal whole mount in which the pigment epithelium had been removed by osmotic shock was used to examine the retinal topography of the ganglion cell layer. There was a foveal density of between 13.0 × 10⁴ cells per mm² (S.D. ± 1.8 × 10⁴ cells per mm²), counted in the retinal whole mount, and 15.0 × 10⁴ cells per mm², counted in transverse sections, which diminished to a peripheral density of 4.5 × 10⁴ cells per mm² (S. D. ± 0.8 × 10⁴ cells per mm²). The total population of axons within the optic nerve was assessed by electron microscopy. Optic axon densities ranged from 2 × 10⁶ axons per mm² in the caudal apex to over 16 × 10⁶ axons per mm² within a specialized region of unmyelinated axons in the rostral apex. The topography of the proportion of unmyelinated axon population (26%) follows closely that of the total population of optic nerve axons. There was a total of 104,452 axons within the optic nerve compared with 102,918 cells within the retinal ganglion cell layer. A close relationship is revealed between ganglion cell soma areas and axon areas where the organization in the optic nerve and retina may reflect some functional retinotopicity.     

Transferrin binding protein is expressed by oligodendrocytes in the avian retina

It has been documented that some axons of ganglion cells in the nerve fiber layer of avian retina are wrapped in a myelin sheath. However, the identity of myelin-forming cells has not been established. In this study we demonstrated immunohistochemical evidence for the existence of a large population of oligodendrocytes in avian retina, using an antiserum against transferrin binding protein (TfBP), the avian homologue of the mammalian GRP 94 family of stress-regulated proteins. TfBP⁺ cells were mostly confined to the ganglion cell and optic nerve fiber layers of the retina, in which they were closely associated with the soma and axons of ganglion cells. The double-labeling experiments clearly show that TfBP is specific to oligodendrocytes. The morphology, distribution, and antigenic properties indicated by our findings suggest that TfBP⁺ cells are retinal oligodendrocytes that may be responsible for the myelination of ganglion cell axons in avian retina. A putative tropic role of TfBP⁺ oligodendrocytes to the ganglion cells is also discussed in conjunction with the physical properties of TfBP and avascular retinae of birds.     

Netrin 1-mediated chemoattraction regulates the migratory pathway of LHRH neurons

Luteinizing hormone-releasing hormone (LHRH) neurons migrate from the vomeronasal organ (VNO) to the forebrain in all mammals studied. In mice, the direction of LHRH neuron migration is dependent upon axons that originate in the VNO, but bypass the olfactory bulb and project caudally into the basal forebrain. Thus, factors that guide this unique subset of vomeronasal axons that comprise the caudal vomeronasal nerve (cVNN) are candidates for regulating the migration of LHRH neurons. We previously showed that deleted in colorectal cancer (DCC) is expressed by neurons that migrate out of the VNO during development [Schwarting et al. (2001) J. Neurosci., 21, 911-919]. We examined LHRH neuron migration in Dcc-/- mice and found that trajectories of the cVNN and positions of LHRH neurons are abnormal. Here we extend these studies to show that cVNN trajectories and LHRH cell migration in netrin 1 (Ntn1) mutant mice are also abnormal. Substantially reduced numbers of LHRH neurons are found in the basal forebrain and many LHRH neurons migrate into the cerebral cortex of Ntn1 knockout mice. In contrast, migration of LHRH cells is normal in Unc5h3rcm mutant mice. These results are consistent with the idea that the chemoattraction of DCC+ vomeronasal axons by a gradient of netrin 1 protein in the ventral forebrain guides the cVNN, which, in turn, determines the direction of LHRH neuron migration in the forebrain. Loss of function through a genetic deletion in either Dcc or Ntn1 results in the migration of many LHRH neurons to inappropriate destinations.     

