Effects of mycophenolic acid alone and in combination with its metabolite mycophenolic acid glucuronide on rat embryos in vitro.

Mycophenolic acid (MPA) is an immunosuppressive agent that acts as a selective, non-reversible inhibitor of the enzyme inosine-5'-monophosphate dehydrogenase (IMPDH). Malformations have been described in children after maternal exposure to mycophenolate. However, the causal link is unclear in most cases because women had been treated with a combination of drugs and birth defects may have other causes. Therefore, it is important to study the action of this drug and its main metabolite on embryonic tissue. We studied the teratogenic potential of MPA and its major metabolite, the mycophenolic acid glucuronide (MPAG) in the rat whole-embryo culture. A total of 147 day 9.5 embryos were cultivated for 48 h in the standard medium containing 85 % serum. We tested MPA at concentrations of 0.1; 0.25; 0.5; 0.75 mg/l (0.31; 0.78; 1.56; 2.34 µM) and MPA glucuronide at concentrations of 3; 10; 30; 100 mg/l (6.04; 20.14; 60.43; 201.43 µM). Both substances are highly protein bound, and MPA glucuronide might displace MPA from protein binding. Therefore, we examined whether the effects of MPA can be enhanced when studied in combination with the glucuronide. Furthermore, the focus was on additional endpoints to the standard evaluation of cultivated embryos, such as development of cranial nerves [trigeminal nerve (V), facial nerve (VII), glossopharyngeal nerve (IX), vagus nerve (X)] after staining with an antibody against 2H3 neurofilament. Ultrastructural changes were evaluated by electron microscopy. At a concentration of 0.75 mg MPA/l medium, all embryos showed dysmorphic changes. Embryos exposed to 0.25 mg MPA/l medium showed impaired development of nerves, and at 0.1 mg/l, no effects were detectable. Concentration-dependent ultrastructural changes, such as signs of apoptosis, were found by electron microscopy. The examination of the metabolite in this assay showed that at a concentration of 100 mg MPAG/l, the embryos exhibited distinct malformations. This is probably caused by MPA, which was detectable at 0.6 % in the material used for our experiments. The combination of the parent compound (0.03; 0.1; 0.25 mg/l) with its metabolite MPAG (3 mg/l) did not cause enhanced toxicity under our experimental conditions. IMPDH, the target enzyme of MPA, could be detected in rat embryos on day 9.5 of embryonic development as well as at the end of the culture period 48 h later. In summary, MPA impairs embryonic development at low, therapeutically relevant concentrations, but the glucuronide does not exhibit such a potential. Activity of MPA is not enhanced by MPAG.

Topographic study on nerve-associated lymphatic vessels in the murine craniofacial region by immunohistochemistry and electron microscopy.

The distribution and fine structure of lymphatic vessels associated with nerves was studied by immunohistochemistry in the murine craniofacial region. The tissue sections and blocks were immunostained for LYVE-1, protein gene product 9.5, CD34 and aquaporin-1 to demonstrate the lymphatic vessels, nerves, blood vessels and water channel protein, respectively. Transmission electron microscopic examination was also performed to investigate the relationship between the lymphatics and nerves. In the nasal area, the lymphatics were found in dura mater on the cribriform plate and beneath the nasal mucosa, this supposedly supplying the cerebrospinal fluid drainage route along the olfactory nerves. The proximal portions of the cranial nerves were equipped with the lymphatics in the epineurium. In the distal portions of the nerves, the lymphatics were distributed in close proximity of the perineural sheath, and thus might contribute to maintenance of microenvironment suitable for the nerves by an absorptive activity of the lymphatic endothelial cells. The present findings suggest that the lymphatic system associated with the cranial nerves provides the pathway for transport of cerebrospinal fluid, tissue fluid, and free cells involved in immune response and tumor metastasis in the craniofacial region.

Cranial visceral afferent pathways through the nucleus of the solitary tract to caudal ventrolateral medulla or paraventricular hypothalamus: target-specific synaptic reliability and convergence patterns.

