Differential and overlapping expression patterns of X-dll3 and Pax-6 genes suggest distinct roles in olfactory system development of the African clawed frog Xenopus laevis.

In Xenopus laevis, the formation of the adult olfactory epithelium involves embryonic, larval and metamorphic phases. The olfactory epithelium in the principal cavity (PC) develops during embryogenesis from the olfactory placode and is thought to respond to water-borne odorants throughout larval life. During metamorphosis, the PC undergoes major transformations and is exposed to air-borne odorants. Also during metamorphosis, the middle cavity (MC) develops de novo. The olfactory epithelium in the MC has the same characteristics as that in the larval PC and is thought to respond to water-borne odorants. Using in situ hybridization, we analyzed the expression pattern of the homeobox genes X-dll3 and Pax-6 within the developing olfactory system. Early in development, X-dll3 is expressed in both the neuronal and non-neuronal ectoderm of the sense plate and in all cell layers of the olfactory placode and larval PC. Expression becomes restricted to the neurons and basal cells of the PC by mid-metamorphosis. During metamorphosis, X-dll3 is also expressed throughout the developing MC epithelium and becomes restricted to neurons and basal cells at metamorphic climax. This expression pattern suggests that X-dll3 is first involved in the patterning and genesis of all cells forming the olfactory tissue and is then involved in neurogenesis or neuronal maturation in putative water- and air-sensing epithelia. In contrast, Pax-6 expression is restricted to the olfactory placode, larval PC and metamorphic MC, suggesting that Pax-6 is specifically involved in the formation of water-sensing epithelium. The expression patterns suggest that X-dll3 and Pax-6 are both involved in establishing the olfactory placode during embryonic development, but subtle differences in cellular and temporal expression patterns suggest that these genes have distinct functions.

Neuronal turnover in the Xenopus laevis olfactory epithelium during metamorphosis.

Metamorphic changes in the amphibian olfactory system present many interesting questions concerning the competing possibilities of neuronal respecification versus replacement. For example, are olfactory neurons retained during this transition with their presumed sensitivity to waterborne versus airborne stimuli respecified, or are olfactory neurons completely replaced? We address this question using the African clawed frog (Xenopus laevis) as a model. The water-sensing nose (principal cavity; PC) of larval X. laevis is respecified into an air-sensing cavity in adults, with changes in odorant receptor gene expression, ultrastructure, and site of innervation of the receptor neurons. The vomeronasal organ (VNO) does not appear to change function, structure, or innervation during metamorphosis. We labeled PC and VNO olfactory receptor neurons with injections of retrogradely transported fluorescent microspheres into the main and accessory olfactory bulbs. Injections were performed in larvae, and animals were allowed to survive through metamorphosis. After metamorphosis, few labeled cells were observed in the PC, whereas the VNO and the olfactory bulbs remained heavily labeled. Animals that were killed before metamorphosis always had extensive label in the PC epithelium regardless of how long the beads were present. This suggests that changes in the PC olfactory epithelium that are seen during metamorphosis are due primarily to turnover of the neurons in this epithelium rather than to respecification of existing neurons. These results also are discussed in terms of natural turnover time of olfactory receptor neurons.

The role of the brain in metamorphosis of the olfactory epithelium in the frog, Xenopus laevis.

Retrograde signaling from the brain to the olfactory sensory epithelium is important for neuronal survival, but the importance of the olfactory bulb in retrograde signaling during the naturally-induced, neuronal plasticity occurring during metamorphosis is unclear. The olfactory system of the African clawed frog (Xenopus laevis) undergoes dramatic rearrangements during metamorphosis, making this an ideal system in which to examine interactions between the brain and the olfactory sensory epithelium. The main olfactory epithelium of larvae, located in the principal cavity (PC), changes at metamorphosis in function, receptor neuron morphology, biochemistry, and axon termination sites. A new, "middle", cavity forms during metamorphosis that assumes all the characteristics of the larval PC. Using a combination of bulbectomy and olfactory transplantation, we investigated changes in expression of a marker protein (E7) and in apical ultrastructure in olfactory receptor neurons either (1) connected to the olfactory bulb, (2) connected to non-olfactory brain regions, or (3) with no apparent central nervous system (CNS) connections. We find that neurons in the middle cavity (MC) lacking connections with the CNS appear mature but neurons in the PC do not. Supporting cells in the PC undergo the changes normally observed during metamorphosis. Neurons connected to non-olfactory brain regions, either after bulbectomy or transplantation, appeared normal with regard to the changes normally expected after metamorphosis. These results suggest that influence from the brain is necessary for metamorphic changes in the X. laevis olfactory epithelium, but that these signals are not confined to the olfactory bulb; non-olfactory brain regions can also support these metamorphic changes.

