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.     

Distribution of acetylcholinesterase and choline acetyltransferase in the main and accessory olfactory bulbs of the hedgehog (Erinaceus europaeus)

The distribution of cholinergic markers was studied in the main olfactory bulb (MOB) and accessory olfactory bulb (AOB) of the western European hedgehog (Erinaceus europaeus) by using choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry. A dense network of AChE-containing and ChAT-immunoreactive fibers was observed innervating all layers of the MOB except the olfactory nerve layer, where neither AChE- nor ChAT-labeled elements were found. The highest density of AChE- and ChAT-positive axons was found in the glomerular layer (GL)/external plexiform layer (EPL) boundary, and in the internal plexiform layer. This general distribution pattern of ChAT- and AChE-stained axons resembled the distribution pattern found in rodents. Nevertheless, some interspecies differences, such as the lack of atypical glomeruli in the hedgehog, were also found. In addition to fibers, a population of noncholinergic and presumably cholinoceptive AChE-active neurons was observed in the hedgehog. All mitral and tufted cells of the hedgehog MOB showed a dark AChE staining unlike previous observations in the mitral and tufted cells of rodents. As in other species previously reported, subpopulations of external tufted cells and short-axon cells were also AChE-active. Finally, a population of small AChE-containing cells was observed in the EPL of the hedgehog MOB. The size, shape, and location of these cells coincided with those of satellite and perinidal cells, two neuronal types described previously in the EPL of the hedgehog and not present in the rodent MOB. The AOB of the hedgehog showed a distribution of AChE- and ChAT-positive fibers similar to the rodent AOB. Nevertheless, a heterogeneous innervation of vomeronasal glomeruli by bundles of AChE- and ChAT-labeled axons found in the hedgehog has not been previously found in any other species. As in the MOB, all mitral cells in the AOB showed a strong AChE activity. These results demonstrate some similarities but also important differences between the distribution of ChAT and AChE in the MOB and AOB of rodents and this primitive mammalian. These variations may indicate a different organization of the cholinergic modulation of the olfactory information in the insectivores.     

Regionalization of Fos immunostaining in rat accessory olfactory bulb when the vomeronasal organ was exposed to urine.

The distribution of Fos-immunoreactive (Fos-ir) cells in the accessory olfactory bulb (AOB) of rats following vomeronasal organ exposure to urine was studied. Following exposure to male and female Wistar rat urine, Fos-ir cells were found in the mitral/tufted cell layer, granule cell layer and periglomerular cell layer of the AOB of female Wistar rat, with the highest number in the granule cell layer. Exposure to water or removal of the vomeronasal organ suppressed the expression of Fos-ir cells. These results suggest that female Wistar rats specifically detect urinary substances derived from male or female Wistar rats via the vomeronasal organ. Exposure of the vomeronasal organ of female Wistar rats to male Wistar urine induced the appearance of many more Fos-ir cells in all layers of the AOB than exposure to female Wistar urine. As for the mitral/tufted cell layer, the density of Fos-ir cells in the rostral portion (Gi2alpha-positive) of all regions of the AOB was about twice as high as that in the caudal portion when male urine was given. The distribution pattern of Fos-ir cells in response to female urine was not identical to that in response to male urine. That is, the density of Fos-ir cells in the caudal portion was slightly larger than that in the rostral portion in the lateral region, while in other regions the density in the rostral portion was higher than that in the caudal portion. It is likely that information from different pheromones is transmitted to the higher brain regions through the different regions of the AOB.     

An autoradiographic investigation of the projection of the vomeronasal organ to the accessory olfactory bulb in the mouse

Autoradiographic labelling was used to investigate the primary olfactory projections in the mouse. Small pieces of gelatin foam soaked in [³H]proline were inserted directly into the cavity of the vomeronasal organ. At survival times from 6 h to 10 days, radioactive label was found over the vomeronasal nerves and the glomeruli of the accessory olfactory bulb. Conversely, when radioactive amino acid was applied to the main olfactory mucosa, the label appeared over the main olfactory nerves and the glomeruli of the main olfactory bulb.In both the main and accessory bulbs there was evidence for a transneuronal uptake of label in the external plexiform and mitral cell layers subjacent to the labelled glomeruli. This transneuronal uptake was heavier at longer survival periods.     

