Biomedical subjects

A J Todd

Publications and source records attributed to A J Todd.

At least 37 records · Page 2Linked to original sources

PubMed1998

GluR1 and GluR2/3 subunits of the AMPA-type glutamate receptor are associated with particular types of neurone in laminae I-III of the spinal dorsal horn of the rat.

GluR1 and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor are expressed at high levels by neurones in laminae I-III of rat spinal dorsal horn, an area which contains numerous, densely packed small neurones. In order to determine whether these subunits are expressed by inhibitory or excitatory neurones, we combined pre-embedding immunocytochemistry with antibodies that recognize either GluR1, or an epitope common to GluR2 and 3, with postembedding detection of gamma-aminobutyric acid (GABA) and glycine. Most (78%) of the neurones with GluR1-immunoreactivity were GABA-immunoreactive, and some of these were also glycine-immunoreactive, whereas nearly all (97%) of the GluR2/3-immunoreactive neurones were not GABA- or glycine-immunoreactive. We carried out double-immunofluorescence and confocal microscopy to provide further information on the neurochemistry of cells that express these subunits. As expected, all neurotensin- and virtually all somatostatin-immunoreactive cells (which are thought to be excitatory interneurones) were GluR2/3- but not GluR1-immunoreactive, whereas parvalbumin-containing cells (most of which are GABAergic) possessed GluR1-, but usually not GluR2/3-immunoreactivity. Neurones that contained nitric oxide synthase (most of which are GABAergic) were more variable, with 57% GluR1-immunoreactive and 41% GluR2/3-immunoreactive. Cholinergic neurones in lamina III (which are also GABAergic) invariably showed each type of GluR-immunoreactivity. These results suggest that neuronal populations in laminae I-III have characteristic patterns of GluR expression: GluR1 is particularly associated with inhibitory neurones, and GluR2 with excitatory neurones. This makes it likely that some of the AMPA receptors present on the inhibitory interneurones lack the GluR2 subunit, and may therefore have significant Ca2+-permeability.

Animals↗

PubMed1998

Cells in laminae III and IV of the rat spinal cord which possess the neurokinin-1 receptor receive monosynaptic input from myelinated primary afferents.

We have previously demonstrated that neurons which have cell bodies in laminae III or IV of the rat spinal cord, dendrites that enter the superficial laminae and which possess the neurokinin-1 receptor receive a major synaptic input from substance P-containing primary afferent axons. In this study we set out to determine whether these cells also receive monosynaptic input from myelinated primary afferents by using transganglionic transport of the B subunit of cholera toxin to identify the central terminals of myelinated afferents from the sciatic nerve. Dual-immunofluorescence and confocal microscopy revealed apparent contacts between labelled primary afferent terminals and all of the neurokinin-1 receptor-immunoreactive cells examined, although these contacts were much less numerous than those which the cells receive from substance P-containing primary afferents. By using a combined confocal and electron microscopic technique we were able to confirm that synapses were present at some of the contacts between primary afferents and neurokinin-1 receptor-immunoreactive neurons. These results suggest that cells of this type will have wide-dynamic range receptive fields, but with a relatively strong input from nociceptors.

Animals↗

PubMed1997

Cells in laminae III and IV of the rat spinal cord that possess the neurokinin-1 receptor and have dorsally directed dendrites receive a major synaptic input from tachykinin-containing primary afferents.

Many neurons with cell bodies in laminae III or IV of the spinal dorsal horn possess the neurokinin 1 receptor and have dorsal dendrites that arborize in the superficial dorsal horn. We have performed a confocal microscopic study to determine whether these cells receive inputs from substance P-containing primary afferents. All neurons of this type received contacts from substance P-immunoreactive axons, and in most cases the contacts onto dorsal dendrites were very numerous. A great majority (90-100%) of substance P-immunoreactive varicosities in contact with these cells were also immunoreactive with antibody to calcitonin gene-related peptide, indicating that they were of primary afferent origin. The density of contacts from substance P-immunoreactive varicosities onto these cells was significantly higher than that seen on cholinergic neurons in lamina III (which do not possess the receptor). Electron microscopy revealed that synapses were present at points of contact between substance P-immunoreactive boutons and dorsal dendrites of cells with the neurokinin 1 receptor. Some cells of this type belong to the spinothalamic tract, and we therefore examined neurons with cell bodies in laminae III or IV that possessed the neurokinin 1 receptor and were labeled retrogradely after thalamic injection of cholera toxin B subunit. These cells also received contacts from substance P-immunoreactive axons on their dorsal dendrites. The results of this study indicate that neurons of this type are a major target for substance P-containing primary afferents.

