Papers by Jonathan Blagburn
Development, Apr 1, 1985
The cercal afferent, giant interneurone pathway in Periplaneta americana was used as a model for ... more The cercal afferent, giant interneurone pathway in Periplaneta americana was used as a model for synapse formation. The morphology of the two identified filiform hair sensory neurones (FHSNs) and of two giant interneurones (GI2 and GI3) was followed throughout embryogenesis by cobalt injection. The FHSN axons enter the CNS at the 45% stage of embryogenesis, branch at 50% and form complete arborizations by 70%. The giant interneurones send out a primary dendrite at 45%. Secondary branches form between 50% and 60% and elaboration of the branching pattern takes place until 80% embryogenesis. At early stages the FHSN axons are within filopodial range of GI dendrites which may use these sensory processes as guidance cues. Synapse formation between the main FHSN axon shafts and GI dendrites was investigated by injection of the latter with HRP. From 55% to 65% the process is initiated by desmosome-like filopodial contacts, with subsequent vesicle clustering and formation of a small synaptic density. Numbers of contacts did not significantly increase after about 70%, but the number of synapses doubled between 65% and 75%, with each GI process becoming postsynaptic to two FHSN synapses and the presynaptic densities lengthening to become bars. From 75% embryogenesis to hatching there is a further small increase in synaptic bar length. In the first instar GI3 is postsynaptic to both FHSN axons, whereas GI2 forms very few synapses with the axon of the lateral FHSN (LFHSN). This imbalance of contacts is present throughout synaptogenesis, apart from some early filopodial contacts. GI3 forms synapses with the lateral side of the LFHSN axon from 60% embryogenesis but these are totally absent at hatching. The growth of glia along this side of the axon during the last 30% of development appears to be associated with degeneration of synapses in this region. Thus, as the dendrites of the GIs grow to form a miniature version of the adult without loss of branches, there is little evidence of an initial overproduction of FHSN-GI synapses. Similarly there is no evidence that GI2 forms &#39;incorrect&#39; synapses with the axon of LFHSN. However, GI3 contacts are removed from an inappropriate region of a correct synaptic partner, LFHSN.
PubMed, Aug 1, 1988
The synaptic connection between the filiform hair sensory neurons (FHSNs) and the giant interneur... more The synaptic connection between the filiform hair sensory neurons (FHSNs) and the giant interneurons (GIs) in the terminal abdominal ganglion of the first-instar cockroach Periplaneta americana has been used as a model system for investigating the mechanism of synaptic specificity. Electrophysiological and electron microscopical techniques were used to determine the normal patterns of connectivity between FHSNs and GIs. It was found that, even during synaptogenesis, no incorrect synapses were formed between the sensory axons and the interneuronal dendrites. Studies of a mutant in which there is a supernumerary filiform hair have given an insight into the role of positional information in determining the shape of a sensory neuron, and its connectivity with the GIs. There is electrophysiological evidence for competitive interactions between the supernumerary sensory axon and the original.