Differential antigen expression during metamorphosis in the tripartite olfactory system of the African clawed frog, Xenopus laevis.

In the adult African clawed frog, Xenopus laevis, olfactory epithelium is housed in three separate nasal cavities: the principal cavity, the middle cavity, and the vomeronasal organ. The sensory epithelium in each of these cavities has distinct cellular features, and presumed physiological and behavioral functions, which arise during metamorphosis. Most notably, the middle cavity is formed de novo, and the principal cavity is transformed from a larval sensory epithelium with water exposure to an adult olfactory epithelium with air exposure. To understand the cellular nature of this plasticity more clearly, we characterized the staining patterns generated in the olfactory system of X. laevis with a new monoclonal antibody, anti-E7. The olfactory epithelium is first stained with anti-E7 during late embryonic development. Transection of the olfactory nerves during metamorphosis eliminates all staining and indicates that the staining is associated with mature or nearly mature olfactory receptor neurons. The antibody diffusely stains the vomeronasal organ throughout development and in adults. In the larval principal cavity, the olfactory receptor neurons are brightly stained, but this cellular staining is lost after metamorphosis. The mucus from Bowman's glands in the principal cavity, however, is intensely stained in adults. The middle cavity, throughout development and in adulthood, has the same staining characteristics as the larval principal cavity. Thus, the E7 antibody can distinguish the three areas of the olfactory epithelium, allowing measurement of sensory epithelium volume, and serves as an excellent marker for the changes in the sensory epithelium that occur during metamorphosis.

Ultrastructure of the olfactory organ in the clawed frog, Xenopus laevis, during larval development and metamorphosis.

Development of the olfactory epithelia of the African clawed frog, Xenopus laevis, was studied by scanning and transmission electron microscopy. Stages examined ranged from hatching through the end of metamorphosis. The larval olfactory organ consists of two chambers, the principal cavity and the vomeronasal organ (VNO). A third sensory chamber, the middle cavity, arises during metamorphosis. In larvae, the principal cavity is exposed to water-borne odorants, but after metamorphosis it is exposed to airborne odorants. The middle cavity and the VNO are always exposed to waterborne odorants. Electron microscopy reveals that in larvae, principal cavity receptor cells are of two types, ciliated and microvillar. Principal cavity supporting cells are also of two types, ciliated and secretory (with small, electron-lucent granules). After metamorphosis, the principal cavity contains only ciliated receptor cells and secretory supporting cells, and the cilia on the receptor cells are longer than in larvae. Supporting cell secretory granules are now large and electron-dense. In contrast, the middle cavity epithelium contains the same cell types seen in the larval principal cavity. The VNO has microvillar receptor cells and ciliated supporting cells throughout life. The cellular process by which the principal cavity epithelium changes during metamorphosis is not entirely clear. Morphological evidence from this study suggests that both microvillar and ciliated receptor cells die, to be replaced by newly generated cells. In addition, ciliated supporting cells also appear to die, whereas there is evidence that secretory supporting cells transdifferentiate into the adult type. In summary, significant developmental additions and neural plasticity are involved in remodeling the olfactory epithelium in Xenopus at metamorphosis.

Influence of olfactory innervation on neurogenesis in the developing olfactory bulb of the frog, Xenopus laevis.

