Intracellular Injection Of Horseradish Peroxidase Research Articles

Topology of thalamic axons suggests two developmental modes during innervation of visual cortex

Event Abstract Back to Event Topology of thalamic axons suggests two developmental modes during innervation of visual cortex Andreas Hauri1*, Nuno Da Costa1, Rodney Douglas1 and Kevan Martin1 1 ETHZ/UZH, Institute of Neuroinformatics, Switzerland How does a single axon grow from its cell body to form the arborization pattern seen in an adult brain? Axonal growth cones are responsible for generating these patterns. They produce these patterns based on the genetic instructions they contain, their position, their local chemical environment and other epigenetic factors. The first step to understand this process it is to establish the metrics and topology of adult axons since any model that wishes to describe axonal growth should achieve the same metrics. This study provides such a description for one of the most well studied axonal projections in the cortex the arborization of the thalamocortical axons in the primary visual cortex of the adult cat. During development the thalamic axons innervating the cortical plate divide into different branches that form their main arborization in layer 4. These arborizations form cluster of synaptic boutons that are the basis of the ocular dominance system. We labeled single axons in anaesthetized cats in vivo using intracellular injection of horseradish peroxidase (HRP) or the anterograde tracer biotinylated dextran amine (BDA). After processing for light and electron microscopy, we reconstructed 10 axons. Using a mean shift cluster algorithm we identified the location of the clusters of boutons formed by these axons in layer 4. We named as the ‘skeleton’ of the axon the branches of the thalamic axon that had their branch-point located outside the cluster, and those that branched inside the cluster the ‘florets’. We analysed the axons for several metric and topological properties like Horten-Strahler and Galton-Watson statistics of the ‘skeleton” and the ‘florets’. These analyses showed properties like branch length, bouton density and probability of branching are different between ‘skeleton’ and ‘florets’. The ‘skeleton’ seems more directed to find an appropriate target location for making connections, e.g. an ocular dominance slab in layer 4, whereas the ‘florets’ optimize the coverage of this location with synaptic boutons. These difference in statistics strongly suggests two different phases of growth during the development of the thalamic arbour. An initial set of instructions that leads to ingrowth which is then substituted by a second inducing synaptogenesis. This research was supported by the EU Grant FP7-216593 "SECO". Conference: Neuroinformatics 2010 , Kobe, Japan, 30 Aug - 1 Sep, 2010. Presentation Type: Poster Presentation Topic: General neuroinformatics Citation: Hauri A, Da Costa N, Douglas R and Martin K (2010). Topology of thalamic axons suggests two developmental modes during innervation of visual cortex. Front. Neurosci. Conference Abstract: Neuroinformatics 2010 . doi: 10.3389/conf.fnins.2010.13.00052 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Jun 2010; Published Online: 11 Jun 2010. * Correspondence: Andreas Hauri, ETHZ/UZH, Institute of Neuroinformatics, Zuerich, Switzerland, haurian@student.ethz.ch Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Andreas Hauri Nuno Da Costa Rodney Douglas Kevan Martin Google Andreas Hauri Nuno Da Costa Rodney Douglas Kevan Martin Google Scholar Andreas Hauri Nuno Da Costa Rodney Douglas Kevan Martin PubMed Andreas Hauri Nuno Da Costa Rodney Douglas Kevan Martin Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Functional connection from Mauthner cell to homologous reticulospinal neurons in the goldfish hindbrain

Crossed vestibular inhibition first described in mammals, also occurs in many species ranging from reptiles to mammals, but was nct observed in frog and toadfish. During a study of the inhibitory pathways of the Mauthner cell (M-cell) of a teleost, two classes of interneurons were individualized. Their identification was made possible by intracellular injection of horseradish peroxidase (HRP) after they had been physiologicaIly identified by the presence of an intracellularly recorded passive hyperpolarizing potential induced by the firing of their adjacent M-cell. Interneurons of the first group are part of the recurrent collateral network of the M-cell; they have some similarities with the Renshaw cells. The second group is composed of commissural vestibule-vestibular neurons. In a recent study of the neurons inhibiting the M-cell of teleosts, it was demonstrated that each bouton established on the M-cell by the commissural and recurrent collateral interneurons functions as an independent all-or-none unit. This result has prompted the investigation of the ultrastructure of these endings to determine if they were morphologically different from those found at other central chemically transmitting synapses.This chapter deals with deals with the relation that was found between the morphology and the function of a set of neurons that are known to inhibit the M-cell, in this case second order vestibulo-vestibular interneurons.