PLOS ONE, Aug 17, 2018
The synapse between auditory Johnston's Organ neurons (JONs) and the giant fiber (GF) of Drosophi... more The synapse between auditory Johnston's Organ neurons (JONs) and the giant fiber (GF) of Drosophila is structurally mixed, being composed of cholinergic chemical synapses and Neurobiotin-(NB) permeable gap junctions, which consist of the innexin Shaking-B (ShakB). Previous observations showed that misexpression of one ShakB isoform, ShakB(N+16), in a subset of JONs that do not normally form gap junctions results in their de novo dye coupling to the GF. Misexpression of the transcription factor Engrailed (En) in these neurons also has this effect, and in addition causes the formation of new chemical synapses. These results, along with earlier studies suggesting that gap junctions are required for the development of some chemical synapses, led to the hypothesis that ShakB would, like En, have an instructive effect on the distribution of mixed chemical/electrical contacts. To test this, we first confirmed quantitatively that ShakB(N+16) misexpression increased the dye-coupling of JONs with the GF, indicating the formation of ectopic gap junctions. Conversely, expression of the 'incorrect' isoform, ShakB(N), abolished dye coupling. Immunocytochemistry of the ShakB protein showed that ShakB(N+16) increased gap junctional plaques in JON axons but ShakB(N) did not. To test our hypothesis, fluorescently-labeled presynaptic active zone protein (Brp) was expressed in JONs and the changes in its distribution on the GF dendrites was assayed with confocal microscopy in animals with misexpression of ShakB(N+16), ShakB(N) or, as a positive control, En. Using different methods of image analysis, we confirmed our previous result that En misexpression increased the chemical synapses with the GF and the amount of GF medial dendrite branching. However, contrary to our hypothesis, misexpression of ShakB did not increase these parameters. Immunostaining showed no association between presynaptic active zones and the new ShakB plaques, further evidence against the hypothesis. We conclude that both subsets of JON form chemical synapses onto the GF dendrites but only one population forms gap junctions, comprised of ShakB(N+16). Misexpression of this isoform in all JONs does not instruct the formation of new mixed chemical/electrical synapses, but results in the insertion of new gap junctions, presumably at the sites of existing chemical synaptic contacts with the GF.
Journal of Neurophysiology, Oct 1, 1995
1. The final steps in synapse formation and stabilization involve the adjustment of strength of c... more 1. The final steps in synapse formation and stabilization involve the adjustment of strength of connections through competitive interactions between neurons contacting a common target. The mechanisms underlying this competition-driven adjustment of synaptic strength are not well understood. We have studied one aspect of this phenomenon using the cercal sensory system of first instar cockroach nymphs. 2. Quantal analysis of excitatory postsynaptic potentials (EPSPs) recorded at the synapse between the lateral filiform hair sensory neuron (L) and giant interneuron 3 (GI3) was carried out to determine whether the reduction in EPSP amplitude observed in the presence of an additional sensory neuron (&quot;space invader neuron&quot; or SIN) was due to pre- or postsynaptic changes. 3. Mean quantal content, not quantal size, was reduced at the L-GI3 synapse in the presence of a SIN. Fitting binomial distributions to the EPSP amplitude histograms gave estimates of the number of releasable quanta or of synaptic sites (n) and the probability of release at these sites (p). The reduction in quantal content is associated with a decrease in the binomial parameter n and not p, suggesting that there is a decrease in the number of contacts, release sites, or quanta available for release, rather than a change in probability of release.
Journal of Insect Physiology, 1986
Simultaneous intracellular recordings were made from filiform hair sensory neurones (FHSNs) and g... more Simultaneous intracellular recordings were made from filiform hair sensory neurones (FHSNs) and giant interneurones, and from pairs of giant interneurones in the terminal ganglion of the first-instar cockroach. Three out of the four filiform hairs on the cerci were immobilized. The remaining mobile hair was free to oscillate in the saline solution bathing the preparation. Hair movements produced regular bursts of action potentials in the FHSN axon. These spikes elicited excitatory postsynaptic potentials (epsps) in some of the giant interneurones. Simultaneous impalements of giant interneurones 2 and 3 were made with different mobile filiform hairs, in order to qualitatively compare the strengths of their excitatory synaptic inputs. Giant interneurone 3 receives strong excitatory input from the lateral FHSNs on both sides of the ganglion and some input from the medial FHSN contralateral to the giant interneurone 3 cell body. Giant interneurone 2 receives strong excitatory input from both medial FHSNs and little discernible influt from the lateral axons. These findings are consistent with estimates of the numbers of FHSN axon, :giant interneurone synapses obtained from ultrastructural studies.
