R7 photoreceptor axon targeting depends on the relative levels of lost and found expression in R7 and its synaptic partners.

As neural circuits form, growing processes select the correct synaptic partners through interactions between cell surface proteins. The presence of such proteins on two neuronal processes may lead to either adhesion or repulsion; however, the consequences of mismatched expression have rarely been explored. Here, we show that the Drosophila CUB-LDL protein Lost and found (Loaf) is required in the UV-sensitive R7 photoreceptor for normal axon targeting only when Loaf is also present in its synaptic partners. Although targeting occurs normally in loaf mutant animals, removing loaf from photoreceptors or expressing it in their postsynaptic neurons Tm5a/b or Dm9 in a loaf mutant causes mistargeting of R7 axons. Loaf localizes primarily to intracellular vesicles including endosomes. We propose that Loaf regulates the trafficking or function of one or more cell surface proteins, and an excess of these proteins on the synaptic partners of R7 prevents the formation of stable connections.

New nerve cells in a developing organism face a difficult challenge: finding the right partners to connect with in order to form the complex neural networks characteristic of a fully formed brain. Each cell encounters many potential matches but it chooses to connect to only a few, partly based on the proteins that decorate the surface of both cells. Still, too many cell types exist for each to have its own unique protein label, suggesting that nerve cells may also use the amount of each protein to identify suitable partners. Douthit, Hairston et al. explored this possibility in developing fruit flies, focusing on how R7 photoreceptor cells ­ present in the eye to detect UV light ­ connect to nerve cells in a specific brain layer. It is easy to spot when the process goes awry, as the incorrect connections will be in a different layer. Experiments allowed Douthit, Hairston et al. to identify a protein baptized 'Lost and found' ­ 'Loaf' for short ­ which R7 photoreceptors use to find their partners. Removing Loaf from the photoreceptors prevented them from connecting with their normal partners. Surprisingly though, removing Loaf from both the eye and the brain solved this problem ­ the cells, once again, formed the right connections. This suggests that R7 photoreceptors identify their partners by looking for cells that have less Loaf than they do: removing Loaf only from the photoreceptors disrupts this balance, leaving the cells unable to find their match. Another unexpected discovery was that Loaf is not present on the surface of cells, but instead occupies internal structures involved in protein transport. It may therefore work indirectly by controlling the movement of proteins to the cell surface. These findings provide a new way of thinking about how nerve cells connect. In the future, this may help to understand the origins of conditions in which the brain is wired differently, such as schizophrenia and autism.

Nerve excitability differences in slow and fast motor axons of the rat: more than just Ih.

The objective was to determine biophysical differences between fast and slow motor axons using threshold tracking and demonstrate confounds related to anesthetic. Nerve excitability of motor axons innervating the slow-twitch soleus (SOL) and fast-twitch tibialis anterior (TA) muscles was tested. The experiments were conducted with pentobarbital sodium (SP) anesthetic and compared with previous results that used ketamine-xylazine (KX). Nerve excitability indices measured with SP show definitive differences between TA and SOL motor axons that extend beyond previous reports. Nerve excitability indices sensitive to changes in Ih indicated an increase in SOL axons compared with TA axons [e.g., S3 t = 7.949 (df = 10), P < 0.001; hyperpolarizing threshold electrotonus (90-100 ms), t = 2.659 (df = 20); P = 0.01; hyperpolarizing I/V slope, t = 4.308 (df = 19); P < 0.001]. SOL axons also had a longer strength-duration time constant [t = 3.35 (df = 20); P = 0.003] and a longer and larger magnitude relative refractory period [RRP (ms) t = 3.53 (df = 12); P = 0.004; Refractoriness at 2 ms, t = 0.0055 (df = 9); P = 0.006]. Anesthetic choice affected many measures of peripheral nerve excitability with differences most apparent in tests of threshold electrotonus and recovery cycle. For example, recovery cycle with KX lacked a clear superexcitable and late subexcitable period. We conclude that KX had a confounding effect on nerve excitability results consistent with ischemic depolarization. Results using SP revealed the full extent of differences in nerve excitability measures between putative slow and fast motor axons of the rat. These results provide empirical evidence, beyond conduction velocity, that the biophysical properties of motor axons vary with the type of muscle fiber innervated. These differences suggest that fast axons may be predisposed to dysfunction during hyperpolarizing stresses, e.g., electrogenic sodium pumping following sustained impulse conduction.NEW & NOTEWORTHY Nerve excitability testing is a tool used to provide insight into the properties of ion channels in peripheral nerves. It is used clinically to assess pathophysiology of axons. Researchers customarily think of motor axons as homogeneous; however, we demonstrate there are clear differences between fast and slow axons in the rat. This is important for interpreting results with selective motor neuronopathy, like aging where fast axons are at high risk of degeneration.