Spatial distribution patterns of excitatory and inhibitory synapses in the dendritic tree differ between jaw-closing and -opening motoneurons

This paper reviews recent data on the spatial distribution of inhibitory and excitatory synapses on the dendritic tree of jaw-closing (JC) and -opening (JO) motoneurons in the cat, in which a combination of techniques employing intracellular injections of horseradish peroxidase and postembedding immunogold labelling was used. The dendritic tree is divided into three segments: primary and distal dendrites and intermediate dendrites between the two segments. The proportion of inhibitory boutons (immunoreactive for GABA and/or glycine) is slightly higher than proportion of excitatory boutons (immunoreactive for glutamate) in JC motoneurons, but this trend is reversed in JO motoneurons. In the two kinds of motoneuron, boutons immunoreactive to glycine alone are more numerous than boutons double-labelled to GABA and glycine, which, in turn, occur more frequently than boutons immunoreactive to GABA alone. In JC motoneurons, the packing density (number of boutons per 100 μm 2) of the inhibitory boutons decreases somatofugally, but this trend is not applicable to the excitatory boutons. In contrast, the packing density of the inhibitory and excitatory boutons in JO motoneurons does not significantly differ among the three dendritic compartments, though it is slightly higher for the excitatory than the inhibitory ones on each dendritic segment. These differences have important implications for synaptic integration in JC and JO motoneurons.

Morphology of physiologically identified otolith-related vestibular neurons in cats

The morphology of physiologically identified otolith nerve-activated vestibular neurons was investigated using intracellular injections of horseradish peroxidase (HRP). Eleven utricular, 11 saccular and three utricular/saccular nerve-activated vestibular neurons were labeled with HRP. All of these neurons except one were secondary neurons, the exception being a convergent neuron. The labeled neurons were pyramidal, elongated and ovoidal in shape. Most of the labeled cells were medium to large (mean diameter: ≥30 μm). There was no apparent correlation between morphology and the different types of otolith nerve-activated vestibular neurons. Thus, it seems likely that the functional type of vestibular neurons cannot be presumed on the basis of their morphology alone.

Structure–function correlation in transient amacrine cells of goldfish retina: Basic and multifractal analyses of dendritic trees in distinct synaptic layers

Amacrine cells generating light-evoked transient ON–OFF responses were stained by intracellular injection of horseradish peroxidase after determining their input–output (voltage response vs. light intensity) profiles. Ten cells specifically having bistratified dendritic trees were analyzed. The cross-sectional area of the dendrites in each sublamina (a and b) of the inner plexiform layer was initially measured. Although some variability was observed, there was no statistically significant overall difference in the cross-sectional areas of the dendritic trees in sublaminae a and b. Also, the amplitudes of the ON and OFF responses, generated by a midrange criterion stimulus, could not be correlated with the cross-sectional areas or the number of branches of the dendrites in sublaminae b and a, respectively. On the other hand, determination of the generalized fractal spectra revealed that the negative (up to –3) and zero-order fractal dimensions of the dendritic trees in sublamina a were consistently higher than those for sublamina b. Furthermore, there was a positive correlation between response amplitude and some part of the generalized fractal dimension in the respective parts of the dendritic trees. It is concluded that dendritic tree characteristics differ in the two halves of the inner plexiform layer and that these can be related to the cells' light-evoked response amplitudes. Furthermore, generalized fractal analysis appears to be a useful method for correlating structure and function in retinal amacrine cells with complex dendritic trees. J Neurosci. Res. 66:1208–1216, 2001. © 2001 Wiley-Liss, Inc.