Previous research on development of the Xenopus olfactory bulb from our laboratory has shown that mitral cells begin to differentiate after olfactory axons make contact with the olfactory bulb, and the number of olfactory axons is correlated with the number of mitral cells throughout development. In embryos, removal of all afferent innervation before the mitral cells begin to differentiate results in a failure of the bulb to form; removal of half the olfactory axons, results in development of half the normal number of mitral cells. At larval stages, transection of the olfactory nerve results in a decrease in the number of neurons in the olfactory bulb. Thus, the olfactory axons play a major role in stimulating or maintaining development of the olfactory bulb neurons. Since we have found that neurogenesis in the bulb continues through metamorphosis, the goal of the current study was to determine whether olfactory axons influence cell genesis and/or neuronal maturation in the larval olfactory bulb. To study cell genesis, we cut the olfactory nerves, and 6 days later, injected the animals with [3H]thymidine. After 6 hr, the animals were killed and the tissue was processed for autoradiography. The number of labeled cells in the ventricular zone of the olfactory bulb was determined in every fifth section through the bulb in control and experimental animals. There was no statistical difference (Mann-Whitney rank sum test) in the number of labeled ventricular cells between controls and experimentals. Thus, olfactory axon innervation does not appear to play a role in precursor cell division during larval development. To study the influence of olfactory axon innervation on the ability of newly generated neurons to mature, we followed the same procedures. However, the animals were killed 21 days after the [3H]thymidine injection. The results from this experiment showed that there are many fewer labeled mitral cells in the experimental animals at 21 days. Together these results suggest that sensory deafferentation influences mitral cell differentiation or survival even during late stages of larval development.

Cellular and molecular interactions in the development of the Xenopus olfactory system.

Development of the olfactory system in Xenopus laevis begins during gastrulation, with the induction of olfactory placodes at the rostral edge of the prospective neural plate. Initial placodal induction appears to involve cerberus, a molecule secreted from the involuting anterior endoderm. Possible downstream genes expressed in the anterior neural ridge and sense plate include the transcription factors Pax-6, X-dll2, X-dll3, and Xotx2. Forebrain development is dependent on the presence of the placode and subsequent innervation by olfactory axons, with the extent of this dependence declining as development advances. During metamorphosis thyroid hormones initiate extensive changes in the olfactory system, including the origins of new regions of the olfactory epithelium and olfactory bulb, and a change in olfactory projection patterns.

Metamorphic remodeling of the primary olfactory projection in Xenopus: developmental independence of projections from olfactory neuron subclasses.

In adult Xenopus, the nasal cavity is divided into separate middle (MC) and principal (PC) cavities; the former is used to smell water-borne odorants, the latter air-borne odorants. Recent work has shown that olfactory neurons of each cavity express a distinct subclass of odorant receptors. Moreover, MC and PC axons project to distinct regions of the olfactory bulb. To examine the developmental basis for this specificity in the olfactory projection, we extirpated the developing MC from early metamorphic (stage 54-57) tadpoles and raised the animals through metamorphosis. In most lesioned animals, the MC partly regenerated. Compared with the unlesioned side, reduction of the region of the glomerular layer of the olfactory bulb receiving MC afferents ranged from 70% to 95%. PC afferents did not occupy regions of the olfactory bulb deprived of MC afferents. These results support a model in which intrinsic cues in the olfactory bulb control the projection pattern attained by ingrowing olfactory axons.

Neurogenesis in the olfactory bulb of the frog Xenopus laevis shows unique patterns during embryonic development and metamorphosis.

We determined the time of origin of neurons in the olfactory bulb of the South African clawed frog, Xenopus laevis. Tritiated thymidine injections were administered to frog embryos and tadpoles from gastrulation (stage 11/12) through metamorphosis (stage 65), paraffin sections were processed for autoradiography, and the distribution of heavily and lightly labeled cells was examined. In the ventral olfactory bulb, we observed that the mitral cells were born as early as stage 11/12 and continued to be generated through the end of metamorphosis. Interneurons (periglomerular and granule cells) were not born in the ventral bulb until stage 41, and birth of these cells also continued through metamorphosis. Labeled cells were observed in the accessory olfactory bulb, beginning at stage 41. In contrast, the cells of the dorsal olfactory bulb were not born until the onset of metamorphosis (stage 54); at this stage in the dorsal bulb, the genesis of mitral cells, interneurons, and glial cells completely overlapped. The results indicate that olfactory axon innervation is not necessary to induce early stages of neurogenesis in the ventral olfactory bulb. On the other hand, the results on the dorsal olfactory bulb are consistent with the hypothesis that innervation from new or transformed sensory neurons in the principal cavity induces neurogenesis in the dorsal bulb.