Cranial visceral afferents activate central pathways that mediate systemic homeostatic processes. Afferent information arrives in the brainstem nucleus of the solitary tract (NTS) and is relayed to other CNS sites for integration into autonomic responses and complex behaviors. Little is known about the organization or nature of processing within NTS. We injected fluorescent retrograde tracers into two nuclei to identify neurons that project to sites involved in autonomic regulation: the caudal ventrolateral medulla (CVLM) or paraventricular nucleus of the hypothalamus (PVN). We found distinct differences in synaptic connections and performance in the afferent path through NTS to these neurons. Anatomical studies using confocal and electron microscopy found prominent, primary afferent synapses directly on somata and dendrites of CVLM-projecting NTS neurons identifying them as second-order neurons. In brainstem slices, afferent activation evoked large, constant latency EPSCs in CVLM-projecting NTS neurons that were consistent with the precise timing and rare failures of monosynaptic contacts on second-order neurons. In contrast, most PVN-projecting NTS neurons lacked direct afferent input and responded to afferent stimuli with highly variable, intermittently failing synaptic responses, indicating polysynaptic pathways to higher-order neurons. The afferent-evoked EPSCs in most PVN-projecting NTS neurons were smaller and unreliable but also often included multiple, convergent polysynaptic responses not observed in CVLM-projecting neurons. A few PVN-projecting NTS neurons had monosynaptic EPSC characteristics. Together, we found that cranial visceral afferent pathways are structured distinctly within NTS depending on the projection target. Such, intra-NTS pathway architecture will substantially impact performance of autonomic or neuroendocrine reflex arcs.

Histogenesis of the oropharyngeal cavity taste buds and the relevant nerves and brain centers in substrate-brooding and mouth-brooding cichlid fish (Cichlidae, Teleostei).

This study follows the histogenesis of the oropharyngeal cavity taste buds, along with the development of the relevant neural centers and gustatory nerves, in two cichlid species: the substrate-brooding Cichlasoma cyanoguttatum and the mouth-brooding Astatotilapia flavijosephi, from fertilization to 20-day-old juveniles, grown at a temperature of 26 degrees C. Significant differences in pace of development were shown between the two social types: Substrate-brooders complete embryogenesis and hatch 48 h after fertilization (HAF) and begin to swim 120 HAF, with the yolk sac disappearing 160 HAF, whereas mouth-brooders hatch 84 HAF and begin to swim 196 HAF, with the yolk sac disappearing 360 HAF. Histogenesis of primordial taste buds occurs 75 HAF and 160 HAF in C. cyanoguttatum and A. flavijosephi, respectively. Accordingly, the related sensory ganglia and nerves (VII, IX, and X) develop much earlier in the substrate-brooded larvae and postlarvae. Nerve and brain development in juvenile A. flavijosephi of 13 mm total length (TL) closely resemble those of 8-mm-TL C. cyanoguttatum. These differences in development continue throughout the early stages of growth. Similar differences are observed in the ripening and increase in number of taste buds and dentition on the jaws and pharyngeal bones. The possible triggers and causes of such differences in development, as well as the inductors of taste bud development, are discussed.

Apparent diffusion coefficient on rat brain and nerves intoxicated with methylmercury.

The effects of methylmercury chloride (MMC) on the degenerative changes in rat brain and cranial nerves were studied. Twelve Wistar rats were divided into two equal groups. The rat model of methylmercury intoxication (MMC group) was made by subcutaneously administering 10 mg mercury/g body weight daily for 7 days. Control group rats were infused with the same amount of normal saline during this period. Magnetic resonance imaging (MRI) measurements were performed before and 14 days after the first MMC administration, using a 4.7-T MR system. No significant focal changes were observed on T1- and T2-weighted MR images regarding the internal structures of the brains of the MMC-intoxicated rats, atrophy of the cerebellum, and dilatation of the arachnoid space around the brain stem of MMC-treated rats, but were demonstrated without edematous change. The apparent diffusion coefficients (ADC) of the cortex, caudate-putamen, and trigeminal nerve were not significantly different between the MMC-treated and control rats. However, the ADC parallel to the optic nerves were significantly increased in the MMC group, in contrast to the unchanged ADC perpendicular to the optic nerves. An electron microscopy study revealed a marked decrease of microtubules and moderate decrease of neurofilaments in the axons of myelinated fibers of optic nerves of the MMC-treated rats. We have thus demonstrated a disturbance in the integrity of microtubules and neurofilaments as a toxic action of MMC in the rat nervous system in vivo, particularly in the optic nerves. The use of ADC values calculated by diffusion-weighted MRI is a promising approach for the evaluation of changes in brains and nerves in methylmercury intoxication research.