Structure-function correlation in transient amacrine cells of goldfish retina: basic and multifractal analyses of dendritic trees in distinct synaptic layers.

Amacrine cells generating light-evoked transient ON-OFF responses were stained by intracellular injection of horseradish peroxidase after determining their input-output (voltage response vs. light intensity) profiles. Ten cells specifically having bistratified dendritic trees were analyzed. The cross-sectional area of the dendrites in each sublamina (a and b) of the inner plexiform layer was initially measured. Although some variability was observed, there was no statistically significant overall difference in the cross-sectional areas of the dendritic trees in sublaminae a and b. Also, the amplitudes of the ON and OFF responses, generated by a midrange criterion stimulus, could not be correlated with the cross-sectional areas or the number of branches of the dendrites in sublaminae b and a, respectively. On the other hand, determination of the generalized fractal spectra revealed that the negative (up to -3) and zero-order fractal dimensions of the dendritic trees in sublamina a were consistently higher than those for sublamina b. Furthermore, there was a positive correlation between response amplitude and some part of the generalized fractal dimension in the respective parts of the dendritic trees. It is concluded that dendritic tree characteristics differ in the two halves of the inner plexiform layer and that these can be related to the cells' light-evoked response amplitudes. Furthermore, generalized fractal analysis appears to be a useful method for correlating structure and function in retinal amacrine cells with complex dendritic trees.

Quantitative ultrastructure of physiologically identified premotoneuron terminals in the trigeminal motor nucleus in the cat

Little is known about the ultrastructure of synaptic boutons contacting trigeminal motoneurons. To address this issue, physiologically identified premotor neurons (n = 5) in the rostrodorsomedial part of the oral nucleus (Vo.r) were labeled by intracellular injections of horseradish peroxidase (HRP) in cats. The ultrastructure of 182 serially sectioned axon terminals from the five neurons was both qualitatively and quantitatively analyzed. In addition, the effects of the glycine antagonist strychnine, GABAA antagonist bicuculline, NMDA antagonist 2-amino-5-phosphonovalerate (APV), and non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on Vo.r-induced postsynaptic potentials in trigeminal motoneurons (n = 11) were examined to evaluate potential signaling substances of the premotor neurons. Labeled boutons made synaptic contacts with either jaw-closing or -opening motoneurons. All the boutons contained pleomorphic vesicles, and most formed a single symmetric synapse either on the somata or on primary dendrites. Morphometric analyses indicated that bouton volume, bouton surface area, apposed surface area, total active zone area, and mitochondrial volume were not different between boutons on jaw-closing and -opening motoneurons. Vesicle number and density, however, were higher for boutons on jaw-closing motoneurons. The five morphological parameters were positively correlated with bouton volume. Vesicle density was the exception, which tending to be negatively correlated. Intravenous infusion of strychnine or bicuculline suppressed Vo.r-induced inhibitory postsynaptic potentials (IPSPs) in jaw-closing motoneurons. Abolition of Vo.r-induced excitatory postsynaptic potentials in jaw-opening motoneurons with APV and CNQX unmasked IPSPs. The present results suggest that premotor neurons in the Vo.r are inhibitory and that positive correlations between the ultrastructural parameters associated with synaptic release and bouton size are applicable to the interneurons, as they are in primary afferents. J. Comp. Neurol. 426:13–30, 2000. © 2000 Wiley-Liss, Inc.

Morphology of wide-field bistratified and diffuse human retinal ganglion cells.