Assessment of axonal recruitment using model-guided preclinical spinal cord stimulation in the ex vivo adult mouse spinal cord.

Spinal cord stimulation (SCS) is used clinically to limit chronic pain, but fundamental questions remain on the identity of axonal populations recruited. We developed an ex vivo adult mouse spinal cord preparation to assess recruitment following delivery of clinically analogous stimuli determined by downscaling a finite element model of clinical SCS. Analogous electric field distributions were generated with 300-µm × 300-µm electrodes positioned 200 µm above the dorsal column (DC) with stimulation between 50 and 200 µA. We compared axonal recruitment using electrodes of comparable size and stimulus amplitudes when contacting the caudal thoracic DC and at 200 or 600 µm above. Antidromic responses recorded distally from the DC, the adjacent Lissauer tract (LT), and in dorsal roots (DRs) were found to be amplitude and site dependent. Responses in the DC included a unique component not seen in DRs, having the lowest SCS recruitment amplitude and fastest conduction velocity. At 200 µm above, mean cathodic SCS recruitment threshold for axons in DRs and LT were 2.6 and 4.4 times higher, respectively, than DC threshold. SCS recruited primary afferents in all (up to 8) caudal segments sampled. Whereas A and C fibers could be recruited at nearby segments, only A fiber recruitment and synaptically mediated dorsal root reflexes were observed in more distant (lumbar) segments. In sum, clinically analogous SCS led to multisegmental recruitment of several somatosensory-encoding axonal populations. Most striking is the possibility that the lowest threshold recruitment of a nonprimary afferent population in the DC are postsynaptic dorsal column tract cells (PSDCs) projecting to gracile nuclei.NEW & NOTEWORTHY Spinal cord stimulation (SCS) is used clinically to control pain. To identify axonal populations recruited, finite element modeling identified scaling parameters to deliver clinically analogous SCS in an ex vivo adult mouse spinal cord preparation. Results showed that SCS first recruited an axonal population in the dorsal column at a threshold severalfold lower than primary afferents. These putative postsynaptic dorsal column tract cells may represent a previously unconsidered population responsible for SCS-induced paresthesias necessary for analgesia.

Temporal identity establishes columnar neuron morphology, connectivity, and function in a Drosophila navigation circuit.

The insect central complex (CX) is a conserved brain region containing 60 + neuronal subtypes, several of which contribute to navigation. It is not known how CX neuronal diversity is generated or how developmental origin of subtypes relates to function. We mapped the developmental origin of four key CX subtypes and found that neurons with similar origin have similar axon/dendrite targeting. Moreover, we found that the temporal transcription factor (TTF) Eyeless/Pax6 regulates the development of two recurrently-connected CX subtypes: Eyeless loss simultaneously produces ectopic P-EN neurons with normal axon/dendrite projections, and reduces the number of E-PG neurons. Furthermore, transient loss of Eyeless during development impairs adult flies' capacity to perform celestial navigation. We conclude that neurons with similar developmental origin have similar connectivity, that Eyeless maintains equal E-PG and P-EN neuron number, and that Eyeless is required for the development of circuits that control adult navigation.

Heterogeneity of retinogeniculate axon arbors.

The retinogeniculate synapse transmits information from retinal ganglion cells (RGC) in the eye to thalamocortical relay neurons in the visual thalamus, the dorsal lateral geniculate nucleus (dLGN). Studies in mice have identified genetic markers for distinct classes of RGCs encoding different features of the visual space, facilitating the dissection of RGC subtype-specific physiology and anatomy. In this study, we examine the morphological properties of axon arbors of the BD-RGC class of ON-OFF direction selective cells that, by definition, exhibit a stereotypic dendritic arbor and termination pattern in the retina. We find that axon arbors from the same class of RGCs exhibit variations in their structure based on their target region of the dLGN. Our findings suggest that target regions may influence the morphologic and synaptic properties of their afferent inputs.