The quantitative relationship between olfactory axons and mitral/tufted cells in developing Xenopus with partially deafferented olfactory bulbs.

Partial deafferentation of the olfactory bulb in Xenopus embryos was performed to analyze the effects of afferent innervation on the development of the central olfactory structure. In an attempt to analyze a possible early inductive role of the olfactory axons, one olfactory placode was removed before differentiation of the neural tube began (stages 26-31). A morphological and quantitative analysis was performed on larvae at the onset of metamorphic climax (stage 58). When the single olfactory nerve innervated one side of the rostral telencephalon, a single olfactory bulb developed on that side and no olfactory bulb formed on the contralateral side. When the nerve innervated the midline of the rostral telencephalon, a smaller-than-normal, fused olfactory bulb developed. Partial deafferentation at these early stages resulted in a significant reduction in the number of olfactory axons (to approximately one-half of control values) and a corresponding decrease in the number of mitral/tufted cells (output neurons of the olfactory bulb). To control for possible damage to the neural tube during olfactory-placode removal, a portion of the neural tube directly beneath one of the olfactory placodes was removed in embryos. In these animals, the neural tube regenerated within 24 h and formed a normal olfactory bulb; olfactory axon and mitral/tufted-cell numbers were not significantly different from controls. In conclusion, olfactory-afferent innervation was critical for differentiation of the olfactory bulb, and decreasing the number of olfactory axons resulted in a reduction in the number of output neurons of the olfactory bulb.

Morphological and quantitative evaluation of olfactory bulb development in Xenopus after olfactory placode transplantation.

We found previously that the number of olfactory axons is correlated with the number of mitral/tufted cells (output neurons of the olfactory bulb) during normal larval development. To examine the significance of this quantitative relationship, we evaluated the effects of transplanting an extra olfactory placode on the development of the larval olfactory bulb. We found that the transplanted tissue retained the normal, pseudostratified, columnar appearance and had the same cell types as normal olfactory epithelium, and the olfactory bulbs had the same laminar organization as control bulbs. With gross examination of the olfactory bulb, the side innervated by the transplant appeared slightly larger than the contralateral side in animals analyzed at a young larval stage (stage 50) and in 2 of the 9 animals examined at late larval stages (57/58). Tissue sections and area measurements, however, revealed that the volume of the olfactory bulbs in animals with a transplant was not significantly different from control values. Our quantitative analysis also showed that in stage-50 animals with a transplant, the total number of olfactory axons (in nerves from the transplanted and host olfactory organs) appeared to be greater than in control animals, but not to a statistically significant level. The number of mitral/tufted cells was not different from controls. In animals examined at stage 57/58, there was no difference from controls in either the total number of olfactory axons, total number of mitral/tufted cells, or convergence ratio of olfactory axons onto mitral/tufted cells. Thus, in the late-stage larvae, the quantitative relationship between olfactory axons and mitral/tufted cells was not altered by the experimental manipulation. These results suggest that the olfactory bulb can regulate the number of afferent fibers.

Morphological study of the effects of intranasal zinc sulfate irrigation on the mouse olfactory epithelium and olfactory bulb.

The effects of intranasal zinc sulfate (ZnSO4) irrigation on the morphology of the olfactory epithelium and olfactory bulb were studied in mice with short survival times (as early as 1 day) and with long survival times (up to 593 days) after the irrigation procedure. As in several previous studies, the olfactory epithelium was completely destroyed within a few days after the ZnSO4 treatment. Within 2-4 days, the septum and turbinates were covered by a new, cuboidal epithelium, the cells of which differed significantly from any cells normally seen in the olfactory epithelium. Slowly, over several months, small areas of the olfactory epithelium regenerated in many of the animals. The ultrastructural changes occurring in the olfactory bulb from 1 to 25 days (the reactive stage) were characterized by degenerating olfactory axons and axon terminals, hypertrophy of astroglial cell processes, and proliferation of or extravasation by phagocytic cells. By 25 days after intranasal ZnSO4 irrigation, the number of reactive glial processes and phagocytic cells returned to normal. In some mice with survival times of 150 days or longer, there was reinnervation of small areas of the olfactory bulb by regenerated olfactory axons. These new olfactory axons innervated only superficial glomeruli or the outer portions of deeper glomeruli, but they formed synaptic contacts with mitral/tufted cells and periglomerular cells that did not differ from control animals. These findings were supported by tract-tracing experiments with 3H-amino acids and by behavioral analysis. In summary, the ultrastructural changes observed in the olfactory bulb in this study were not significantly different from those observed after surgical lesions of the olfactory epithelium or nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of the olfactory nerve in the clawed frog, Xenopus laevis: II. Effects of hypothyroidism.