Journal of comparative neurology, Dec 8, 1990
The synapses between the filiform hair sensory afferents and giant interneurons (GIs) 1-6 of embr... more The synapses between the filiform hair sensory afferents and giant interneurons (GIs) 1-6 of embryonic and first instar cockroaches, Periplaneta americana, were used to investigate the role of neuronal anatomy in determining synaptic specificity. The pattern of afferent-to-GI synapses was first determined by intracellular recording of excitatory postsynaptic potentials (EPSPs). The lateral (L) axon synapses only with GIs 3, 4, and 6, while the medial (MI axon synapses with the contralateral dendrites of all six GIs but with the ipsilateral dendrites only of GIs 1, 2, and 4. The three-dimensional anatomy of the filiform afferents and GIs was determined by injection of cobalt. There is little anatomical segregation of the filiform afferents; consequently, there is no correlation between the anatomy of the GIs and their synaptic inputs. The M axon and ipsilateral GI3 were studied in more detail by light and electron microscopy. Despite the presence of an anterior M axon branch which loops around the ipsilateral GI3 neurite at a distance of 2 p,m, no synapses are formed between them. This lack of synapses is not due to the presence of physical barriers. Investigation of filiform afferents and GIs in embryonic ganglia shows that at no stage are the afferents sufficiently separated for their anatomy to be a n important factor in determining the specificity of the synaptic inputs of the GIs. It was postulated that two pairs of complementary cell surface labels would be sufficient to code for this specificity, and that, in GIs 3, 5, and 6, spatial differences in the expression of these labels allow the M axon to distinguish ipsilateral dendrites from contralateral.
Journal of comparative neurology, Jun 8, 1992
An attempt is made to relate the distribution of filiform hairs on the cercus of the second insta... more An attempt is made to relate the distribution of filiform hairs on the cercus of the second instar cockroach, Periplaneta americana, to the morphology and patterns of synaptic connectivity of their afferents. We studied the most distal 25 of the 39 filiform hairs which are commonly present. Filiform afferent arborizations were stained by cobalt filling from the cell body in the cercus. Three fundamental arbor types were found, two similar to those of the first instar medial (M) and lateral (L) afferents, and a third, novel type. L‐type arbors could be divided into four subtypes. The most obvious correlate of arbor type is the circumferential position of the hair on the cercus. The proximodistal position of the sensillum within each cereal segment is also a determinant of its arbor. By comparison of hair positions and afferent morphologies, we were able to ascribe homologies between the second instar hairs and members of adult longitudinal hair columns. The patterns of monosynaptic connections between afferents and giant interneurons (GIs) 1, 2, 3, 5, and 6 were determined by recording synaptic potentials in GIs evoked by direct mechanical displacement of individual filiform hairs. Latency from stimulus onset to the rise phase of the first excitatory postsynaptic potential (EPSP) was used as the criterion of monosynapticity. The EPSP amplitudes of the two original L and M afferents are halved in the second instar, in the absence of a significant decrease in GI input resistance. The other afferents can be divided into two basic classes: those which input to GI5 (M‐type), and those which input to GI3 and GI6 (L‐type). The former is correlated with a central or medial position, while the latter is associated with a group of afferents situated laterally on the cercus. In segments 3 and 4, input to GIs 1 and 2 also correlates with a medial cereal position, however, in the more proximal segments 5 and 6, afferents at all positions input to these interneurons. The occurrence of afferents of identical morphology and similar connectivity in equivalent positions in different segments suggests that each sensory neuron is determined by its two‐dimensional position within a segment. The presence of afferents with the same morphology which display proximodistal differences in synaptic connectivity, and of other afferents which have M‐type connectivity despite L‐type morphology, means that anatomy is generally a poor predictor of synaptic connectivity.