Fragile X granules are a family of axonal ribonucleoprotein particles with circuit-dependent protein composition and mRNA cargos.

Local axonal protein synthesis plays a crucial role in the formation and function of neuronal circuits. Understanding the role of this mechanism in specific circuits requires identifying the protein composition and mRNA cargos of the ribonucleoprotein particles (RNPs) that form the substrate for axonal translation. FXGs (Fragile X granules) are axonal RNPs present in a stereotyped subset of mature axons in the intact brain that contain one or more of the Fragile X related (FXR) proteins (FMRP, FXR2P, and FXR1P) along with mRNA and ribosomes. Here we performed a systematic survey of the FXR protein composition and mRNA association of FXGs in the brain. We have identified four FXG types that can be categorized based on their FXR protein complement. All FXGs contain FXR2P, with FMRP and/or FXR1P present in circuit-selective subsets. Individual neuronal cell types predominantly express a single FXG type, with FMRP-containing FXGs the most prevalent in forebrain neurons. All FXG types associate with ribosomes and mRNA, but the specific mRNA cargos are a function of FXG type, brain region and neuron class. Transcripts for ß-catenin and its regulator APC associate with a subset of forebrain FXGs. Moreover, both these transcripts can colocalize within individual FXGs, suggesting that the axonal translation of functionally related proteins may be coordinately regulated with high spatiotemporal resolution. Cell type-dependent expression of specific RNP types with distinct mRNA cargos, such as FXGs, presents a potential mechanism for regulating local translation and its output in a circuit-dependent manner.

Subrepellent doses of Slit1 promote Netrin-1 chemotactic responses in subsets of axons.

BACKGROUND: Axon pathfinding is controlled by guidance cues that elicit specific attractive or repulsive responses in growth cones. It has now become clear that some cues such as Netrin-1 can trigger either attraction or repulsion in a context-dependent manner. In particular, it was recently found that the repellent Slit1 enables the attractive response of rostral thalamic axons to Netrin-1. This finding raised the intriguing possibility that Netrin-1 and Slit1, two essential guidance cues, may act more generally in an unexpected combinatorial manner to orient specific axonal populations. To address this major issue, we have used an innovative microfluidic device compatible not only with dissociated neuronal cultures but also with explant cultures to systematically and quantitatively characterize the combinatorial activity of Slit1 and Netrin-1 on rostral thalamic axons as well as on hippocampal neurons. RESULTS: We found that on rostral thalamic axons, only a subthreshold concentration of the repellent Slit1 triggered an attractive response to a gradient of Netrin-1. On hippocampal neurons, we similarly found that Slit1 alone is repulsive and a subthreshold concentration of Slit1 triggered a potent attractive or repulsive behavioral response to a gradient of Netrin-1, depending on the nature of the substrate. CONCLUSIONS: Our study reveals that at subthreshold repulsive levels, Slit1 acts as a potent promoter of both Netrin-1 attractive and repulsive activities on distinct neuronal cell types, thereby opening novel perspectives on the role of combinations of cues in brain wiring.

Classifying GABAergic interneurons with semi-supervised projected model-based clustering.