Quantitative and morphological data were obtained on developing olfactory axons in normal and hypothyroid larvae of the African clawed frog Xenopus laevis. Hypothyroid larvae were produced by rearing the animals, beginning at stage 48, in a 0.01% solution of propylthiouracil (PTU), a treatment that blocks synthesis of thyroid hormone. These PTU-treated larvae were compared to their age-matched siblings when these siblings reached stage 52 (premetamorphic larvae; prior to synthesis of thyroid hormone), stage 57 (late premetamorphic larvae; after the onset of thyroid hormone synthesis), or stage 58 (larvae at the onset of metamorphic climax; thyroid hormone levels continue to rise). The number of olfactory axons did not differ between stage 52 control animals and the age-matched, PTU-treated animals, but there were only about half the number of axons in the PTU-treated animals that were age-matched to the stage 57 or 58 controls. Thus, PTU had no effect on olfactory axon number prior to the normal rise in thyroid hormone levels. But PTU significantly reduced the normal increase in olfactory axon number compared to stage 58 control larvae, whose thyroid hormone levels are high. While PTU also produced some changes in several other body measurements, the effect on the olfactory axons was the most consistent and prominent. The results presented here support our previous findings that thyroid hormone plays a significant role in the development of the olfactory system in Xenopus.

Development of the olfactory bulb in the clawed frog, Xenopus laevis: a morphological and quantitative analysis.

The relationship between olfactory axons and the cells of the olfactory bulb during normal development was analyzed to determine whether olfactory afferent axons could play a role in the induction of olfactory bulb formation. The morphology of the olfactory bulb in Xenopus larvae from stages 26 to 58 and in adult frogs was analyzed with light and electron microscopy. Axons were first observed beneath the basal lamina of the neural tube at stages 30 and 32; at stage 32, neurons in this area of the neural tube began to differentiate. Synapses of olfactory axons onto young neuronal processes were observed as early as stages 36 and 38. By stage 44, all of the layers of the olfactory bulb were present. The basic structure of the mature form of the olfactory bulb was apparent as early as stage 48/49 and remained constant throughout late larval life and even into adulthood, with only the size increasing. To determine the numerical relationship between olfactory axons from both main and vomeronasal epithelia and mitral/tufted cells in the main and accessory olfactory bulbs, a quantitative study was also performed in which the number of olfactory axons and the number of mitral/tufted cells were estimated for larval stages (stages 50 to 58) and adults. The number of axons increased with stage, with a 16-fold increase between stage 58 and adulthood. The number of mitral/tufted cells increased with stage, with only a 2.3-fold increase between stage 58 and adults. There is a correlation between axon number and mitral/tufted cell number during larval development that is consistent with the hypothesis that olfactory axons influence olfactory bulb development. The convergence ratio of olfactory axons onto mitral/tufted cells was 5:1 in larvae and increased to 34:1 in adults; this increase probably results in increased olfactory sensitivity in adult frogs.

Development of the olfactory nerve in the African clawed frog, Xenopus laevis: I. Normal development.

Quantitative and morphological data were obtained on developing olfactory axons in the African clawed frog, Xenopus laevis, during late premetamorphosis (stages 48-54), prometamorphosis (stages 55-57), and halfway through metamorphic climax (stages 58-62). Larval axons throughout these stages of development did not change with respect to morphology or diameter and were similar in all respects to olfactory axons described in other vertebrate species. The number of axons in the olfactory nerve increased throughout development, more rapidly after stage 54. Based on comparisons of the number of axons in proximal and distal regions of the nerve, there also appeared to be more axons growing into the olfactory nerve at early metamorphic climax than during premetamorphosis. Through the onset of metamorphic climax, the number of olfactory axons was correlated with other measures of body growth. In the later stages of climax, however, the number of olfactory axons continued to rise, whereas body weight, length, and width, as well as olfactory nerve length, decreased. Not all animals developed at the same rate, but for all quantitative measurements in this study, stage was a better predictor of any given parameter than age of the animal. Rearing conditions affected the rate of development but did not have a significant effect on most of the features analyzed quantitatively. Although most of the new olfactory axons in these larval animals probably represent addition of fibers resulting from development, the ensheathing glial cells at all stages showed evidence of phagocytic activity, suggesting that there might be turnover of olfactory receptor cells during larval development. The results presented here provide a baseline for future reports on various factors that may influence normal development in this system.