To study the detailed morphology of human retinal ganglion cells, we used intracellular injection of horseradish peroxidase and Neurobiotin to label over 1,000 cells in an in vitro, wholemount preparation of the human retina. This study reports on the morphology of 119 wide-field bistratified and 42 diffuse ganglion cells. Cells were analyzed quantitatively on the basis of dendritic-field size, soma size, and the extent of dendritic branching. Bistratified cells were similar in dendritic-field diameter (mean +/- S.D. = 682 +/- 130 microm) and soma diameter (mean +/- S.D. = 18 +/- 3.3 microm) but showed a broad distribution in the extent of dendritic branching (mean +/- S.D. branch point number = 67 +/- 32; range = 15-167). Differences in the extent of branching and in dendritic morphology and the pattern of branching suggest that the human retina may contain at least three types of wide-field bistratified cells. Diffuse ganglion cells comprised a largely homogeneous group whose dendrites ramified throughout the inner plexiform layer. The diffuse cells had similar dendritic-field diameters (mean +/- S.D. = 486 +/- 113 microm), soma diameters (mean +/- S.D. = 16 +/- 2.3 microm), and branch points numbers (mean +/- s.D. = 92 +/- 32). The majority had densely branched dendritic trees and thin, very spiny dendrites with many short, fine, twig-like thorny processes. Five of the diffuse cells had much more sparsely branched dendritic trees (<50 branch points) and less spiny dendrites, suggesting that there are possibly two types of diffuse ganglion cells in human retina. Although the presence of a diversity of large bistratified and diffuse ganglion cells has been observed in a variety of mammalian retinas, little is known about the number of cell types, their physiological properties, or their central projections. Some of the human wide-field bistratified cells in the present study, however, show morphological similarities to monkey large bistratified cells that are known to project to the superior colliculus.

Quantitative ultrastructure of physiologically identified premotoneuron terminals in the trigeminal motor nucleus in the cat

Little is known about the ultrastructure of synaptic boutons contacting trigeminal motoneurons. To address this issue, physiologically identified premotor neurons (n = 5) in the rostrodorsomedial part of the oral nucleus (Vo.r) were labeled by intracellular injections of horseradish peroxidase (HRP) in cats. The ultrastructure of 182 serially sectioned axon terminals from the five neurons was both qualitatively and quantitatively analyzed. In addition, the effects of the glycine antagonist strychnine, GABA(A) antagonist bicuculline, NMDA antagonist 2-amino-5-phosphonovalerate (APV), and non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on Vo.r-induced postsynaptic potentials in trigeminal motoneurons (n = 11) were examined to evaluate potential signaling substances of the premotor neurons. Labeled boutons made synaptic contacts with either jaw-closing or -opening motoneurons. All the boutons contained pleomorphic vesicles, and most formed a single symmetric synapse either on the somata or on primary dendrites. Morphometric analyses indicated that bouton volume, bouton surface area, apposed surface area, total active zone area, and mitochondrial volume were not different between boutons on jaw-closing and -opening motoneurons. Vesicle number and density, however, were higher for boutons on jaw-closing motoneurons. The five morphological parameters were positively correlated with bouton volume. Vesicle density was the exception, which tending to be negatively correlated. Intravenous infusion of strychnine or bicuculline suppressed Vo.r-induced inhibitory postsynaptic potentials (IPSPs) in jaw-closing motoneurons. Abolition of Vo. r-induced excitatory postsynaptic potentials in jaw-opening motoneurons with APV and CNQX unmasked IPSPs. The present results suggest that premotor neurons in the Vo.r are inhibitory and that positive correlations between the ultrastructural parameters associated with synaptic release and bouton size are applicable to the interneurons, as they are in primary afferents.