Distribution of [3H]RNA in goldfish optic tectum following intraocular or intracranial injection of [3H]uridine. Evidence of axonal migration of RNA in regenerating optic fibers.

The distribution of [³H]RNA in the goldfish optic tectum following eitherintra-ocular orintracranial injection of [³H]uridine during optic fiber regeneration has been studied by light (LMA) and electron (EMA) microscopic autoradiography.In one group of 4 fish both optic nerves were crushed, and 18 days later [³H]uridine was injected into the right eye. A second group of 5 fish, in which only one optic nerve had been crushed, received intracranial injections of [³H]uridine 18 or 22 days after the crush. All fish were sacrificed 24 days after crushing the optic nerves, a time when regenerating optic fibers have entered the tectum and are establishing functional reconnections. Tecta were fixed in situ with glutaraldehyde, dissected out, and samples were processed for LMA and EMA. Controls were carried out to ensure that [³H]RNA was the only radioactive component present in the tissue after fixation.The distribution of silver grains related to [³H]RNA in intraocularly injected goldfish was different from that following intracranial injection. Following intraocular injection virtually all the [³H]RNA was located in the layers of the left optic tectum (contralateral to the side of intraocular injection) where the regenerating optic fibers course and terminate, whereas virtually no radioactivity was present in the right optic tectum. EMA quantitative analysis of the labeled layers of the left optic tectum revealed that perikarya of cells, most of which are glial cells, had a density of grains related to [³H]RNA of 20–28 g/100 sq.μm; axonal growth cones had a density of 14–24 g/100 sq.μm. Grain densities over non-axonal cell structures were markedly lower, ranging between 3 and 6 g/sq.μm. Grains located over axons and growth cones accounted for 50–60% of all counted grains.Inintracranially injected goldfish, either 2 or 6 days after injection, silver grains were clustered over leptomeninges as well as vessels and parenchymal cells of the tectal strata containing the regenerating optic fibers. In the stratum opticum a high grain density was seen over glial cells, whereas virtually no grains were present over the fascicles of regenerating axons. EMA quantitative analysis revealed a grain density over glial and other parenchymal cells of the stratum opticum of 67 g/100 sq.μm, whereas densities over growth cones and regenerating axons were 1.3 g/100 sq.μm and 1.8 g/100 sq.μm respectively. Grains located over axons and growth cones accounted for 3.3% of all counted grains.On the basis of the present and previous findings it is suggested that followingintraocular injection of [³H]uridine the [³H]RNA present inside regenerating optic axons is transported from the ganglion cells of the retina; on the other hand, the [³H]RNA present in surrounding glial cells is the result of local utilization of [³H]RNA precursors which also migrate from the retina along with the [³H]RNA.It is also concluded that 2 and 6 days followingintracranial injection of [³H]uridine no substantial tranfer of [³H]RNA from glial cells to regenerating optic fibers occurs in the goldfish optic tectum.     

Dendritic and axonal organization of mitral and tufted cells in the rat olfactory bulb

The output cells of the main olfactory bulb, the mitral and tufted cells, can be categorized into subclasses on the basis of their intrabulbar dendritic and axonal characteristics. Their form was studied in rats following labeling by iontophoretic injection of horseradish peroxidase into the external plexiform layer (EPL). The fact that these extracellular injections labeled small numbers of neurons permitted reconstruction of individual cells. The injection depth within the EPL determined the type of cells labeled. Secondary dendrites of each cell type lay in one of three partially overlapping zones in the EPL. The deepest zone contained the secondary dendrites of one group of mitral cells (Type I), which had the deepest and longest dendrites of the output cells. An intermediate zone of the EPL contained the secondary dendrites of middle tufted and a second class of mitral cells (Type II). The superficial zone, adjacent to the glomerular layer, contained the relatively short, asymmetric dendritic fields of external tufted cells. The few labeled internal tufted cells had secondary dendrites in either the intermediate or deep zones. Every cell type, except the Type I mitral cells, had axon collaterals in the internal plexiform and upper granule cell layers. No cell types had axons re-entering the EPL. These results for output cells combined with our previous observations on granule cells point to a functional sublaminar organization of the EPL that has not previously been proposed.     