Development of GABA immunoreactivity in brainstem auditory nuclei of the chick: ontogeny of gradients in terminal staining.

The development of gamma-aminobutyric acid-immunoreactivity (GABA-I) in nucleus magnocellularis (NM) and nucleus laminaris (NL) of the chick was studied by using an antiserum to GABA. In posthatch chicks, GABA-I is localized to small, round punctate structures in the neuropil and surrounding nerve cell bodies. Electron microscopic immunocytochemistry demonstrates that these puncta make synaptic contact with neuronal cell bodies in NM; thus, they are believed to be axon terminals. GABAergic terminals are distributed in a gradient of increasing density from the rostromedial to the caudolateral regions of NM. The distribution of GABA-I was studied during embryonic development. At embryonic days (E) 9-11, there is little GABA-I staining in either NM or NL. Around E12-14, a few fibers are immunopositive but no gradient is seen. More GABA-I structures are present at E14-15. They are reminiscent of axons with varicosities along their length, preterminal axonal thickenings and fiber plexuses. At E15, terminals become apparent circumscribing neuronal somata and are also discernible in the neuropil of both nuclei. In E16-17 embryos, terminals are the predominantly labeled GABA-I structures and they are uniformly distributed throughout NM. The density of GABAergic terminals increases in caudolateral regions of NM such that by E17-19, there is a gradient of increasing density of GABA-I terminals from the rostromedial to caudolateral regions of NM. The steepness of this gradient increases during development and is the greatest in posthatch (P) chicks. Cell bodies labeled with the GABA antiserum are located around the borders of both NM and NL and in the neuropil between these two nuclei. Occasionally, GABA-I neurons can be found within these auditory brainstem nuclei in both embryonic and posthatch chicks. Nucleus angularis (NA) contains some GABAergic cells. The appearance of GABA-I terminals around E15 is correlated in time with the formation of end-bulbs of Held on NM neurons. Thus, the ontogeny of presumed inhibitory inputs to chick auditory brainstem nuclei temporally correlates with, and could modulate the development of, excitatory auditory afferent structure and function.

Effect of testosterone on input received by an identified neuron type of the canary song system: a Golgi/electron microscopy/degeneration study.

Combinations of the Golgi stain, anterograde degeneration, and electron microscopy are used to further characterize the hormone-sensitive "type IV" neuron of the forebrain nucleus robustus archistriatalis (RA) of adult female canaries. Anterograde degeneration was used to "stain," at the electron-microscopic level, the axon terminals of neurons projecting to RA from hyperstriatum ventralis, pars caudalis (HVc) and from the lateral magnocellular nucleus of the anterior neostriatum (L-MAN). The HVc neurons projecting to RA type IV cells form synapses predominantly on the dendritic spines of those cells, while L-MAN neurons that project to RA type IV cells form a 2.5:1 mixture of shaft and spine synapses. There were about 1000 synapses from HVc neurons (about 30% of all spine synapses) on typical type IV cells and about 50 synapses from L-MAN neurons. Earlier work had shown that in female canaries the dendrites of type IV neurons of the avian song control nucleus RA increase in total length after systemic testosterone treatment, and that this increase in dendritic length was accompanied by the development of malelike song. We now show that testosterone treatment also increases the number of dendritic spines present in type IV neurons. Presumably this is accompanied by an increase in the number of synaptic inputs received by type IV cells. Earlier evidence suggested that the testosterone-induced addition of extra dendritic length to type IV cells occurred at existing dendritic tips. We tested the hypothesis that these added peripheral ends received a special subset of inputs, which might then account for the change in behavior, and found it to be false. Mapping and counts of degenerating synapses resulting from lesion of HVc and L-MAN suggest that under the influence of hormone, new synapses are added throughout the dendritic tree, with no special distribution or change in ratio of inputs occurring at the tip of dendrites. Under the influence of testosterone, each type IV cell may receive only "more of the same" inputs it received before onset of treatment. We speculate on how such changes in circuitry may relate to song stability and learning.