The continuing search for outer hair cell afferents in the guinea pig spiral ganglion

Antidromic stimulation of the stump of the VIIIth nerve was combined with microelectrode recording in the spiral ganglion of the guinea pig cochlea in an attempt to identify a sub-population of neurons with long-latency antidromic action potentials that might correspond to the thin unmyelinated afferent neurons emanating from the outer hair cells. The techniques used were similar but not identical to those employed in an earlier study by Brown (1994). By far the largest population of cells contacted had short antidromic latencies (0.58±0.12 ms, 76 units) and also responded to acoustic stimulation. These were assumed to be type I afferents emanating from inner hair cells. Eight cells had antidromic latencies larger than 1 ms, all but one of which had a zero spontaneous rate. All eight of these longer-latency cells were unresponsive to acoustic stimulation despite the fact that short-latency neurons in the same cochleas showed robust responses to sound before and after they were contacted. Four of these longer-latency cells had their antidromic thresholds accurately measured and two had significantly higher thresholds to electrical stimulation (0.1 ms duration) than type I cells in the same animal while two had similar electrical thresholds. Attempts to trace the eight long-latency neurons to the outer hair cells using intracellular injection of horseradish peroxidase were unsuccessful. On the basis of present evidence, we cannot conclude definitively that the long-latency neurons found in the spiral ganglion belong to the outer hair cell afferent population.

Morphology and intercellular communication in glial cells of intramural ganglia from the guinea-pig urinary bladder

Neurons in most peripheral ganglia are surrounded by satellite glial cells (SCs), but these cells have so far received little attention. We used immunohistochemistry and intracellular injections of tracers to characterize SCs in the intramural ganglia of the guinea-pig urinary bladder, which are part of the parasympathetic system. Intracellular injections of horseradish peroxidase (HRP) revealed two morphological types: cells that surrounded neurons and are SCs proper, and bipolar cells with processes that projected into the nerve fiber bundles connecting the ganglia. SCs were immunopositive for glutamine synthetase (GS) and S100β and immunonegative for glial fibrillary acidic protein (GFAP). Injections of Lucifer yellow (LY) or biocytin (molecules known to cross gap junctions) into single SCs showed that these cells have a very low degree of intercellular coupling. A mean of 0.31 and 0.71 cells were coupled to the injected cells, using LY and biocytin, respectively. It appears that SCs in the bladder ganglia are distinct from central and enteric glial cells in the small degree of their coupling and in the absence of GFAP immunostaining.

Morphologic characteristics of physiologically defined neurons in the cat trigeminal nucleus principalis

Although the principalis nucleus (Vp) contains trigeminothalamic and internuclear tract cells, the functional and morphologic differences between the two kinds of neurons have remained unsettled. The present study was aimed to address these problems by using the intracellular horseradish peroxidase injection technique in the cat. Of 20 neurons stained, 7 and 13 were located in the dorsomedial subnucleus (Vpd) and ventrolateral subnucleus (Vpv) of Vp, respectively. The Vpd neurons received input from the intraoral structures only but the Vpv neurons from the intraoral or facial structures. Nineteen neurons could be divided as class I and class II, based on the branching pattern of their stem axons. Class I (eight neurons) had an ascending stem axon without branching. Class II was divided into two subclasses (IIa and IIb). Class IIa (eight neurons) had an ascending stem axon from which branches were given off. Their branches formed a local-circuit restricted to the lower brainstem. Class IIb (three neurons) had a stem axon that formed the local-circuit only. The dendritic morphology was indistinguishable between different classes of neurons and between the subdivisions. Although the dendritic arborization pattern was governed by the location of the somata, it was suggested to be also important elements for determining primary afferent arborizations. In the brainstem nuclei, the jaw-closing motor nucleus received the highest density of projections from class II neurons with the receptive field involving the periodontal ligaments. The present study provides new findings that Vp neurons could be divided into three distinct populations and suggests that each population exerts a distinct function with respect to sensory discrimination, sensorimotor reflexes, or both.