Rhombomere origin plays a role in the specificity of cranial motor axon projections in the chick.

Guidance of cranial motor axons to their targets conforms to a segmental plan in the chick embryo. Trigeminal motor neurons lie within rhombomeres 2 and 3 and project via an exit point in rhombomere 2 to innervate the first branchial arch. Facial motor neurons lie within rhombomeres 4 and 5 and grow out via an exit point in rhombomere 4 to innervate the second branchial arch. We have investigated the axial level-specific matching of motor neurons and branchial arches using donor to host transplantation in avian embryos. Previous work has shown that rostrocaudal reversal of a single hindbrain segment (rhombomere 3) leads to misprojection of a contingent of trigeminal axons via the facial nerve exit point. Using the same experimental manipulation in chick embryos and quail-chick chimaeras, we have analysed the pathways of these aberrant projections. We have found that in the majority of embryos analysed from stage 19 to 31, trigeminal axons from the transplanted rhombomere projected towards second branchial arch muscles, in addition to their normal first arch muscle targets. However, from stage 32 to 36, aberrant projections to second arch-derived muscles were detected only in a small minority of embryos. These experiments show that trigeminal motor neurons show a lack of specificity in their early projection into the periphery but that inappropriate projections may be later eliminated. This suggests that segmental mechanisms intrinsic to the hindbrain specify motor neurons with respect to their eventual innervation pattern.

Cell movements, neuronal organisation and gene expression in hindbrains lacking morphological boundaries.

Rhombomeres are segmental units of the hindbrain that are separated from each other by a specialised zone of boundary cells. Retinoic acid application to a recently segmented hindbrain leads to disappearance of posterior rhombomere boundaries. Boundary loss is preceded by changes in segmental expression of Krox-20 and Cek-8 and followed by alterations in Hox gene expression. The characteristic morphology of boundary cells, their expression of follistatin and the periodic accumulation of axons normally associated with boundaries are all lost. In the absence of boundaries, we detect no change in anteroposterior dispersal of precursor cells and, in most cases, no substantial cell mixing between former rhombomeric units. This is consistent with the idea that lineage restriction can be maintained by processes other than a mechanical barrier composed of boundary cells. Much of the early organisation of the motor nuclei appears normal despite the loss of boundaries and altered Hox expression.

Immunohistochemical and Ultrastructural characterization of the terminal nerve ganglion cells of the ayu, Plecoglossus altivelis (Salmoniformes, Teleostei).

BACKGROUND: Little is known on the cytological properties of the terminal nerve ganglion (TNG) cells in teleosts (Demski, 1993. Acta Anat., 148:81-95). MATERIALS AND METHODS: To characterize the TNG cells of a salmonoid fish, Plecoglossus altivelis, we adopted immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The majority of the TNG cells formed a compact mass halfway between the olfactory sac and the olfactory bulb, whereas a few cells were scattered in the ventromedial region of the olfactory bulb. The cell had a voluminous perikaryon that was positive to antisera against gonadotropin-releasing hormone (GnRH), molluscan cardioexcitatory tetrapeptide (FM-RFamide), and neuropeptide Y (NPY). Immunostaining of consecutive sections with each antiserum showed the coexistence of these antigens in the same cells and their processes. Most of the processes originating from the cells projected centrally to the basal forebrain, including the optic nerve. With TEM, the cells revealed a peptidergic nature, i.e., the presence of abundant granular endoplasmic reticula and well-developed Golgi bodies in association with vesicles that were 70-100 nm in diameter. Occasionally, the cells adjoined one another directly without the intervention of glial sheets. Synaptic contacts were frequent in the proximal region of the processes, where thin lateral processes of the cells and axon terminals of unknown origin were intermingled with each other. Terminal buttons being engulfed by the soma were commonly seen. CONCLUSIONS: The TNG cells of the salmonoid fish share many cytological characteristics with the cells of the nucleus olfactoretinalis of advanced teleosts such as acanthopterygians.

An in vitro evaluation of the effects of local magnetic-susceptibility-induced gradients on anisotropic water diffusion in nerve.

