The olfactory bulb and central pathways.
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The transcriptional regulator Gli3 and the secreted signal Shh influence induction, patterning, and differentiation at several sites of mesenchymal/epithelial (M/E) interaction including the limbs, heart, face, and forebrain. We asked whether loss of function of these two genes has specific consequences for early differentiation of the primary olfactory pathway-which comprises both craniofacial and forebrain structures and depends on M/E induction during initial stages of development. Loss of Gli3 or Shh function does not compromise several aspects of olfactory receptor neuron (ORN) and olfactory ensheathing cell maturation; however, directed outgrowth of ORN axons and their initial targeting to the telencephalon is altered. In Gli3 mutant extra toes-Jackson (Xt(J)Xt(J)) embryos, ORN axons defasciculate and project aberrantly near the forebrain. They rarely enter the central nervous system, and their association with mesenchymal laminin is disrupted. In Shh-/-embryos, ORN axons exit a single olfactory epithelium (OE) that develops centrally within an altered mesenchymal environment in a dysmorphic proboscis. These axons project as a single nerve toward the mutant forebrain; however, their trajectory varies according to the position of the proboscis relative to the forebrain. These alterations in axon outgrowth probably reflect compromised inductive interactions in the olfactory primordia because neither Gli3 nor Shh are expressed in olfactory neurons. Thus, two genes that influence induction and subsequent differentiation of craniofacial structures and the forebrain have distinct consequences for ORN axon growth during the initial genesis of the olfactory pathway.
At least two different G-protein-mediated transduction cascades, the adenylate cyclase and phospholipase C (PLC) pathway, process chemosensory stimuli for various species. In squid olfactory receptor neurons (ORNs), physiological studies indicate that both pathways may be present; however, confirmation of the transduction molecules at the protein level is absent. Here we provide evidence that the G-proteins involved in both adenylate cyclase and PLC pathways are present in squid ORNs (Lolliguncula brevis). We used immunoblotting to show that Galpha(olf), Galpha(q), and a downstream effector, enzyme PLC140, are present in the squid olfactory epithelium (OE). To localize these proteins to one or more of the five morphological cell types described for squid OE, paraformaldehyde-fixed olfactory organs were cryosectioned (10 microm), double-labeled for Galpha(olf), Galpha(q), or PLC140, and imaged. Analysis of serial sections from entire olfactory organs for epithelial area and patterns of immunofluorescence revealed a region of highest immunoreactivity at the anterior half of the organ. At the cellular level, type 1 cells could not be distinguished morphologically and were not included in the analysis. The three labeling patterns observed in type 2 cells were Galpha(q) alone, PLC140 alone, and colocalization of Galpha(q) and PLC140. Subsets of cell types 3, 4, and 5 showed colocalization of Galpha(olf) with Galpha(q) but not with PLC140. These data suggest that the PLC pathway predominates in type 2 cells; however, coexpression of Galpha(olf) with Galpha(q) in cell types 3, 4, and 5 suggests that both pathways may participate in olfactory transduction in non-type 2 squid ORNs.
The sublethal effects of copper on the sensory physiology of juvenile coho salmon (Oncorhynchus kisutch) were evaluated. In vivo field potential recordings from the olfactory epithelium (electro-olfactograms) were used to measure the impacts of copper on the responses of olfactory receptor neurons to natural odorants (L-serine and taurocholic acid) and an odorant mixture (L-arginine, L-aspartic acid, L-leucine, and L-serine) over a range of stimulus concentrations. Increases in copper impaired the neurophysiological response to all odorants within 10 min of exposure. The inhibitory effects of copper (1.0-20.0 micrograms/L) were dose-dependent and they were not influenced by water hardness. Toxicity thresholds for the different receptor pathways were determined by using the benchmark dose method and found to be similar (a 2.3-3.0 micrograms/L increase in total dissolved copper over background). Collectively, examination of these data indicates that copper is broadly toxic to the salmon olfactory nervous system. Consequently, short-term influxes of copper to surface waters may interfere with olfactory-mediated behaviors that are critical for the survival and migratory success of wild salmonids.