Inhibitory connections between antagonistic motor neurones of the crayfish walking legs

The inhibitory relationship between two antagonistic groups of motor neurones (MNs) that control the second leg joint of the crayfish Procambarus clarkii, was investigated in an in vitro preparation of the ventral nerve cord. Paired intracellular recordings were used to test the hypothesis that reciprocal inhibitory connections between levator (Lev) and depressor (Dep) MNs are direct. The injection of depolarising current into a Lev MN induces a hyperpolarising response in the Dep MN. This inhibitory relationship does not require spikes in the presynaptic MN, because it persists when spikes are suppressed by the sodium channel blocker tetrodotoxin (TTX). This reciprocal inhibition is graded, and both the amplitude and the time constant of the hyperpolarising response increase with increasing amount of depolarising current injected into an antagonistic MN. Although this inhibition is slow (synaptic delay around 10 ms), it is probably supported by a direct glutamatergic synapse from the antagonistic glutamatergic MN because it persists in the presence of the gamma-amino-butyric acid (GABA) synthesis inhibitor 3-mercapto-propionic acid (3-MPA). This hypothesis is reinforced by the demonstration of close appositions between antagonistic MNs by using a confocal microscope, and by the presence of glutamate-immunoreactive synapses on the neurites of MNs labelled for electron microscopy by intracellular injection of horseradish peroxidase.

Morphology of human retinal ganglion cells with intraretinal axon collaterals.

Ganglion cells with intraretinal axon collaterals have been described in monkey (Usai et al., 1991), cat (Dacey, 1985), and turtle (Gardiner & Dacey, 1988) retina. Using intracellular injection of horseradish peroxidase and Neurobiotin in in vitro whole-mount preparations of human retina, we filled over 1000 ganglion cells, 19 of which had intraretinal axon collaterals and wide-field, spiny dendritic trees stratifying in the inner half of the inner plexiform layer. The axons were smooth and thin (approximately 2 microm) and gave off thin (<1 microm), bouton-studded terminal collaterals that extended vertically to terminate in the outer half of the inner plexiform layer. Terminal collaterals were typically 3-300 microm in length, though sometimes as long as 700 microm, and were present in clusters, or as single branched or unbranched varicose processes with round or somewhat flattened lobular terminal boutons 1-2 microm in diameter. Some cells had a single axon whereas other cells had a primary axon that gave rise to 2-4 axon branches. Axons were located either in the optic fiber layer or just beneath it in the ganglion cell layer, or near the border of the ganglion cell layer and the inner plexiform layer. This study shows that in the human retina, intraretinal axon collaterals are associated with a morphologically distinct ganglion cell type. The synaptic connections and functional role of these cells are not yet known. Since distinct ganglion cell types with intraretinal axon collaterals have also been found in monkey, cat, and turtle, this cell type may be common to all vertebrate retinas.

Morphological analysis of cat masseteric motoneurons after intracellular staining with horseradish peroxidase

Intracellular injection of horseradish peroxidase (HRP) into 58 masseteric motoneurons identified by antidromic activation was performed in cats under pentobarbital anesthesia. Monosynaptic EPSPs were evoked by masseteric nerve stimuli in 52 cells, and were absent in the remaining six cells. The antidromic nature of the evoked spikes was confirmed by IS-SD separation observed at high frequency (50 Hz) stimulation. Motoneurons with monosynaptic excitation from masseter afferents showed IPSPs following stimulation of lingual and inferior alveolar nerves. Motoneurons which did not show monosynaptic excitation from masseter afferents showed no IPSPs from the above nerves. There were no differences in cell size or the number of stem dendrites between motoneurons with and without monosynaptic EPSPs. No recurrent collaterals were observed in any motor axons. Motoneurons with monosynaptic EPSPs were located at all rostrocaudal levels throughout the trigeminal motor nucleus, whereas motoneurons without such EPSPs were encountered only at the middle level. Dendrites of motoneurons with monosynaptic EPSPs did not extend into the medial portion of the nucleus where motoneurons innervating the anterior belly of the digastric muscle were located. In contrast, motoneurons without monosynaptic EPSPs had dendrite branches extending well into the medial part. The results show that there are two subpopulations of masseteric motoneurons that differ in peripheral inputs as well as dendritic morphology.