Distribution of dendrites of mitral, displaced mitral, tufted, and granule cells in the rabbit olfactory bulb

To determine the dendritic fields, mitral, displaced mitral, middle tufted, and granule cells in the rabbit olfactory bulb were stained by intracellular injection of HRP. The secondary dendrites of mitral cells were distributed mostly in the inner half of the external plexiform layer (EPL). Those of displaced mitral cells extended mainly into the middle and superficial sublayers in the EPL. The secondary dendrites of middle tufted cells were distributed mostly in the superficial portion of the EPL. Mitral cells extended their secondary dendrites in virtually all directions within a plane tangential to the mitral cell layer (MCL) and thus had a disklike projection field with a radius of about 850 microns. Displaced mitral cells had similar dendritic projection fields in the tangential plane but with somewhat distorted shapes. The secondary dendrites of middle tufted cells had a tendency to extend in particular directions. From the projection pattern of the gemmules on the peripheral processes, granule cells were classified into three types. Type I granule cells had gemmules both in the superficial and in the deep sublayers of the EPL. The peripheral processes of Type II granule cells were confined to the deep half of the EPL. The gemmules of Type III granule cells ere distributed in the superficial half of the EPL. The differing dendritic ramification among mitral, displaced mitral, and middle tufted cells suggests the separation of the dendrodendritic synaptic interactions with granule cells in different sublayers in the EPL. It also suggests a functional separation of the sublayers of the EPL.     

Axonal transport and transcellular transfer of nucleosides and polyamines in intact and regenerating optic nerves of goldfish: speculation on the axonal regulation of periaxonal cell metabolism

The axonal transport, metabolism, and transcellular transfer of uridine, adenosine, putrescine, and spermidine have been examined in intact and regenerating optic nerves of goldfish. Following intraocular injection of labeled nucleosides, axonal transport was determined by comparing left-right differences in tectal radioactivity, and transcellular transfer was indicated by light autoradiographic analysis. The results demonstrated axonal transport, transcellular transfer, and periaxonal cell utilization of both nucleosides in intact axons and severalfold increases of all of these processes in regenerating axons. Experiments in which the metabolism of the nucleosides was studied resulted in data which suggested that uridine and adenosine, when delivered to the tectum by axonal transport, are protected from degradation and thus are relatively more available for periaxonal cell utilization than nucleosides reaching these cells via the blood. In intact axons, the majority of the nonmetabolized radioactivity was present as UMP, UDP, and UTP following [3H]uridine injections, whereas the majority of the radioactivity following [3H]adenosine injections was present as adenosine, with the phosphorylated derivatives constituting a smaller proportion. During nerve regeneration, the relative proportion of nucleosides to nucleotides was reversed, with uridine being the principal labeled compound in the first case, and AMP, ADP, and ATP being the major labeled compounds in the latter case. The nucleosides also were found to be different from each other in that adenosine, but not uridine, can be taken up by optic axons and transported retrogradely from the tectum to retinal ganglion cell bodies in the eye. Following intraocular injection of [3H]spermidine, radioactivity was transported to the optic tectum and transferred to tectal cells in the vicinity of the regenerating axons. Following [3H]putrescine injections, silver grains were found over periaxonal glia, but preliminary findings suggest that they are not present over tectal neurons nor over radial glial cells in the periependymal layers. Analysis of tectal radioactivity showed in each case that it was composed primarily of the injected compounds. These studies indicate that, following axonal transport, the polyamines do not remain within regenerating axons but are transferred to cells surrounding the axon. On the basis of these and previous findings, we speculate that the axonal transport and transcellular transfer of uridine, adenosine, polyamines, and perhaps other small molecules are means of communication between axons and periaxonal cells; that the axon can affect RNA and protein synthesis in periaxonal cells by regulating the availability of these small molecules; and that, during nerve regeneration, the increased metabolic needs of periaxonal cells are met by an increased axonal supply of precursors (adenosine and uridine) and other molecules (polyamines) critical for protein synthesis.     