Excitotoxin lesions do not mimic the alteration of somatostatin in Huntington's disease.

Huntington's disease is accompanied by severe neuronal death in the striatum, but despite this cell loss, there is a marked increase in the striatal concentration of somatostatin-like immunoreactivity (SLI). We attempted to examine the mechanism of this increase by using kainic or ibotenic acid to effect a unilateral lesion in the rat neostriatum. Graded doses of toxin cause a proportional decrease in the concentration of somatostatin-like immunoreactivity to a maximum of 50% of control, which is stable over an interval of 3 months. The increased somatostatin-like immunoreactivity in Huntington's disease is not mimicked by the excitotoxin lesions in rats. In addition we find that unilateral kainic acid lesions in the striatum reduce SLI in the contralateral striatum as well, although histologic evidence and assay of choline acetyltransferase activity indicate that damage is confined to the injected side. Immunocytochemistry demonstrates a loss of somatostatin-containing neurons on the lesioned side but no discernible loss in the contralateral striatum. The bilateral decrease in SLI following unilateral lesions suggests damage to a somatostatin projection to the contralateral striatum or a compensatory interaction between the two striatal nuclei.

Ultrastructural characterization of synaptic terminals formed on newly generated neurons in a song control nucleus of the adult canary forebrain.

The fine structure of synaptic terminals contacting neurons generated in the forebrain of adult male canaries was investigated by autoradiography and electron microscopy. The procedure for labeling the new neurons included pretreating adult canaries with 3H-thymidine and sacrificing them 23-45 days later. Neurons were identified as newly generated by the presence of 3H-thymidine in the cell nucleus. The new neurons in the nucleus hyperstriatum ventralis, pars caudalis (HVc) were identified by autoradiography and light microscopy and examined with electron microscopy. Several types of synaptic terminals contacted the cell body and proximal dendrites of the newly formed neurons. Synaptic junctions were formed by terminals that contained spherical, agranular vesicles, large dense-core vesicles and spherical, agranular vesicles, and pleomorphic or flattened synaptic vesicles. Terminals that contained spherical vesicles were most often associated with asymmetric synaptic densities, and terminals that contained pleomorphic or flattened vesicles formed symmetric junctions. New neurons were also contacted by small terminals that contained few vesicles and had little pre- or postsynaptic density associated with the junction; these terminals may be a special type or may be in the process of developing their synaptic contact with the new neuron. In addition, rare terminals that appeared to be degenerating or to contain debris from other degenerating neural elements contacted new neurons. In summary, these data indicate that the new neurons, which are known to be inserted into existing neural networks, receive synaptic input from at least three different sources.

High-resolution 2-deoxyglucose autoradiography in quick-frozen slabs of neonatal rat olfactory bulb.

We have used rapid freezing and freeze-substitution fixation to permit electron microscopic study of [3H]2-deoxyglucose autoradiographs. The techniques minimize diffusion of label into processing fluids and, by inference, migration of label within tissue. Slabs of olfactory bulbs from 12-day-old rats were quick-frozen after one hour of exposure to physiological olfactory stimuli. In light microscopic autoradiographs at low magnification, the neuropil of individual olfactory glomeruli appeared uniformly labeled with different levels of labeling in different glomeruli. At higher magnification, glomerular neuropil labeling consisted of small unlabeled regions surrounded by label clusters, suggesting greater deoxyglucose uptake by olfactory nerve terminals as compared with their postsynaptic dendrites. Periglomerular neurons were labeled differentially. Some microglia and glia precursor cells were heavily labeled in all bulbar laminae. The ultrastructure of cells and neuropil in all bulbar laminae was well-preserved. Cell processes and organelles could be identified in both stained sections and unstained electron microscopic autoradiographs. These experiments demonstrate the feasibility of combining quick-freezing with freeze substitution, in order to extend the resolution of studies using diffusable tracers such as 2-deoxyglucose. The results suggest that this is a promising method for assessing several controversies concerning deoxyglucose incorporation and neuronal and glial metabolism.