Immunohistochemistry by using monoclonal antibodies named A5 and B2, which specifically recognize cell surface proteins the neuropilin and the plexin, respectively, revealed that olfactory axons in Xenopus tadpoles were classified into several subgroups by virtue of the expression levels of these two cell surface molecules. The vomeronasal axons expressed the plexin but not the neuropilin. The plexin-positive and neuropilin-negative vomeronasal axons form a discrete fiber bundle, even after they joined with the principal olfactory axons. However, the principal olfactory axons were divided into at least two subclasses; the neuropilin-predominant axons which expressed high levels of the neuropilin and low levels of the plexin, and the plexin-predominant axons which expressed high levels of the plexin and low levels of the neuropilin. Within the olfactory nerve the pathways for these two principal olfactory axon subclasses were initially intermingled with each other, but were gradually segregated throughout their courses from the nose to the cerebrum. Eventually, the neuropilin-predominant and the plexin-predominant principal olfactory axon subclasses projected to specified glomeruli in topographically related regions within the main olfactory bulb. Neuroanatomical tracings of the olfactory projection also confirmed the gradual segregation of the pathways for the principal olfactory axons. These results allow us to speculate that both the neuropilin and the plexin are involved in axon interactions, and play roles in the organization of the precise patterns of the olfactory pathway and projection.
Mammals can discriminate among a large number (> 10,000) of unique odorants. The most highly supported explanation for this ability is that olfactory neurons express a large number of seven transmembrane receptors that are not spatially organized at the level of the olfactory epithelium, but whose axonal projections form a distinct pattern within the olfactory bulb. The odor-induced signaling pathway in olfactory neurons includes a Gs-like protein (G(olf)) that activates a specific adenylyl cyclase (type III) isoform, resulting in elevations of cyclic AMP and subsequent activation of a cyclic nucleotide-gated channel. The channel also can be regulated by cyclic GMP. Recently, an olfactory neuron-specific guanylyl cyclase was discovered in rodents, and subsequently a large family of sensory neuronal guanylyl cyclases was identified in nematodes. These guanylyl cyclases are concentrated in the plasma membrane of the dendritic cilia and contain extracellular domains that retain many of the primary sequence characteristics of guanylyl cyclases known to be receptors for various peptides. Thus, the guanylyl cyclases appear to represent a second family of odorant/pheromone receptors.
We have investigated possible roles of the Eph family receptor tyrosine kinases and their ligand ephrins in the developing primary olfactory nerve pathway in the moth Manduca sexta. The Manduca homologs of the Eph receptor (MsEph) and ephrin ligand (MsEphrin) are most closely related to Drosophila Eph and ephrin, respectively. In situ labeling with Fc-fusion probes, in which IgG Fc was linked to the extracellular domain of MsEph (Eph-Fc) or MsEphrin (ephrin-Fc), reveals that both Eph receptors and ephrins are expressed on axons of olfactory receptor cells (ORCs) during their ingrowth to the primary center, the antennal lobe (AL). Interestingly, Eph receptors and ephrins are differentially distributed among identifiable glomeruli such that glomeruli with high receptor staining show little or no ligand staining, and vice versa, suggesting a complementary Eph-ephrin expression by subsets of ORC axons innervating a particular set of glomeruli. In contrast, neither Eph receptors nor ephrins are detectable in intrinsic components of the AL. In vitro, ephrin-Fc and Eph-Fc, when present homogeneously in the substratum, inhibit neurite outgrowth from olfactory epithelial explants. Moreover, in patterned substratum, neurites growing on the standard substratum turn or stop after encountering the test substratum containing ephrin-Fc. These in vitro observations indicate that MsEphrin can act as an inhibitor/repulsive cue for ORC axons. Based on results from in situ and in vitro experiments, we hypothesize that Eph receptors and ephrins mediate axon sorting and fasciculation through repulsive axon-axon interactions.
Stimulation of one side of the olfactory system during training with odor-milk pairings in neonatal rats results in their ability to recall an odor memory by using the trained but not the untrained side of the brain. In 12-day-old rats, olfactory learning can be recalled by stimulation of either the trained or untrained side. The development of bilateral recall reflects the maturation of olfactory commissural pathways that provide access to the olfactory memory stored on the contralateral side. Furthermore, the commissural pathways need not be present at the time of memory formation but can establish new and specific access to already existing olfactory memories.