Distribution of VIP- and NPY-like immunoreactivities in rat main olfactory bulb

The distribution of vasoactive intestinal peptide (VIP)- and neuropeptide Y (NPY)-like immunoreactivities in the Sprague-Dawley rat main olfactory bulb was analyzed using the peroxidase-antiperoxidase light microscopic immunocytochemical technique. VIP-like immunoreactivity was most prominently localized within a large number of intermediate-sized neurons whose perikarya and extensively branched varicose processes remained confined to the external plexiform layer (EPL). A few small short-axon type neurons in the mitral cell layer and granule cell layer (GRL) and even fewer large neurons in the glomerular layer (GL)/EPL border region contained immunoreactivity for VIP as well. Neuropeptide Y-like immunoreactivity (NPY-I) was principally localized within sparsely distributed large multipolar neurons of the deep GRL and within axons distributed with diminishing density from deep to superficial GRL. In addition, dense NPY-I was localized within very few large superficial short-axon type neurons of the GL/EPL border region. The restricted laminar and cellular distribution of NPY-I and VIP-I suggests that both peptides may act to modulate granule cell activity, and therefore, indirectly, olfactory bulb output.     

Noradrenaline-induced enhancement of oscillatory local field potentials in the mouse accessory olfactory bulb does not depend on disinhibition of mitral cells

The olfactory bulb differs from other brain regions by its use of bidirectional synaptic transmission at dendrodendritic reciprocal synapses. These reciprocal synapses provide tight coupling of inhibitory feedback from granule cell interneurons to mitral cell projection neurons in the accessory olfactory bulb (AOB), at the first stage of vomeronasal processing. It has been proposed that both the mGluR2 agonist DCG-IV and noradrenaline promote mate recognition memory formation by reducing GABAergic feedback on mitral cells. The resultant mitral cell disinhibition is thought to induce a long-lasting enhancement in the gain of inhibitory feedback from granule to mitral cells, which selectively gates the transmission of the learned chemosensory information. However, we found that local infusions of both noradrenaline and DCG-IV failed to disinhibit AOB neural activity in urethane-anaesthetised mice. DCG-IV infusion had similar effects to the GABA(A) agonist isoguvacine, suggesting that it increased GABAergic inhibition in the AOB rather than reducing it. Noradrenaline infusion into the AOB also failed to disinhibit mitral cells in awake mice despite inducing long-term increases in power of AOB local field potentials, similar to those observed following memory formation. These results suggest that mitral cell disinhibition is not essential for the neural changes in the AOB that underlie mate recognition memory formation in mice.     

Acrylamide exposure preferentially impairs axonal transport of glycoproteins in myelinated axons.

The right L5 dorsal root ganglion of adult rats exposed to acrylamide (40 mg/kg body weight/day for nine consecutive days) was injected with either [3H]methionine or [3H]glucosamine. After allowing incorporation into macromolecules and axonal transport to proceed for 5 hr, the distribution of radioactivity in cross sections and longitudinal sections of sciatic nerve was determined by autoradiography. Control and treated animals showed no difference in distribution of label within the sciatic nerve with respect to rapidly transported proteins labelled with [3H]methionine. In control animals the distribution of rapidly transported glycoproteins labelled with [3H]glucosamine was similar to that found for [3H]methionine-labelled proteins. In contrast, acrylamide-exposed rats had a very different distribution of labelled glycoproteins; there was a marked paucity of label in the myelinated axons. We interpret this result as indicating that acrylamide preferentially inhibits glycosylation or axonal transport of glycoproteins in neurons bearing myelinated axons.     

Different granule cell populations innervate superficial and deep regions of the external plexiform layer in rat olfactory bulb

Studies on the morphological organization of the main olfactory bulb have indicated that there are subpopulations of granule cells with different dendritic patterns in the external plexiform layer (EPL). Small, extracellular injections of horseradish peroxidase (HRP) were made iontophoretically into superficial and deep parts of the EPL and the granule cell layer (GCL) in adult rats. Superficial EPL injections principally labeled superficial granule cell somata, whereas deep EPL injections labeled both superficial and deep granule cell somata. Injections in the superficial GCL labeled granule cell dendritic processes extending across the entire EPL. However, deep GCL injections labeled few granule cell dendrites in the superficial EPL, but labeled many such processes in the deep EPL. These results were the same in material processed with the Hanker-Yates procedure, where the morphology of individual neurons could be studied, and in the more sensitive tetramethyl benzidineprocedure. Serial reconstructions of individual granule cells were made from both HRP and Golgi-Kopsch material. The distal dendrites of deep granule cells reached only as far as the deep EPL, where they branched extensively and had many dendritic spines. The dendrites of superficial granule cells, however, reached the most superficial part of the EPL where they ramified most extensively. The superficial granule cells typically had a higher spine density in the superficial part of the EPL than in the deep part. On the basis of these results, we conclude that the superficial granule cells predominantly innervate the superficial EPL and that the deep granule cells exclusively innervate the deep EPL. Granule cells are believed to exert inhibitory influences on the bulbar output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses. Since the secondary dendrites of the tufted cells ramify in the superficial EPL and the dendrites of most mitral cells ramify in deep EPL, the superficial and deep granule cells may preferentially modulate the responses of tufted and mitral cells, respectively.     