It has been suggested that the anistropy of the water-diffusion coefficient measured in nerve and in white matter could arise from locally anisotropic background gradients induced by the static field, B0. By utilizing 1) pulse sequences, which minimize the effects of background gradients, and 2) changes in sample orientation, which would maximize the change in the magnitude of these gradients if present, it is shown that in four excised nerves the background gradients do not play a measurable role in the anisotropy of the water-diffusion coefficient at a field strength of 2.35 T. The excised nerves evaluated were the olfactory, trigeminal, and optic nerves of the garfish and the sciatic nerve of the frog.

Developmental increases in expression of neurofilament mRNA selectively in projection neurons of the lamprey CNS.

Neurofilaments of the sea lamprey are unique in being homopolymers of a single subunit (NF-180). Digoxigenin-labeled RNA probes complementary to NF-180 were used to determine the distribution and timing of expression of neurofilament message in the brain and spinal cord of the lamprey. In the brainstem, detection of NF-180 mRNA was restricted to neurons with axons projecting to the spinal cord or the periphery. The majority of brainstem neurons, whose axons project locally, did not express NF-180 within the detection limits of this technique. NF-180-positive neurons included cells with a wide range of axon diameters, suggesting neurofilament mRNA expression was linked to axon length rather than caliber. To further evaluate this hypothesis, expression was studied in animals of different developmental stages between larvae and adults. In younger (shorter) larvae, the large Mauthner and rhombencephalic Müller cells did not express NF-180 mRNA, even though their axons are among the largest caliber in the animal and extend the entire length of the spinal cord. In contrast, many other reticulospinal neurons, whose axons are smaller in diameter than those of the Müller and Mauthner cells, expressed NF-180 message throughout larval development. Furthermore, neurons of the cranial motor nuclei did not express NF-180 until later developmental stages and the extraocular motor neurons did not label until metamorphosis. Therefore, while detectable neurofilament mRNA expression in the lamprey is restricted to neurons with long axons, its expression in this population of neurons appears to be developmentally regulated by factors still not determined. It is postulated that need for NF message is determined by a balance between the volume of axon to be filled and the rate of turnover of NF in that axon.

Cranial nerves and brain fiber systems of the medaka fry as observed by a whole-mount staining method.

The cranial nerves and the brain fiber systems of the medaka (Oryzias latipes) fry are revealed by a whole-mount staining method. Newly hatched fry of an albino strain of the medaka were fixed, partially digested with trypsin, treated in 1% Triton X-100, and finally immunohistochemically stained using anti-neurofilament protein (70K+160K+210K) antibodies. Since both head skin and eyes were colorless in the albino fish, the three-dimensional distribution of of nerve fibers in the brain could be readily observed in whole specimens without interference of pigment cells. All cranial nerves and main fiber systems in the adult fish were differentiated in the fry brain. Using this method, the distribution of nerves to the ocular muscles and the periorbital pit organs was shown.

Pathology of roots, spinal cord and brainstem in syringomyelia-like syndrome of Tangier disease.

We report here a post-mortem examination of a 46-year-old patient who died after a 23-year-long syringomyelia-like syndrome of Tangier disease. The L5 dorsal root and the superficial peroneal nerve showed fiber loss and lipid vacuole accumulation in Schwann cell cytoplasm. The L5 ventral root had moderate fiber loss without lipid vacuoles. In the cervical roots, fiber loss was intense and there were no foamy Schwann cells. Motor neuron loss was severe in the cervical spinal cord and the facial nerve nucleus and slight at the lumbar level. Under electron microscopy, some neurons of the lower spinal cord showed atypical inclusions. These data suggest that an unknown metabolic defect is responsible for a primary neuronopathy. Lipid accumulation in Schwann cells, resulting from fiber degeneration is probably transient, accounting for the absence of foamy cells in regions with longstanding involvement.

PNS-CNS transitional zone of the first cranial nerve.

This study examined the ultrastructure of the region of transition where fascicles of olfactory axons leave the peripheral nervous system (PNS) to enter the central nervous system (CNS), the so-called PNS-CNS transitional zone. Adult rats were transcardially perfused with a solution of 1% glutaraldehyde and 1% paraformaldehyde, decapitated, and the heads decalcified over a period of several weeks in a solution of 1% glutaraldehyde in 0.1 M tetrasodium ethylenediamine tetraacetic acid; the latter solution was changed daily. It was found that astrocytes did not form the glia limitans at the nerve entry zone, unlike the situation that exists in other cranial and spinal nerves. Rather, the glia limitans in this region of the olfactory bulb was formed by a special type of glial cell, referred to as an ensheathing cell. Ensheathing cells are found only in the nerve fiber layer of the olfactory bulb. They possess a mixture of Schwann cell and astrocytic features and are more likely to be of placodal than of CNS origin. The meningeal coverings of the olfactory nerve rootlets and of the olfactory bulb are also described and the functional implications of the findings discussed.