Anterograde and retrograde axonal tracing methods have been combined with transection of the stria terminalis to investigate the centrifugal afferent connections of the accessory olfactory bulb in the mouse. Injection of tritiated proline into the postero-medial cortical amygdaloid nucleus (C3) gives rise to anterograde autoradiographic labelling of a pathway terminating in the internal granular layer of the accessory olfactory bulb (AOB). Transection of the ipsilateral stria terminalis completely abolishes labelling of this pathway. Injections further rostral, in the bed nucleus of the accessory olfactory tract (bnAOT) and medial amygdaloid nucleus (M), give rise to labelling of a second ipsilateral afferent pathway to the AOB which terminates in the internal plexiform layer (IPL) and is unaffected by strial transection. Injections of wheat germ lectin-HRP conjugate into the AOB confirm that it receives afferents from the ipsilateral bnAOT, M and C3, and from a few cells in the contralateral C3. Transection of the ipsilateral stria terminalis prevents retrograde labelling of any cells in the ipsilateral C3, but does not affect labelling of cells in M or bnAOT (or contralateral C3). The conjugate is also transported anterogradely in this system, labelling the efferent projections of the AOB to bnAOT, M and C3. It is concluded that the AOB receives at least two sets of ipsilateral afferents: one set from C3, via the stria terminalis, terminating in the internal granular layer, and a second set from M and/or bnAOT terminating in the IPL and probably running in the accessory olfactory tract.
Synapses within deutocerebral glomeruli between GABA-immunoreactive, putatively inhibitory local interneurons and uniglomerular projection (output) neurons were demonstrated by means of a combination of GABA-immunogold labeling and intracellular HRP injection. The following connections were identified. 1) GABA-immunoreactive (GABAir) neurons form output synapses in a dyadic fashion onto a uniglomerular projection neuron and, in addition, a second GABAir neuron. A uniglomerular projection neuron in turn forms dyadic output synapses onto two GABAir neurons. Several examples of reciprocal connections have been identified between, first, GABAir neurons and uniglomerular projection neurons, and, second, GABAir neurons themselves. 2) GABAir neurons are serially connected with uniglomerular projection neurons via interposed GABAir processes. In some cases, also the first GABAir process of such a polysynaptic connection formed an output synapse onto the projection neuron. Such serial connections may form the structural basis for both, the feedforward inhibition as well as the feedforward disinhibition of uniglomerular projection neurons by GABAergic neurons. The reciprocal contacts may serve as control devices that modulate the output activity of the projection neurons.
The efferent projections of the telencephalon in the tiger salamander were examined by the Nauta and Fink-Heimer methods following unilateral hemispherectomies, rostral hemispheric ablations and pallial lesions. The cerebral hemisphere connects with most areas of the contralateral hemisphere via the pallial, anterior and habenular commissures. The descending fibers travel in the medial and lateral forebrain bundles and in the tracts comprising the stria medullaris. Degenerating fibers and terminals were present throughout the diencephalon but were more abundant ipsilaterally. Fibers reach the pretectum and optic tectum via dorsal and ventral pathways. There is a heavy projection to the midbrain tegmentum and a sparse projection to the tectum via the ipsilateral lateral forebrain bundle. This tract continues into the medulla oblongata and the cervical spinal cord. Rostral and dorsal hemispheric ablations revealed that the majority of fibers forming the olfacto-peduncular tract originate in the ventral, rostral one-third of the hemisphere. It was also determined that the majority of the descending efferent fibers located in the lateral forebrain bundle originate from the caudal lateral hemispheric wall, and that these fibers form connections characteristic of mammalian corticofugal and striatofugal systems. The cytoarchitecture and connections of the caudal lateral hemispheric wall suggest that it is homologous to parts of motor isocortex and amygdala of amniotes.
We have isolated and characterized the cDNAs for nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) from the terrestrial slug Limax marginatus, and examined the presence and distribution of their mRNAs in the central nervous system using histological techniques and a reverse transcription-polymerase chain reaction method. Our results showed that both bursting and nonbursting neurons in the procerebral lobes contain the mRNAs for both NOS and sGC. We further found that the oscillation frequency of electrical activity in the procerebral lobes increases with increasing intracellular concentrations of cyclic GMP (cGMP). Taken together with previous data on the NO-induced cGMP-like immunoreactivity and on the anatomical distribution of neurites and the localization of synapses of bursting and nonbursting neurons, our present results suggest that NO-induced changes in cGMP concentration modulate the oscillation frequency in the procerebral lobes by acting on the olfactory input pathways, but possibly not on the output pathways, in slugs. .