Regeneration of motor axons in the rat sciatic nerve studied by labeling with axonally transported radioactive proteins.

Labeling regenerating axons with axonally transported radioactive proteins provides information about the location of the entire range of axons from the fastest growing ones to those which are trapped in the scar. We have used this technique to study the regeneration of motor axons in the rat sciatic nerve after a crush lesion. From 2 to 14 days after the crush the lumbar spinal cord was exposed by laminectomy and multiple injections of [3H]proline were made stereotactically in the ventral horn. Twenty-four hours later the nerves were removed and the distribution of radioactivity along the nerve was measured by liquid scintillation counting. There was a peak of radioactivity in the regenerating axons distal to the crush due to an accumulation of label in the tips of these axons. After a delay of 3.2 +/- 0.2 (S.E.) days, this peak advanced down the nerve at a rate of 3.0 +/- 0.1 (S.E.) mm/day. The leading edge of this peak, which marks the location of the endings of the most rapidly growing labeled fibers, moved down the nerve at a rate of 4.4 +/- 0.2 mm/day after a delay of 2.1 +/- 0.2 days; this is the same time course as that of the most rapidly regenerating sensory axons in the rat sciatic nerve, measured by the pinch test. Another peak of radioactivity at the crush site, presumed to represent the ends of unregenerated axons or misdirected sprouts, declined rapidly during the first week, and more slowly thereafter.     

Localization of acetylcholinesterase in the main and accessory olfactory bulbs of the mouse by light and electron microscopic histochemistry

Experiments were conducted to examine by light and electron microscopy the localization of acetylcholinesterase (AChE) in the main (MOB) and accessory (AOB) olfactory bulbs of the normal mouse. Evidence from the literature for cholinergic innervation of the mammalian olfactory bulb was then assessed in light of possible correlation between reported sites of termination of centrifugal fibers to the olfactory bulb and the localization of AChE. AChE-positive nerve fibers were concentrated in the periglomerular region and internal plexiform layer of the MOB. Stained fibers were also present in the granule cell, mitral cell, and external plexiform layers as well as within glomeruli. A few neurons in all layers of the MOB contained AChE reaction product. Unlike the MOB, AChE-positive fibers were not present in the glomerular layer of the AOB. AChE-positive fibers were concentrated in the inner plexiform layer, whereas fewer stained fibers were observed in the external plexiform and mitral cell layer and granule cell layer. Lightly stained neurons were found in the deeper portions of the external plexiform and mitral cell layer and granule cell layer. Ultrastructurally, AChE reaction product in the MOB and AOB was predominantly associated with small unmyelinated axons. Reaction product was also observed adjacent to axon terminals and dendrites. Occasionally within the MOB, AChE activity was found within periglomerular, tufted, short-axon, mitral, and granule cells. In the AOB, however, intracellular AChE activity was observed within some mitral/tufted cells and only a few granule cells. In conclusion, the AChE reaction product was mainly associated with axons in regions of the MOB where centrifugal fibers have been reported. Accessory olfactory bulb AChE localization was different from that of the MOB, suggesting a different pattern of cholinergic input to the AOB. The small amounts and sites of intraneuronal AChE reaction product in cells of the olfactory bulb indicate cholinoceptive rather than cholinergic function.     