The Journal of Neuroscience, Feb 1, 2002
Journal of Comparative Physiology A-neuroethology Sensory Neural and Behavioral Physiology, Nov 1, 1989
Direct evidence for monosynaptic connections between filiform hair sensory axons and giant intern... more Direct evidence for monosynaptic connections between filiform hair sensory axons and giant interneurons (GIs) in the first instar cockroach, Periplaneta americana, was obtained using intracellular recording and HRP injection followed by electron microscopy. GIs 1-6 all receive monosynaptic input from at least one filiform afferent axon. GI1, GI2 and GI5 receive input only from the medial (M) axon, while GI3, GI4 and GI6 receive input from both M and lateral (L) axons. The dendrites of GI3 and GI6 which are contralateral to the cell bodies receive input from both axons whereas the smaller ipsilateral dendritic fields have synapses only from the L axon. GI5 has M axon input only onto its contralateral dendrites. In 50% of preparations GI7 receives weak input from the ipsilateral L axon. There is no obvious relationship between the morphology of the giant interneurons and the pattern of input they receive from the filiform afferents.
The Journal of Experimental Biology, Nov 1, 1984
and connections between anatomically and physiologically defined axons have been described (King ... more and connections between anatomically and physiologically defined axons have been described (King & Wyman, 1980). To date though, synapses have not been visualized in an insect central pathway in which aspects of the anatomy, physiology, pharmacology and associated behaviour have been investigated. The cereal afferent, giant interneurone synapses in the sixth abdominal (A6) ganglion of the cockroach Periplaneta americana are well suited for such studies. Using cobalt staining (Pitman, Tweedle & Cohen, 1972) of single neurones, seven giant interneurones (GIs 1-7) can be identified on the basis of their morphology (Harrow, Hue,
Journal of Chemical Neuroanatomy, Dec 1, 2012
We have previously shown that application of fibroblast growth factor-2 (FGF-2) to cut optic nerv... more We have previously shown that application of fibroblast growth factor-2 (FGF-2) to cut optic nerve axons enhances retinal ganglion cell (RGC) survival in the adult frog visual system. These actions are mediated via activation of its high affinity receptor FGFR1, enhanced BDNF and TrkB expression, increased CREB phosphorylation, and by promoting MAPK and PKA signaling pathways. The role of endogenous FGF-2 in this system is less well understood. In this study, we determine the distribution of FGF-2 and its receptors in normal animals and in animals at different times after optic nerve cut. Immunohistochemistry and Western blot analysis were conducted using specific antibodies against FGF-2 and its receptors in control retinas and optic tecta, and after one, three, and six weeks post nerve injury. FGF-2 was transiently increased in the retina while it was reduced in the optic tectum just one week after optic nerve transection. Axotomy induced a prolonged upregulation of FGFR1 and FGFR3 in both retina and tectum. FGFR4 levels decreased in the retina shortly after axotomy, whereas a significant increase was detected in the optic tectum. FGFR2 distribution was not affected by the optic nerve lesion. Changes in the presence of these proteins after axotomy suggest a potential role during regeneration.
Journal of Comparative Physiology A-neuroethology Sensory Neural and Behavioral Physiology, Nov 1, 1991
Mutant first instar cockroaches (Periplaneta americana) with supernumerary filiform hair sensilla... more Mutant first instar cockroaches (Periplaneta americana) with supernumerary filiform hair sensilla on their cerci were used to study the effects of cell body position on axonal morphology and synaptic connections. The wild-type cercus has two hairs, one lateral (L) and the other medial (M), each with an underlying sensory neuron. Silver-intensified cobalt fills show that the supernumerary lateral neuron (SIN) in the mutant has the same shape of arborization as L, and electrophysiological recording shows that it forms synaptic connections with the same subset of giant interneurons (GIs) as L in the terminal ganglion: GI3 and GI6. The supernumerary medial neuron (SUM) has the same axonal morphology as M and synapses with the same GIs as does M: ipsilateral GIs 1 and 2 and contralateral GIs 1, 2, 3, 5 and 6. In 0.1% of approximately 8000 animals screened, a supernumerary hair arose on the cercal midline (C hair). The C neuron sends its axon to the CNS in the same branch of the cercal nerve as the L and SIN, and has a similar arborization. However, the C neuron forms synapses with the same GIs as do M and SuM. Electron microscopy of horseradish peroxidase-injected neurons was used to confirm that the C afferent forms a monosynaptic connection to GI2. It was concluded that the position of the sensory neuron cell body does control its axonal morphology and synaptic connectivity, but that these characteristics are produced by independent mechanisms.