OBJECTIVES: A recently introduced pragmatic scheme promises to be a useful catalog of interneuron names. We sought to automatically classify digitally reconstructed interneuronal morphologies according to this scheme. Simultaneously, we sought to discover possible subtypes of these types that might emerge during automatic classification (clustering). We also investigated which morphometric properties were most relevant for this classification. MATERIALS AND METHODS: A set of 118 digitally reconstructed interneuronal morphologies classified into the common basket (CB), horse-tail (HT), large basket (LB), and Martinotti (MA) interneuron types by 42 of the world's leading neuroscientists, quantified by five simple morphometric properties of the axon and four of the dendrites. We labeled each neuron with the type most commonly assigned to it by the experts. We then removed this class information for each type separately, and applied semi-supervised clustering to those cells (keeping the others' cluster membership fixed), to assess separation from other types and look for the formation of new groups (subtypes). We performed this same experiment unlabeling the cells of two types at a time, and of half the cells of a single type at a time. The clustering model is a finite mixture of Gaussians which we adapted for the estimation of local (per-cluster) feature relevance. We performed the described experiments on three different subsets of the data, formed according to how many experts agreed on type membership: at least 18 experts (the full data set), at least 21 (73 neurons), and at least 26 (47 neurons). RESULTS: Interneurons with more reliable type labels were classified more accurately. We classified HT cells with 100% accuracy, MA cells with 73% accuracy, and CB and LB cells with 56% and 58% accuracy, respectively. We identified three subtypes of the MA type, one subtype of CB and LB types each, and no subtypes of HT (it was a single, homogeneous type). We got maximum (adapted) Silhouette width and ARI values of 1, 0.83, 0.79, and 0.42, when unlabeling the HT, CB, LB, and MA types, respectively, confirming the quality of the formed cluster solutions. The subtypes identified when unlabeling a single type also emerged when unlabeling two types at a time, confirming their validity. Axonal morphometric properties were more relevant that dendritic ones, with the axonal polar histogram length in the [π, 2π) angle interval being particularly useful. CONCLUSIONS: The applied semi-supervised clustering method can accurately discriminate among CB, HT, LB, and MA interneuron types while discovering potential subtypes, and is therefore useful for neuronal classification. The discovery of potential subtypes suggests that some of these types are more heterogeneous that previously thought. Finally, axonal variables seem to be more relevant than dendritic ones for distinguishing among the CB, HT, LB, and MA interneuron types.

Distribution and functional organization of glomeruli in the olfactory bulbs of zebrafish (Danio rerio).

Odor molecules are transduced by thousands of olfactory sensory neurons (OSNs) located in the nasal cavity. Each OSN expresses a single functional odorant receptor protein and projects an axon from the sensory epithelia to an olfactory bulb glomerulus, which is selectively innervated by only one or a few OSN types. We used whole-mount immunocytochemistry to study the neurochemistry and anatomical organization of glomeruli in the zebrafish olfactory system. By employing combinations of antibodies against G-protein α subunits, calcium-binding proteins, and general neuronal markers, we selectively labeled various OSN types, their axonal projections to glomeruli, and the detailed anatomical distributions of individual glomeruli in different regions of the olfactory bulb. In this way we identified ≈140 glomeruli in each olfactory bulb of mature zebrafish. A small subset (27) of these glomeruli was unambiguously identifiable in nearly all animals examined. These units were large and, located mainly in the medial olfactory bulbs. Most glomeruli, however, were comparatively small, anatomically indistinguishable, and located in coarsely circumscribed regions; almost all of these latter glomeruli were innervated by OSNs that were labeled with anti-G(α s/olf) and/or anti-calretinin antibodies. Collectively, our results provide a uniquely detailed description of a vertebrate olfactory system and highlight anatomically distinct parallel neural pathways that mediate early aspects of olfactory processing in the zebrafish.

A universal property of axonal and dendritic arbors.

Axonal and dendritic arbors can be characterized statistically by their spatial density function, a function that specifies the probability of finding a branch of a particular arbor at each point in a neural circuit. Based on an analysis of over a thousand arbors from many neuron types in various species, we have discovered an unexpected simplicity in arbor structure: all of the arbors we have examined, both axonal and dendritic, can be described by a Gaussian density function truncated at about two standard deviations. Because all arbors are characterized by density functions with this single functional form, only four parameters are required to specify an arbor's size and shape: the total length of its branches and the standard deviations of the Gaussian in three orthogonal directions. This simplicity in arbor structure can have implications for the developmental wiring of neural circuits.

Odorant receptor gene choice and axonal projection in the mouse olfactory system.

In the mouse olfactory system, each olfactory sensory neuron (OSN) expresses a single type of odorant receptor (OR) out of approximately 1,000 in a monoallelic manner. Furthermore, OSNs expressing the same OR converge their axons to a specific set of glomeruli on the olfactory bulb. These two basic principles are fundamental to the peripheral olfactory system, and are regulated by the expressed OR protein itself. Singular OR gene choice is ensured by the combination of stochastic enhancer-promoter interaction and negative-feedback regulation by OR proteins. In the axonal projection, OR-derived cyclic AMP signals and neuronal activity determine the expression levels of axon guidance/sorting molecules, and thereby direct glomerular positioning and axon sorting.