Neurogenesis in the vomeronasal epithelium of adult garter snakes. 2. Reconstitution of the bipolar neuron layer following experimental vomeronasal axotomy

Postnatal neurogenesis and reconstitution of the neuronal layer of the vomeronasal epithelium have been demonstrated in adult garter snakes following vomeronasal axotomy. Two weeks following axotomy the vomeronasal epithelium was depleted of its bipolar layer but the basal, undifferentiated cells were actively proliferating. In subsequent weeks the undifferentiated cell layer continued to increase its cell population through mitosis and began to occupy the neuron-depleted zone of the receptor cell column. Four weeks following axotomy the denervated epithelium contained an expanded Ud cell layer which occupied the basal one-half to two-thirds of the receptor cell column. The cells at the base of the expanded Ud cell layer were morphologically similar to Ud cells in a normal epithelium whereas the cells at the apex of the columns resembled normal differentiating neurons. A few necrotic cells could still be detected within the apical, cell-depleted zone. By the eighth post-operative week the receptor cell column was fully occupied with cells formed as a result of Ud cell proliferation. The most apical cells, 6-10 cells deep, were morphologically similar to normal bipolar neurons with a dendritic process reaching the lumen of the VN organ. The remaining cells were morphologically similar to normal differentiating or Ud cells. Sixteen weeks following axotomy a larger portion of cells in the receptor cell column were fully differentiated bipolar neurons. The Ud cell population was reduced and, as in the normal epithelium, occupied only the basal portion of each receptor cell column. The regenerated neurons of the VNO were capable of synthesizing and transporting macromolecules to the telencephalon as demonstrated by autoradiography following intraepithelial injections of [3H]proline. Newly formed axons terminated in the accessory olfactory bulb within 8 weeks following axotomy. These results support the view that the basal Ud cells were the source of neurons in the regenerating vomeronasal organ and demonstrate a dynamic process of neuronal proliferation, differentiation and maturation in the denervated vomeronasal epithelia of adult garter snakes.     

Electron microscope autoradiographic evidence for specific transneuronal transport in the mouse accessory olfactory bulb

The distribution of radioactive material was examined autoradiographically 8 h after application of [3H] proline to the vomeronasal organ in mice. Labelled material was transported along the axons of the vomeronasal nerves to their terminals in the glomerular layer of the accessory olfactory bulb (AOB). A lesser but consistent amount of radioactivity was found in the external plexiform layer (EPL) of the AOB. Electron microscopic autoradiography was used to determine which of the components of the EPL contained this labelled material. The method of proportional grain counts showed that the highest concentration of silver grains lay over the mitral cell dendrites, which are the elements immediately postsynaptic to the vomeronasal nerve axons. However, a fairly high proportion of grains also lay over the peripheral processes of granule cells. By application of a method of 'crossfire analysis' (which is explained in detail) it was possible to show that the observed grain distribution is best explained by the assumption that the radioactive material is confined to mitral cells, and the labelling over granule cell processes is due to crossfire from these sources. Im one animal at 5 days after [3H]proline administration label was found to have extended from mitral cells to granule cells, suggesting that the transsynaptically transported radioactive material, which was confined to the mitral cells at 8 h, may have become further redistributed at longer survivals. In a control experiment, [3H]proline was applied directly to the surface of the AOB. This gave rise to a completely different distribution of radioactivity in the EPL: radioactive material was present in all tissue components.     

Short‐term synaptic plasticity at the main and vomeronasal olfactory receptor to mitral cell synapse in frog

Synaptic responses resulting from stimulation of the main olfactory and vomeronasal (VN) nerves were measured in main and accessory olfactory bulb (AOB) of frog, Rana pipiens, to test the hypothesis that properties of these synapses would reflect the distinct differences in the time course of odour delivery to each of these olfactory structures. Paired‐pulse depression dominated responses to repetitive stimulation of the main olfactory nerve for interstimulus intervals (ISI) up to several seconds. Inhibition of voltage‐gated Ca²⁺ channels by GABAb receptors contributes significantly to this inhibition of transmitter release, particularly for ISI > 0.5 s. In contrast, the monosynaptic connection between VN sensory neurons and mitral cells in the AOB showed enhancement with pairs or short trains of stimuli for ISI of 0.5 to > 10 s. A small inhibitory effect of GABAb receptors on presynaptic Ca²⁺ influx and release was only evident when a large proportion of the VN axons were stimulated simultaneously but even with inhibition present an overall enhancement of release was observed. Increasing the number of conditioning stimuli from one to five increased residual [Ca²⁺] and enhancement but a direct correlation between residual [Ca²⁺] and either the magnitude or the time course of enhancement was not observed. Enhanced transmitter release from VN afferent terminals results in effective integration of sustained low‐frequency activity, which may play a role in the detection of low‐intensity odourant stimuli by the VN system.