Afferent Pathways↗

PubMed1997

A method for combining confocal and electron microscopic examination of sections processed for double- or triple-labelling immunocytochemistry.

Double-labelling immunocytochemical techniques are important for revealing synaptic connections between different populations of neurons within the central nervous system. This article describes a new method in which confocal laser scanning microscopy and electron microscopy are performed on the same Vibratome section which has been processed for immunocytochemistry. Two or three primary antibodies are initially detected with fluorescent secondary antibodies and observed with the confocal microscope. The primary antibodies are then revealed by an immunoperoxidase technique (with diaminobenzidine), and the material is prepared for electron microscopy. By comparing the resulting electron micrographs with the images acquired from the confocal microscope, it is possible to recognise each immunoreactive structure seen with the electron microscope in the original confocal images, and therefore to determine which type(s) of immunoreactivity each structure contains. This method has been used to demonstrate that some neurons in the spinal dorsal horn which possess the neurokinin-1 receptor receive axosomatic synapses from boutons that contain substance P and calcitonin gene-related peptide.

Animals↗

PubMed1997

An ultrastructural study of the glycine transporter GLYT2 and its association with glycine in the superficial laminae of the rat spinal dorsal horn.

The glycine transporter GLYT2 is present in axonal boutons throughout the spinal cord, and its laminar distribution matches that of glycine-enriched axons, which are presumed to be glycinergic. In order to determine whether boutons which possess GLYT2 are glycine-enriched, we have carried out pre-embedding immunocytochemistry with antibody raised against GLYT2, and combined this with post-embedding detection of glycine, in the rat. GLYT2 immunoreactivity was present in boutons which formed symmetrical axodendritic, axosomatic or axoaxonic synapses, and was often seen in peripheral axons of type II synaptic glomeruli. One hundred and fifty GLYT2-immunoreactive boutons were analysed quantitatively, and in 142 (94.6%) of these the density of gold particles representing glycine-like immunoreactivity exceeded the background level (over presumed glutamatergic boutons) by at least a factor of two. Within immunoreactive boutons, the GLYT2 reaction product was associated with the plasma membrane, but often appeared as discrete clumps and was generally excluded from the region of the active sites of synapses. These results confirm that GLYT2 is associated with glycine-enriched axonal boutons in the superficial dorsal horn. They also suggest that GLYT2 is unevenly distributed on the plasma membrane of these boutons, and raise the possibility that it may be excluded from synaptic clefts.

Amino Acid Transport Systems, Neutral↗

PubMed1996

Colocalization of GABA, glycine, and their receptors at synapses in the rat spinal cord.

To determine whether GABA and glycine can act as cotransmitters at synapses in the rat spinal cord, we have compared the ultrastructural distribution of GABAA-receptor beta 3 subunit with that of the glycine receptor-associated protein gephyrin and combined this with postembedding detection of GABA and glycine. We also used a dual-immunofluorescence method to confirm that gephyrin was associated with the glycine-receptor alpha 1 subunit throughout the cord. GABAA beta 3-subunit immunoreactivity was restricted primarily to synapses, and at a majority of these synapses the presynaptic axon was GABA-immunoreactive. Many synapses showed both GABAA beta 3 and gephyrin immunoreactivity, and at most of these synapses GABA and glycine were enriched in the presynaptic axon. These results strongly support the idea that cotransmission by GABA and glycine occurs in the spinal cord.

Animals↗

PubMed1996

Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat.

In order to determine whether spinothalamic neurons in the lumbar spinal cord of the rat process neurokinin-1 (substance P) receptors, we injected cholera toxin B subunit into the thalamus and carried out dual-labelling immunocytochemistry to search for neurons that were immunoreactive with antibodies to cholera toxin and neurokinin-1 receptor. We examined 356 spinothalamic neurons in transverse sections and found that 35% of these were neurokinin-1 receptor-immunoreactive. Double-labelled cells made up the majority of the spinothalamic population in lamina I and the lateral spinal nucleus, and were also present in laminae III-V and the area around the central canal. On the side contralateral to the injection site, 77% of spinothalamic neurons in lamina I also showed neurokinin-1 receptor immunoreactivity, while 33% of those in laminae III-V and 14% of the ventromedial group possessed the receptor. Several of the double-labelled neurons with cell bodies in laminae III and IV had dendrites which could be followed dorsally into the superficial dorsal horn. These results indicate that substance P released from nociceptive primary afferents into the superficial dorsal horn is likely to act on spinothalamic tract neurons in lamina I, and also on those with cells bodies in laminae III-IV and long dorsal dendrites.