Distinct deep short-axon cell subtypes of the main olfactory bulb provide novel intrabulbar and extrabulbar GABAergic connections.

A universal feature of neuronal microcircuits is the presence of GABAergic interneurons that control the activity of glutamatergic principal cells and each other. In the rat main olfactory bulb (MOB), GABAergic granule and periglomerular cells innervate mitral and tufted cells, but the source of their own inhibition remains elusive. Here, we used a combined electrophysiological and morphological approach to investigate a rather mysterious cell population of the MOB. Deep short-axon cells (dSACs) of the inframitral layers are GABAergic and have extensive and characteristic axonal ramifications in various layers of the bulb, based on which unsupervised cluster analysis revealed three distinct subtypes. Each dSAC subtype exhibits different electrical properties but receives similar GABAergic and glutamatergic inputs. The local axon terminals of all dSAC subtypes selectively innervate GABAergic granule and periglomerular cells and evoke GABA(A) receptor-mediated IPSCs. One subpopulation of dSACs (GL-dSACs) creates a novel intrabulbar projection from deep to superficial layers. Another subpopulation (GCL-dSACs) is labeled by retrogradely transported fluorescent microspheres injected into higher olfactory areas, constituting a novel projection-cell population of the MOB. Our results reveal multiple dSAC subtypes, each specialized to influence MOB activity by selectively innervating GABAergic interneurons, and provide direct evidence for novel intrabulbar and extrabulbar GABAergic projections.

Quantification of synaptic density in corticostriatal projections from rat medial agranular cortex.

Medial agranular cortex (AGm) has a prominent bilateral projection to the dorsocentral striatum (DCS). We wished to develop a normal baseline by which to assess neuronal plasticity in this corticostriatal system in rats with neglect resulting from a unilateral lesion in AGm, followed by treatment with agents that promote sprouting and functional recovery in other systems. Injections of biotinylated dextran amine were made into AGm in normal rats, and unbiased sampling was used to quantify the density of axons and axonal varicosities present in DCS (the latter represent presynaptic profiles). Labeling density in contralateral DCS is approximately half of that seen in ipsilateral DCS (this ratio is 0.50 for axons, 0.55 for varicosities). The ratio of varicosities is stable over a greater than seven-fold range of absolute densities. There is no consistent relationship between the absolute density of axons and axon varicosities; however, the ratio measures are strongly correlated. We conclude that changes in the contralateral/ipsilateral ratio of axon density after experimental treatments do reflect changes in synaptic density, but axon varicosities are likely to be the most sensitive anatomical parameter by which to assess plasticity at the light microscopic level.

Two distinct subtypes of serotonergic fibers classified by co-expression with vesicular glutamate transporter 3 in rat forebrain.

Although virtually all of serotonin (5-HT) neurons in the midbrain raphe nuclei of rats are known to express vesicular glutamate transporter 3 (VGLUT3), VGLUT3-positive 5-HT fibers have been identified only in the cerebral cortex and hippocampus. Thus, our understanding of forebrain sites where 5-HT-glutamate interaction may be potentially managed by such possible glutamatergic 5-HT fibers themselves, is still largely fragmentary from a morphological point of view. To address this issue, we analyzed the rat forebrain by immunohistochemistry and chemical lesion experiment of 5-HT neurons by intracerebroventricular injection of a neurotoxin, 5,7-dihydroxytriptamine. Contrary to expectation, the double-label immunofluorescence staining revealed that the incidence of VGLUT3-positive 5-HT fibers is generally low over the forebrain, demonstrating occasional fibers with one or two double-labeled varicosities. The most extreme example was the nucleus of the lateral olfactory tract (LOT), which seemed to be devoid of double-labeled fibers despite high densities of 5-HT fibers and VGLUT3-positive fibers. In sharp contrast, robust plexuses of VGLUT3-positive 5-HT fibers were found in the dorsal, but not ventral, part of the lateral septum. The lesion experiment carried out to explore whether VGLUT3 exists in 5-HT fibers showed that in lesioned rats VGLUT3-positive fibers almost completely disappear from the septal region but seemed unchanged in the LOT. The present study shows that midbrain raphe-derived 5-HT fibers can be classified into two subtypes depending on co-expression with VGLUT3 staining in the forebrain.