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In the rodent brain, insulin-like growth factor I (IGF-I) messenger RNA is transiently expressed in sensory projection neurons during periods of synaptogenesis and neuronal growth. Transgenic (Tg) mice with brain IGF-I overexpression and ectopic brain expression of IGF-binding protein-1 (IGFBP-1), an inhibitor of IGF-I actions, show changes in brain size and myelination. We used these mouse models to evaluate in vivo IGF-I effects on sensory pathway development by conducting anatomical studies in the S1 barrel field. Brain size, cortical area, and barrel field dimensions were increased in IGF-I and reduced in IGFBP-1 Tg mice compared with those in wild-type (wt) mice. The brain and cerebral cortex of Tg mice with the highest transgene expression were the most altered in size. Cortex and barrel field size changes were not precisely proportional, because in some Tg mice barrels were relatively more affected than the cortex, whereas in others the opposite was observed. Brain IGF-I overexpression increased the average number of neurons per barrel, neuronal cell body cross-sectional area, and barrel neuropil volume, whereas brain expression of IGFBP-1 reduced each. Neuronal density was greatly reduced in IGF-I Tg mice and increased in IGFBP-1 Tg mice. No differences in body weight, whisker pad and follicle areas, and whisker pad innervation density were found among Tg and wt mice. These observations indicate that IGF-I enhances neuronal growth in developing sensory pathways and support the concept that modified availability of local trophic factors, such as IGF-I, changes brain, neocortical, and S1 relative dimensions by altering neuronal survival and neuropil elaboration. Study of the S1 cortex provides an excellent model to probe the in vivo mechanisms of IGF actions.
During metamorphosis insects undergo dramatic changes in both form and behaviour. Cell birth and death, as well as neurone respecification all contribute to the overall reorganization of the nervous system. Within the visual and chemosensory processing areas of the insect brain large numbers of newly-generated adult neurones are incorporated into the larval nervous system. In the abdominal ganglia, however, identified larval neurones are retained to assume a new adult role. This respecification of motor neurone function involves not only the acquisition of a new target muscle, but also the reorganization of dendritic morphology, and alterations in the interconnections between neurones. For example, an identified abdominal motor neurone in the hawkmoth, Manduca sexta, grows new dendritic processes and changes its synaptic relationship with an abdominal stretch receptor such that an interaction that was inhibitory during larval life, becomes excitatory in the adult. In another example, identified sensory neurones that evoke a larval flexion behaviour, later participate in the defense gin trap reflex that is characteristic of the pupa. In both instances the formation of new pathways is a two-step process in that the new circuits do not become behaviourally relevant as they are formed, but instead are activated abruptly at the appropriate time. For the gin trap reflex an identified peptide hormone is responsible for activating the circuit.
Within the rat medial temporal lobe, transient modulations of neural cell adhesion molecule (NCAM) polysialylation have been observed to follow spatial learning. These have been attributed to neuroplastic events associated with the processing of information destined for long term memory consolidation. To determine if similar events are associated with avoidance learning, we investigated change in polysialylated cell number in the entorhinal, perirhinal, and piriform cortex, following acquisition of a passive avoidance task in the rat. Direct quantification of polysialylated neurons in layer II of these cortical regions revealed a significant increase in polysialylated cell frequency at 12 h following passive avoidance training. Unlike spatial learning, the increased expression of polysialylated neurons persisted for up to 24-48 h following training. In the more dorsal aspect of the perirhinal/entorhinal cortex, this increase was found to be specific to learning, as it was not observed in animals rendered amnesic with scopolamine. By contrast, change in polysialylated cell frequency in the ventral aspect of the medial temporal lobe was only partially reduced by amnesic doses of scopolamine. The persisting activation of NCAM polysialylation in the more dorsal aspects of the perirhinal and entorhinal cortex is suggested to reflect the need for more extensive synaptic alterations, as compared to those required for the consolidation of spatial learning. Moreover, the neuroplastic modulations observed in the more ventral regions of the entorhinal and perirhinal cortex appear to be a unique aspect of avoidance conditioning that reflects the activation of alternative learning strategies associated with motivational and/or contextual parameters of the task.