Synaptic organization of substance P, glutamate and GABA-immunoreactive boutons on functionally identified neurons in cat spinal deeper dorsal horn

In order to determine how nociceptive input conveyed by the C-fibers terminating in superficial laminae of the spinal cord reaches the wide dynamic range (WDR) cells in deeper dorsal horn, which functions as ascending projection pathway, the morphological features of some WDR cells in the deeper dorsal horn of the cat lumbar spinal cord were studied by intracellular injection of horseradish peroxidase and physiological characterization. One of the fully stained neurons with somata in lamina V and dendrites that entered lamina II were examined by electron microscopy. Immunogold staining of ultrathin sections through the labeled proximal dendrites in lamina II revealed that these dendrites received numerous synapses from substance P and glutamate immunoreactive (IR) axons, which were considered originating from C-fibers. In addition, many GABA-IR terminals were found presynaptic to the labeled dendrites. The results, therefore, suggest that the information carried by primary afferent can be sent from the superficial dorsal horn to the deeper laminae through monosynaptic contacts between C-fiber terminals and the long dorsal dendrites of some WDR cells in the deeper laminae, and that GABAergic system is involved in postsynaptic control to modulate the transmission of nociceptive sensory information.

Synaptic connections between identified neuron types in the antennal lobe glomeruli of the cockroach, Periplaneta americana: II. Local multiglomerular interneurons.

Synapses between three types of antennal lobe neurons, namely, local multiglomerular interneurons, antennal receptor neurons, and gamma-aminobutyric acid (GABA)-immunoreactive neurons, were studied by means of a combination of three different markers. The interneurons were labeled by intracellular injection of horseradish peroxidase (HRP) into a single soma or a small group of neurons. Antennal receptor cells were marked by experimentally induced anterograde degeneration, and GABA-containing neurons were identified by postembedding immunogold staining. The following types of connections were found: Local interneurons receive input synapses from 1) degenerated receptor neuron axons, 2) GABA-immunogold-labeled neurites, and 3) non-GABA-immunoreactive neurons. The interneurons form output synapses onto the same three neuron groups. Contacts were also found between HRP-labeled interneurons themselves. The majority of synapses were dyadic. In most cases, only one postsynaptic neuronal process of the dyads was labeled and, thus, was identified. Polysynaptic connections were found between GABA-immunoreactive neurites, HRP-labeled interneuron processes, and nonlabeled neurites or between HRP-labeled interneuron processes and two interconnected GABA-immunoreactive processes. The present findings provide anatomical evidence for an earlier suggested monosynaptic connection between afferent receptor fibers and local, at that time putative, GABAergic interneurons. They further reveal that local multiglomerular interneurons are synaptically interconnected. The interneurons, in addition, form serial connections via more than one GABA-immunoreactive neuron with non-GABA-immunoreactive and putative projection neurons. Such polysynaptic connections would be a substrate for a feed-forward "disinhibition" of projection neurons, which has been suggested on the basis of electrophysiological findings.

Proportions of ON-Center versus OFF-Center Cells in Retinal Ganglion Cells with Regenerated Axons of Adult Cats

Axons of retinal ganglion cells (RGCs) of adult cats were transected and regenerated by the transplantation of peripheral nerves. Two months after transplantation dendritic arbors of RGCs with regenerated axons (r-RGCs) were visualized with intracellular injections of horseradish peroxidase (HRP). The HRP-filled r-RGCs were classified as α, β, γ, or unclassified cells. The r-RGCs were then sectioned vertically and their dendritic ramification in the inner plexiform layer (IPL) was examined to distinguish them as OFF-or ON-center cells. Seventy-nine cells extended their dendrites into either sublamina a (OFF) or sublamina b (ON) of the IPL. Two γ cells spread dendrites into both ON and OFF sublaminae. The numbers of ON-center cells were greater than those of OFF-center cells in three classes: 22 of 24 β cells, 4 of 6 γ cells, and 16 of 18 unclassified cells. These data confirmed the predominance of ON-center cells among X/β and W cells in previous physiological recordings from regenerated axons of r-RGCs. However, only 4 cells were ON-center among 33 r-RGCs with α cell morphology. The result apparently conflicts with the predominance of ON-center Y cells in physiological sampling. Possible explanations for the apparent discrepancy are discussed.