Fiber composition of the rat sciatic nerve and its modification during regeneration through a sieve electrode.

Recovery after peripheral nerve transection is seldom complete, the outcome depending both on lesion and repair conditions, and on the type and neurochemical properties of axons. The interposition between the stumps of a perforated, or regenerative electrode (RE) is a promising avenue in the use of chronic nerve bioimplants, but represents an additional challenge to regeneration. We applied stereological methods to ultrathin and immunostained semithin sections to examine quantitatively the axon types that make up the sciatic nerve in control adult rats, and their changes 2 months after an RE implant. The number of myelinated axons (MAx) increased proximal to RE, but fell to 10% a few millimeters distal. This decrease affected more severely motor fibers, characterized by immunoreactivity to cholinacetyltransferase (ChAT+), than sensory (ChAT-) fibers. Regenerating MAx and myelin sheaths also changed notably in thickness. Unmyelinated axons (UAx) showed a moderate reduction in number distal to the implant. This reduction affected more tyrosine hydroxylase-immunoreactive axons (mostly vaso- and pilomotor fibers), than axons expressing ChAT and/or vasoactive intestinal peptide (mostly sudomotor fibers). Taken together with previous findings [Negredo, P., Castro, J., Lago, N., Navarro, X., Avendaño, C., 2004. Differential growth of axons from sensory and motor neurons through a regenerative electrode: a stereological, retrograde tracer, and functional study in the rat. Neuroscience 128, 605-615.], this study shows that regeneration through the RE is much less successful for MAx than UAx, that motor axons regenerate more poorly than sensory axons, and that some subclasses of sympathetic fibers regenerate better than others. The study also proves the value of the combined methodological approach presented here to assess the fiber composition of a nerve under normal, pathological or experimental conditions.

Electron microscopic examination of the myelinated axons of corpus callosum in perfused young and old rats.

In this study, the myelinated axons of parts of the corpus callosums of young and old rats were examined under the electron microscope and a grading system was performed for quantitating the ultrastructural pathological changes of these axons. Except the old splenium group, the only ultrastructural pathological change, observed in the myelinated axons was the separation in myelin configuration. In addition to this finding, in the old splenium group, in some of the myelinated axons, an interruption was observed in the myelin configuration. Additionally, these ultrastructural pathological findings were present in the larger sized myelinated axons of the corpus callosum.

Forebrain connectivity of the prefrontal cortex in the marmoset monkey (Callithrix jacchus): an anterograde and retrograde tract-tracing study.

The cortical and subcortical forebrain connections of the marmoset prefrontal cortex (PFC) were examined by injecting the retrograde tracer, choleratoxin, and the anterograde tracer, biotin dextran amine, into four sites within the PFC. Two of the sites, the lateral and orbital regions, had previously been shown to provide functionally dissociable contributions to distinct forms of behavioral flexibility, attentional set-shifting and discrimination reversal learning, respectively. The dysgranular and agranular regions lying on the orbital and medial surfaces of the frontal lobes were most closely connected with limbic structures including cingulate cortex, amygdala, parahippocampal cortex, subiculum, hippocampus, hypothalamus, medial caudate nucleus, and nucleus accumbens as well as the magnocellular division of the mediodorsal nucleus of the thalamus and midline thalamic nuclei, consistent with findings in the rhesus monkey. In contrast, the granular region on the dorsal surface closely resembled area 8Ad in macaques and had connections restricted to posterior parietal cortex primarily associated with visuospatial functions. However, it also had connections with limbic cortex, including retrosplenial and caudal cingulate cortex as well as auditory processing regions in the superior temporal cortex. The granular region on the lateral convexity had the most extensive connections. Based on its architectonics and functionality, it resembled areas 12/45 in macaques. It had connections with high-order visual processing regions in the inferotemporal cortex and posterior parietal cortex, higher-order auditory and polymodal processing regions in the superior temporal cortex. In addition it had extensive connections with limbic regions including the amygdala, parahippocampal cortex, cingulate, and retrosplenial cortex.