Golgi analyses of dendritic organization among denervated olfactory bulb granule cells

In the vertebrate olfactory bulb, the primary projection neurons, mitral and tufted cells, have reciprocal dendrodendritic synapses with respective subpopulations of anaxonic interneurons called granule cells. In the neurological murine mutant Purkinje Cell Degeneration (PCD), all mitral cells are lost during early adulthood. As a consequence, a subpopulation of granule cells is deprived of both afferent input and efferent targets. The effect of this event on the morphology and sublaminar distribution of granule cells was studied with light microscopic Golgi procedures in affected homozygous recessive PCD mutants and normal heterozygous littermate controls. In the control mice, a minimum of three subpopulations were identified predominantly on the basis of the topology of apical dendrites and their spinous processes within the external plexiform layer (EPL) of the olfactory bulb: type I had dendrites extending across the full width of the EPL and a homogeneous distribution of spines; type II had dendritic arbors confined to the deeper EPL; type III had apical dendrites that arborized extensively within the superficial EPL with no arbors or spines present in the deeper EPL. Prior studies suggest that type II cells form connections with mitral cells; type III cells form connections with tufted cells; and type I cells may integrate information from both populations of projection neurons. In the mutant PCD mice, the classification of subpopulations of granule cells proved difficult due to a compression of dendritic arbors within the EPL. Dendritic processes followed a more horizontal tangent relative to the radial orientation seen in control mice. The length of dendritic branches was reduced by approximately 20% with a corresponding decrease in the number of spines. The density of spines (#/1 micron of dendrite) was constant in both controls and mutants at approximately 0.21. Truncation of the dendrites in the PCD mutants appeared to occur at terminal portions because the number of dendritic bifurcations was equal in both groups of mice. The data are discussed in terms of subpopulations of granule cells in the mouse olfactory bulb, the sublaminar organization of olfactory bulb circuits, and the capacity for survival and plasticity in the reciprocal dendrodendritic circuits mediated by the granule cell spines.     

Reutilization of precursor following axonal transport of [3H]proline-labeled protein.

In further studies on axonally transported protein in the goldfish visual system, the turnover of rapidly transported [3H]proline-labeled protein was examined. It was found that: (1) a fraction of the rapidly transported protein has a relatively short half-life; (2) [3H]proline released following proteolysis of transported protein is efficiently reutilized for tectal protein synthesis, as inferred from an increased labeling of nuclear protein in the contralateral tectum (COT) relative to that in the ipsilateral tectum (IOT); (3) a small amount of [3H]proline arrives in the COT by axonal flow of the free amino acid; and (4) [3H]leucine and [3H]asparagine are less efficiently reutilized than [3H]proline. These findings may relate to the phenomenon of transneuronal transfer of radioactivity which has been observed with [3H]proline as precursor. The extensive reutilization of [3H]proline may account for part or all of the labeling at secondary synaptic sites. The results suggest that asparagine may be highly suitable for radioautographic identification of primary neuronal fields.     

Species differences in the distribution of substance P and tyrosine hydroxylase immunoreactivity in the olfactory bulb

These studies document species differences in the distribution of the peptide substance P and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) within a central nervous system region of a number of mammalian species including the mouse, rat, guinea pig, rabbit, cat, and two species of hamster (Chinese and Syrian). Substance P-containing neuronal perikarya were observed in the main olfactory bulb (MOB) of both species of the hamster, but not in the MOB of the other species examined. In the accessory olfactory bulb (AOB), however, neuronal staining was observed in all species except the mouse. The number of stained somata and their intensity varied such that label was most prominent in the rat followed in decreasing order by the rabbit, guinea pig, cat, and hamster. The mouse displayed no perikaryal staining. Stained somata in AOB were found in the internal granule cell layer with dendritic processes ramifying through the internal plexiform layer to arborize within the mitral cell layer. The distribution of substance P-stained neurons in the MOB also differed between the two hamster strains. In the Syrian hamster, neurons were primarily juxtaglomerular. In the Chinese hamster, labeled perikarya were found in both the juxtaglomerular region and within the superficial aspect of the external plexiform layer (EPL). The mean longest diameter of the majority of substance P-labeled neurons in both species was greater than 10 micron, suggesting that they were tufted cells. Those in the EPL of the Chinese hamster were the largest (17 micron). Species differences also were observed in the distribution of substance P-positive axons and terminals within the MOB. Label was distributed primarily in the internal granule cell layer of the Syrian hamster and the internal plexiform layer of the Chinese hamster. Tyrosine hydroxylase staining was similar among species with the exception of the Syrian hamster. In the latter species, an additional large population of neurons was found within the external plexiform layer. In all other species, TH-stained neurons were found scattered throughout the MOB and occasionally the AOB but were not numerous in the EPL. Although most TH neurons were larger than 10 microns, in all species a population of smaller TH cells was observed primarily in the glomerular layer, suggesting that most neurons labeled with TH are tufted cells but that some may be periglomerular cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lack of glia-axon transfer of proteins in the normal optic system of goldfish