Transfer of horseradish peroxidase from oligodendrocyte to axon in the myelinating neonatal rat optic nerve: artefact or transcellular exchange?

In this paper we make the surprising observation that intracellular injection of horseradish peroxidase (HRP) into a single myelinating oligodendrocyte also resulted in localised HRP labelling at the nodes of Ranvier of some axons of the unit. It appeared that HRP had been transferred to the nodal axoplasm from the paranodal loops of the HRP-filled oligodendrocyte. Three HRP-filled oligodendrocytes from isolated optic nerves of 14-day-old rats were analysed by serial section electron microscopy, and HRP was observed in the axonal cytoplasm at three of the nodes of Ranvier delineated by one of the cells. At labelled nodes, HRP was of a uniform intensity throughout the nodal axoplasm. Axonal labelling gradually diminished along the paranodal regions and was not evident in the contiguous internodal axoplasm beyond 20 microns from the node. The myelin sheaths, paranodal loops, and axons appeared normal at labelled nodes, and the paranodal loops and astrocyte perinodal processes adjacent to those of the HRP-filled oligodendrocyte unit did not contain HRP. There was no evidence of extracellular HRP or tissue damage in the surrounding neuropil, and axons neighbouring those enwrapped by the HRP-filled oligodendrocyte did not contain HRP. The possibility that axonal labelling was an artefact of either iontophoretic injection or tissue preparation is discussed. This provocative finding is not definite proof of exchange, but the balance of evidence supports the possibility that there was transcellular exchange of HRP at paranodes between the labelled oligodendrocyte and some of the axons in the unit. The rarity of HRP transfer to axons suggests that it may be a transient or labile event. It is not clear whether oligodendrocyte to axon macromolecular exchange has real physiological and/or pathological significance.

Distribution of synapses on two local auditory interneurones, ON1 and ON2, in the prothoracic ganglion of the cricket: relationships with GABA-immunoreactive neurones.

In the prothoracic ganglia of the cricket Gryllus bimaculatus two local auditory interneurones, ON1 and ON2, were labelled for electron microscopy by intracellular injection of horseradish peroxidase following physiological characterisation. The neurones branch in the median ventral association centre and the root of nerve 5 on both sides of the ganglion. As they are very similar in shape and position they may share a common embryological origin. Differences are found in the details of the fine branching pattern and in their physiology as ON1 is tuned particularly to low sound frequencies of 4-5 kHz whereas ON2 is more sensitive to frequencies above 8 kHz. Although the ON1 neurones inhibit each other and are involved in the inhibition of other auditory neurones they were not labelled by antibodies against the inhibitory transmitter GABA and their vesicles differ significantly from those in neurones that are. The same is true of the ON2 neurones whose vesicles also differ significantly from those in ON1 supporting light-microscope evidence that they may use different transmitters. The distribution of input and output synapses on the ipsilateral and contralateral branches of ON1 and ON2, and the proportion of the synapses made from and onto neuropilar processes immunoreactive for GABA was determined. In ON1 94% of the input synapses were received on the ipsilateral branches and 62% of the outputs made from the contralateral branches. This confirms previous physiological evidence that input is received ipsilaterally and output made contralaterally but the presence of some contralateral input and a significant ipsilateral output was unsuspected. Thirty percent of the input synapses on the ipsilateral side and 75% on the contralateral side were made from GABA-immunoreactive processes but processes postsynaptic to ON1 were rarely immunoreactive. The distribution of input synapses on ON2 was similar with 90% received on ipsilateral branches but a higher proportion of outputs (83%) was made from the contralateral side than in ON1. Thirty one percent of ipsilateral inputs were GABA-immunoreactive but only 14% on the contralateral side.