Descending brainstem projections of the pedunculopontine tegmental nucleus in the rat.

Descending brainstem projections from the pedunculopontine tegmental nucleus (PPN) were studied in the rat by use of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) and the retrograde tracer lectin-conjugated horseradish peroxidase (HRP-WGA). Results of these experiments demonstrated prominent bilateral projections to the pontomedullary reticular nuclei, but direct connections to the motor and sensory nuclei of the cranial nerves could not be ascertained. The PPN fibers terminated mainly in the pontine reticular nuclei oralis and caudalis and in ventromedial portions (pars alpha and pars ventralis) of the gigantocellular reticular nucleus. A smaller number of labeled fibers distributed to more dorsal regions of the gigantocellular nucleus, lateral para-gigantocellular, ventral reticular nucleus of the medulla and lateral reticular nucleus. Although a significant number of PHA-L labeled fibers was seen in two cases in the contralateral medial portion of the facial nucleus, and all cases exhibited a sparse predominantly ipsilateral projection to the lateral facial motor neurons, the retrograde tracing experiments have revealed that these facial afferents originated in the nuclei surrounding the PPN. The results are discussed in the context of PPN involvement in motor functions. It is suggested that the PPN may participate in a complex network involved in the orienting reflex.

Organization of choline acetyltransferase-containing structures in the cranial nerve motor nuclei and lamina IX of the cervical spinal cord of the rat.

The cholinergic structures in the cranial nerve motor nuclei and lamina IX of the cervical spinal cord of the rat were examined immunohistochemically with a monoclonal antibody to choline acetyltransferase (ChAT). The brainstem motor nuclei were classified into 3 groups according to the way of distribution of ChAT-positive structures in the neuropil of the nucleus. 1. The oculomotor, trochlear and abducent nuclei contained moderately ChAT-positive perikarya and dendrites. A small number of ChAT-positive bouton-like structures were found in the neuropil, but they were not in contact with ChAT-positive perikarya and dendrites. 2. Motoneurons in the facial, hypoglossal and trigeminal motor nuclei were moderately to strongly ChAT-positive. There were in the neuropil numerous ChAT-positive bouton-like structures and many of them were in contact with ChAT-positive perikarya and dendrites. The same pattern of organization of ChAT-positive structures was observed in lamina IX of the cervical spinal cord. In the nucleus ambiguus, the perikarya and proximal dendrites of about half population of motoneurons contacted with ChAT-positive bouton-like structures while the rest of motoneurons did not. ChAT-positive bouton-like structures in contact with motoneurons are interpreted as the cholinergic synaptic terminals, and this suggests that motoneurons in this group are cholinoceptive as well as cholinergic. 3. Neuronal perikarya and dendrites in the dorsal nucleus of the vagus showed weak to moderate positivity of ChAT. The neuropil of this nucleus was free from any distinctly ChAT-positive structures.

Dense-cored vesicle-containing components of the terminal nerve of sharks and rays.

This paper describes electron microscopic observations of dense-cored vesicle-containing axons, cell bodies, and endings of the terminal nerve in several elasmobranchs. The vesicles are found in two apparent cell types, one with a polymorphic nucleus and another with an oval nucleus. The types may correspond to cells producing one each of two neuropeptides (LHRH and FMRF-amide) that have previously been localized in the nerve. Dense-cored vesicles are found in many unmyelinated fibers in both the terminal nerve proper and its major ganglia. Some of these form complicated structures with interdigitation and wrapping of membranes. Vesicle-containing fibers branch from the nerve, run along nearby blood vessels, and appear to end adjacent to endothelial cells which demonstrate vesicular activity. The observations suggest terminal nerve neurosecretion into the cerebral circulation. Synapses are found in and near the ganglia where they appear to be axodendritic, with multiple contacts in some cases.

Histochemical and electron microscopical demonstration of the sympathetic nerve fibers joining to the fourth and the sixth cranial nerves in rats.