An ultrastructural autoradiographic study of the goldfish optic tectum was carried out to determine whether proteins synthesized in glial cells are transferred into adjacent optic axons. Goldfish were injected intracranially over the optic tecta with [3H]leucine and fixed by perfusion 30 min, 4 and 24 h later. All unincorporated precursors were removed by repeated washings with fixatives, and tissue slices from the optic tecta were embedded and processed for electrom microscopy autoradiography (EMA). The densities of the silver grains were determined over optic axons and their myelin sheaths. The densities over the axons were lower than those over the myelin sheaths at all time intervals. The density of the intraaxonal grains, in absolute terms as well as relative to that of the myelin, was highest in the 4 h experiment. Analysis of the distribution of the grain densities over the myelin sheath and over concentric axonal compartments was carried out at this time interval to determine whether the grain density over the axon represented intraaxonal [3H]proteins or was only the result of grains 'scattered' from [3H]proteins located in the surrounding myelin sheaths. When the experimental distribution of the grain densities over the axon was compared with the theoretical distribution expected over the axon if only the myelin sheaths were labeled, no significant difference was found. This indicates that the silver grains present in the axons were scattered from the adjacent myelin sheath and did not represent intraaxonal radioactivity. It is therefore concluded that in our system there is not a quantitatively significant transfer of proteins between glial cells and adjacent axons.     

Conditional genetic labeling of mitral cells of the mouse accessory olfactory bulb to visualize the organization of their apical dendritic tufts

Chemical information captured through the vomeronasal sensory neurons is transmitted to mitral cells in the accessory olfactory bulb (AOB). Morphological characterization of AOB mitral cells is crucial to reveal the mechanisms underlying pheromone and other chemical information processing. Here, we developed a conditional genetic approach to visualize single AOB mitral cells in mice and analyzed the distribution of their apical dendritic tufts and cell bodies. We found that there is a subpopulation of superficial AOB mitral cells with small cell bodies and simple dendritic arborization. We also showed that segregation of the anterior and posterior AOB appears incomplete at the level of the mitral cell positions. Furthermore, we demonstrated that significant population of the anterior AOB mitral cells has multiple apical dendritic tufts organized in a quite small range along the dorsal–ventral axis. These findings have important implications in how various chemical signals may be processed in the AOB.     

Transneuronal transfer of radioactive substances during postnatal development of the hamster visual system

One day after intraocular injection of [³H]proline, significant amounts of axonally transported radioactive protein were present in the contralateral visual cortex of neonatal hamsters, postnatal age 14 days (P14) and older. Transneuronal transfer from the optic nerve to geniculocortical neurons was greatest in animals injected at the time of normal eye-opening, P14 to P16, and decreased with age. No significant accumulation of [³H]proline-derived radioactivity was found in the visual cortex of prefunctional (P12) animals or in old adults, although primary transport to the lateral geniculate body did not differ significantly from P14 animals. Accumulation in the nonvisual retrosplenial cortex showed a similar developmental pattern. In contrast, cortical incorporation of blood-borne [³H]-proline decreased steadily with age from P8 to adult. The failure to demonstrate transneuronal transfer of [³H]proline-labeled material in the prefunctional visual system was not due to a generalized inability to transfer rapidly transported protein, because significant radioactivity was found in visual cortex of P10, prefunctional hamsters 1 day after intraocular injection of [³H]fucose. It is suggested that enhanced transneuronal transfer of [³H]proline-derived label at the time of eye-opening depends primarily on events occurring in the retinal ganglion cell, e.g., changes in the synthetic fate of precursors and in the processes by which axonally transported materials are released from the optic nerve. Nonspecific developmental changes in uptake and reincorporation may also contribute to the observed pattern of transneuronal transfer.