PLOS ONE, Sep 9, 2016
After lesions to the mammalian optic nerve, the great majority of retinal ganglion cells (RGCs) d... more After lesions to the mammalian optic nerve, the great majority of retinal ganglion cells (RGCs) die before their axons have even had a chance to regenerate. Frog RGCs, on the other hand, suffer only an approximately 50% cell loss, and we have previously investigated the mechanisms by which the application of growth factors can increase their survival rate. Retinoic acid (RA) is a vitamin A-derived lipophilic molecule that plays major roles during development of the nervous system. The RA signaling pathway is also present in parts of the adult nervous system, and components of it are upregulated after injury in peripheral nerves but not in the CNS. Here we investigate whether RA signaling affects long-term RGC survival at 6 weeks after axotomy. Intraocular injection of all-trans retinoic acid (ATRA), the retinoic acid receptor (RAR) type-α agonist AM80, the RARβ agonist CD2314, or the RARγ agonist CD1530, returned axotomized RGC numbers to almost normal levels. On the other hand, inhibition of RA synthesis with disulfiram, or of RAR receptors with the pan-RAR antagonist Ro-41-5253, or the RARβ antagonist LE135E, greatly reduced the survival of the axotomized neurons. Axotomy elicited a strong activation of the MAPK, STAT3 and AKT pathways; this activation was prevented by disulfiram or by RAR antagonists. Finally, addition of exogenous ATRA stimulated the activation of the first two of these pathways. Future experiments will investigate whether these strong survival-promoting effects of RA are mediated via the upregulation of neurotrophins.
Journal of Neuroscience Research, Nov 15, 2008
Application of basic fibroblast growth factor (FGF-2) to the optic nerve after axotomy promotes t... more Application of basic fibroblast growth factor (FGF-2) to the optic nerve after axotomy promotes the survival of retinal ganglion cells (RGCs) in the frog, Rana pipiens, and results in a rapid up-regulation of BDNF and TrkB synthesis by the RGCs. Here we investigate whether this upregulation is maintained in the long term, and whether it is required for FGF-2's survival effect. At 6 weeks after axotomy and FGF-2 treatment we found more RGCs immunopositive for BDNF protein and higher intensity of BDNF and TrkB immunostaining, accompanied by increases in BDNF and TrkB mRNA in RGCs. Application of fluorescently-labeled siRNA targeted against BDNF to the cut RGC axons showed that it was transported to the cell bodies. Axonal siRNA treatment eliminated the increases in BDNF immunostaining and mRNA that were induced by FGF-2, and had no effect on TrkB mRNA. This reduction in BDNF synthesis by siRNA greatly reduced the long term survival effect of FGF-2 on RGCs. This, taken together with previous results, suggests that, while FGF-2 may initially activate survival pathways via ERK signaling, its main long-term survival effects are mediated via its upregulation of BDNF synthesis by the RGCs.