The localization of sympathetic fibers on the floor of the cranium was studied in rats using amine fluorescence histochemistry, neuropeptide-Y (NPY) immunohistochemistry, and electron microscopy. The vast majority of amine fluorescent fibers joined the abducent nerve and were localized in the peripheral zone under the perineurium. After advancing along this nerve for some distance, the fibers diverged into many bundles that converged to form the cavernous plexus at a rostral end of the trigeminal ganglion. On the dorsal surface of the trigeminal ganglion, one or two medium-calibered fluorescent bundles ran inside or in close proximity to the trochlear nerve, while many small-calibered, brightly fluorescent bundles also extended longitudinally in the epidural connective tissue. In rats that had undergone nerve severance, NPY-immunoreactive fibers were detected at the cut ends of the abducent and trochlear nerve. The differing amounts of NPY accumulated at the rostral and the caudal stumps indicated the direction of the NPY-bearing fibers. Electron microscopy confirmed the presence of unmyelinated fibers in both the abducent and trochlear nerves.

Changes of the ratio between myelin thickness and axon diameter in human developing sural, femoral, ulnar, facial, and trochlear nerves.

Previous studies on sural nerves were extended to human femoral, ulnar, facial and trochlear nerves. As asynchronous development of axon diameter and myelin sheath thickness was noted in all nerves studied. Whereas axons reach their maximal diameter by or before 5 years of age, maximal myelin sheath thickness is not attained before 16-17 years of age, i.e., more than 10 years later. The slope of the regression lines for the ratio between axon diameter and myelin thickness is significantly steeper in older than in younger individuals; it also differs if small and large fibers with more or less than 50 myelin lamellae are evaluated separately. The number of Schmidt-Lanterman incisures during later stages of development is related to myelin thickness, but the length of the spiral of the myelin lamella, thought to unrolled, in relation to its width, i.e., internodal length, varies considerably during development. The changes of the relationship between axons and myelin sheath thickness during normal human development have to be taken into account if hypomyelination is considered as a significant pathological phenomenon in peripheral neuropathies, especially in children. The implications of the present findings concerning conduction velocity of peripheral nerve fibers and other electrophysiologic parameters are discussed.

Neuritis of the cauda equina in the horse.

Ultrastructural lesions of the cranial nerves and their ganglia and the autonomic nervous system from 5 cases of neuritis of the cauda equina in the horse are described. They include lysosomal inclusions within the semilunar, geniculate and sympathetic chain ganglia, granulomatous involvement of the coeliaco-mesenteric ganglion and accumulation of axonal organelles in unmyelinated fibres of the great splanchnic nerve, sympathetic chain and oesophageal vagus.

The nervus terminalis in the mouse: light and electron microscopic immunocytochemical studies.

The distribution of gonadotropin-releasing hormone (GnRH)-containing neurons and fibers in the olfactory bulb was studied with light and electron microscopic immunohistochemistry in combination with retrograde transport of "True Blue" and horseradish peroxidase and lesion experiments. GnRH-positive neurons are found in the septal roots of the nervus terminalis, in the ganglion terminale, intrafascicularly throughout the nervus terminalis, in a dorso-ventral band in the caudal olfactory bulb, in various layers of the main and accessory olfactory bulb, and in the basal aspects of the nasal epithelium. Electron microscopic studies show that the nerve fibers in the nervus terminalis are not myelinated and are not surrounded by Schwann cell sheaths. In the ganglion terminale, "smooth" GnRH neurons are seen in juxtaposition to immunonegative neurons. Occasionally, axosomatic specializations are found in the ganglion terminale, but such synaptic contacts are not seen intrafascicularly in the nervus terminalis. Retrograde transport studies indicate that certain GnRH neurons in the septal roots of the nervus terminalis were linked to the amygdala. In addition, a subpopulation of nervus terminalis-related GnRH neurons has access to fenestrated capillaries whereas other GnRH neurons terminate at the nasal epithelium. Lesions of the nervus terminalis caudal to the ganglion terminale result in sprouting of GnRH fibers at both sites of the knife cut. The results suggest that GnRH in the olfactory system of the mouse can influence a variety of target sites either via the blood stream, via the external cerebrospinal fluid or via synaptic/asynaptic contacts with, for example, the receptor cells in the nasal mucosa.