Impaired axonal regeneration by isolectin B4-binding dorsal root ganglion neurons in vitro.

The subpopulation of dorsal root ganglion (DRG) neurons recognized by Griffonia simplicifolia isolectin B4 (IB4) differ from other neurons by expressing receptors for glial cell line-derived neurotrophic factor (GDNF) rather than neurotrophins. Additionally, IB4-labeled neurons do not express the laminin receptor, alpha7-integrin (Gardiner et al., 2005), necessary for optimal axonal regeneration in the peripheral nervous system. In cultures of dissociated DRG neurons of adult mice on laminin, robust spontaneous neurite outgrowth from IB4-negative neurons occurs and is strongly enhanced by previous axotomy. In contrast, IB4-labeled neurons show little neurite outgrowth and do not express GAP 43, even after axotomy or culture with GDNF. Moreover, growth of their axons through collagen gels is impaired compared with other DRG neurons. To determine whether the sparse neurite outgrowth of IB4-labeled neurons is attributable to lack of integrin expression, DRG cultures were infected with a herpes simplex 1 vector encoding alpha7-integrin, but its forced expression failed to promote neurite outgrowth in either IB4-labeled or other DRG neurons or in cultured adult retinal ganglion cells. Forced coexpression of both alpha7-integrin and GAP 43 also failed to promote neurite outgrowth in IB4-labeled neurons. In addition, cultured sciatic nerve segments were found to release much lower levels of GDNF, demonstrated by ELISA, than nerve growth factor. These findings together with their impaired intrinsic axonal regeneration capacity may contribute to the known vulnerability of the IB4-labeled population of DRG neurons to peripheral nerve injury.

Axonal bias at a representational border in adult rat somatosensory cortex (S1).

The cortex is a highly organized structure and this organization is integral to cortical function. However, the circuitry underlying cortical organization is only partially understood, thus limiting our understanding of cortical function. Within the somatosensory cortex, organization is manifest as a map of the body surface. At the level of the cortical circuitry the horizontal connections of Layer 2/3 express a physiological bias that reflects discontinuities within the somatosensory map. Both excitation and inhibition are smaller when evoked from across a representational border, as compared to when they are evoked from within the representation. This physiological bias may be due to a bias in either the strength or number of synapses and/or the number of axons that cross this border and the extent of their arborization. In this study we used both an anterograde (Phaseolus vulgaris leucoagglutinin) and a retrograde (cholera toxin B) tracer to examine Layer 2/3 horizontal projections in rat S1. We determined that there is a bias in the amount of horizontal axonal projections that cross the forepaw/lower jaw border as compared to projections remaining within an individual representation. This bias in axonal projection and the correlated bias in excitation and inhibition may underlie the expression of the representational border.

Medullary dorsal horn neurons providing axons to both the parabrachial nucleus and thalamus.

It has often been suggested that the trigemino- and spino-thalamic pathways are highly implicated in sensory-discriminative aspects of pain, whereas the trigemino- and spino-parabrachial pathways are strongly implicated in affective/emotional aspects of pain. On the other hand, the superficial laminae of the spinal dorsal horn, where many nociceptive neurons are distributed, have been reported to contain projection neurons innervating both the parabrachial nucleus (PBN) and thalamus by way of axon collaterals (Hylden et al., 1989). For the medullary dorsal horn (caudal subnucleus of spinal trigeminal nucleus: Vc), however, the existence of such neurons has not been reported. Thus, in the present study, we examined whether the Vc might contain projection neurons sending their axons to both the thalamus and PBN. Dual retrograde labeling with fluorescence dyes was attempted. In each rat, tetramethylrhodamine-dextran amine and Fluoro-gold were stereotaxically injected into the PBN and thalamic regions, respectively. The proportion of the dually labeled Vc cells in the total population of all labeled Vc cells was about 20%. More than 90% of the dually labeled neurons were distributed in lamina I (marginal zone), less than 10% of them were located in lamina II (substantia gelatinosa), and only a few (about 1%) were found in lamina III (magnocellular zone). The results indicate that some Vc neurons in the superficial laminae mediate nociceptive information directly to the PBN and thalamus by way of axon collaterals and that the vast majority of them project to the ipsilateral PBN and contralateral thalamus.