Communicative & Integrative Biology, Nov 1, 2009
Journal of comparative neurology, Nov 26, 2002
In this study we used immunocytochemistry to investigate the distribution of brain‐derived neurot... more In this study we used immunocytochemistry to investigate the distribution of brain‐derived neurotrophic factor (BDNF) and its receptor tyrosine kinase (trkB) in retina and optic tectum of the frog Rana pipiens during regeneration after axotomy. We also measured changes in BDNF mRNA in retina and tectum. Retrograde labeling was used to identify retinal ganglion cells (RGCs) prior to quantification of the BDNF immunoreactivity. In control animals, BDNF was found in the majority of RGCs and displaced amacrine cells and in some cells in the inner nuclear layer (INL). After axotomy, BDNF immunoreactivity was reduced in RGCs but increased in the INL. BDNF mRNA levels in the retina remained high before and after axotomy. Three months after axotomy, after reconnection to the target, the staining intensity of many of the surviving RGCs had partially recovered. In the control tectum, BDNF staining was present in ependymoglial cells and in neurons throughout layers 4, 6, 8, and 9. After axotomy, BDNF staining in tectal neurons became more intense, even though mRNA synthesis was transiently down‐regulated. In control retinas, trkB receptor immunostaining was present in most RGCs; no significant changes were observed after axotomy. In control tectum, trkB was detected only in ependymoglial cells. After axotomy, many neuronal cell bodies were transiently labeled. Our data are consistent with the hypothesis that a considerable fraction of the BDNF normally present in RGCs is acquired from their targets in the tectum. However, there are also intraretinal sources of BDNF that could contribute to the survival of RGCs. J. Comp. Neurol. 454:456–469, 2002. © 2002 Wiley‐Liss, Inc.
Journal of Neurochemistry, 2006
Application of basic fibroblast growth factor (FGF-2) to the optic nerve after axotomy promotes t... more Application of basic fibroblast growth factor (FGF-2) to the optic nerve after axotomy promotes the survival of retinal ganglion cells (RGCs) in the frog, Rana pipiens. Here we investigate the effects of FGF-2 treatment upon the synthesis of brain-derived neurotrophic factor (BDNF) and its receptor, tyrosine receptor kinase B (TrkB). Axotomy alone increased the amounts of BDNF and TrkB mRNA in RGCs after 1 week and 48 h, respectively; FGF-2 treatment to the nerve accelerated and increased this up-regulation of both. FGF-2 also increased the amounts of phosphorylated cAMP response element binding protein (pCREB) in the retina. Blocking extracellular-regulated kinase (ERK) activation with PD98059 or U0126 prevented the FGF-2-induced up-regulation of BDNF transcription but had no effect on TrkB. However, blocking protein kinase A (PKA) with H89 or Rp-8-Cl-cAMPS reduced the up-regulation of both BDNF and TrkB, and reduced pCREB. In addition, H89 inhibited ERK activation, indicating cross-talk between the pathways. Finally, axonal application of blocking antibody against the FGF receptor 1 (FGFR1) prevented the FGF-2-induced up-regulation of BDNF and TrkB. Our results suggest that FGF-2 acts on RGCs via FGFR1, activating the ERK pathway and CREB to increase BDNF synthesis, and PKA and CREB to increase TrkB synthesis.
Current Biology, Nov 1, 2008
The Journal of Experimental Biology, Aug 1, 2011
Unlike goal-oriented displacements, which are commonly analyzed using circular statistics (Batsch... more Unlike goal-oriented displacements, which are commonly analyzed using circular statistics (Batschelet, 1981), ETs have traditionally been analyzed using linear statistics of escape angle (y-axis) as a function of stimulus angle (x-axis) (e.g. Camhi and Tom, 1978; Eaton et al., 1981; Stern et al., 1997) (Fig.1A). This type of analysis is based on testing the significance of the regression line of y versus x. Although, graphically, it may be possible to qualitatively assess the dependence of escape angle on the stimulus angle, the testing of regression through points that are mainly located in two Cartesian sectors (-x and y; x and-y) may result in significance even in the absence of a significant relationship within each Cartesian sector. A more meaningful test of the relationship between escape angle and stimulus angle is to analyse each Cartesian sector separately, superimposing responses to left and right stimuli in a single graph (e.g. Eaton and Emberley, 1991). However, neither option allows interpretation of the escape behaviour in terms of the various circular patterns of ETs that are theoretically possible [fig.2 in Domenici et al. (Domenici et al.,
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Papers by Jonathan Blagburn