Animals↗

PubMed1996

The mu-opioid receptor (MOR1) is mainly restricted to neurons that do not contain GABA or glycine in the superficial dorsal horn of the rat spinal cord.

The mu-opioid receptor MOR1 is present on primary afferent axons and a population of neurons in the superficial dorsal horn of the rat spinal cord. In order to determine which types of neuron possess the receptor we carried out pre-embedding immunocytochemistry with antibody to MOR1 and combined this with a post-embedding method to detect GABA and glycine in the rat. MOR1 immunoreactivity was seen on many small neurons in lamina II and a few in the dorsal part of lamina III. Although immunostaining was mainly restricted to the cell bodies and dendrites of these neurons, in some cases it was possible to see their axons, and a few of these entered lamina III. One hundred and thirty-nine MOR1-immunoreactive cells were tested with GABA and glycine antibodies, and the great majority of these (131 of 139; 94%) were not GABA or glycine immunoreactive, while the remainder showed GABA but not glycine immunoreactivity. These results suggest that most of the cells in the superficial dorsal horn which possess MOR1 are excitatory interneurons. They support the hypothesis that part of the action of mu-opioid agonists, such as morphine, involves the inhibition of excitatory interneurons which convey input from nociceptors to neurons in the deep dorsal horn, thus interrupting the flow of nociceptive information through polysynaptic pathways in the spinal cord.

Animals↗

PubMed1996

GABA and glycine in synaptic glomeruli of the rat spinal dorsal horn.

The superficial dorsal horn of rat spinal cord contains two types of synaptic glomerulus, which are centred around the terminals of unmyelinated and myelinated primary afferents respectively. Both types of glomerulus contain GABAergic axons and dendrites, which are thought to originate from local inhibitory interneurons. Some of the dendrites contain synaptic vesicles, and may be presynaptic to the central axon of dendroaxonic synapses. In order to determine whether GABAergic structures in the two types of glomerulus are derived from different populations of interneurons, a quantitative post-embedding immunogold study of GABA- and glycine-immunoreactivity in a rat dorsal horn was performed. In type I glomeruli, all of the peripheral axons and most vesicle-containing dendrites were GABA-immunoreactive, but only one of 32 axons and none of the vesicle-containing dendrites was glycine-immunoreactive. In contrast, most of the peripheral axons and some of the vesicle-containing dendrites in type II glomeruli possessed both GABA- and glycine-immunoreactivity. This strongly suggests that different types of inhibitory interneuron in the dorsal horn are associated with the two types of glomerulus. It is therefore likely that different populations of interneurons mediate presynaptic inhibition of unmyelinated and myelinated primary afferents.

Animals↗

PubMed1995

Colocalization of glycine and GABA in synapses on spinomedullary neurons.

Spinomedullary neurons of the postsynaptic dorsal column pathway in adult cats were retrogradely labelled with horseradish peroxidase. Postembedding immunogold reactions were performed with antisera which recognise GABA or glycine to determine if synaptic boutons in contact with these neurons contain both transmitters. Analysis of series of ultrathin sections revealed that synaptic profiles with strong immunogold reactions for GABA usually also displayed strong immunogold reactions for glycine. Pre-embedding immunocytochemistry was performed on sections containing labelled cells with a monoclonal antibody which recognises the glycine receptor-associated protein, gephyrin. Many synapses onto postsynaptic dorsal column neurons were associated with gephyrin-like immunoreactivity and these typically contained irregularly shaped vesicles. Immunogold reactions showed that synaptic profiles apposed to gephyrin-immunoreactive junctions contained GABA and glycine. The evidence suggests that glycine is a neurotransmitter at synapses on spinomedullary neurons and that it is colocalized with GABA.

Animals↗

PubMed1995

Galanin is contained in GABAergic neurons in the rat spinal dorsal horn.

In order to determine which types of neuron in laminae I-III of the rat spinal dorsal horn contain the peptide galanin, pre-embedding immunocytochemistry with antiserum to galanin was combined with post-embedding detection of GABA- and glycine-like immunoreactivities. Sixty-eight galanin immunoreactive neurons in laminae I-III selected from four rats were examined, and in each case semi-thin sections through the cell body were tested with a monoclonal antibody to GABA and an antiserum to glycine. All of the 68 galanin-immunoreactive neurons tested were GABA-immunoreactive, while only one of them (in lamina III) was glycine-immunoreactive. This suggests that galanin is contained in inhibitory interneurons, and that (like enkephalin, neuropeptide Y and thyrotropin-releasing hormone) it is mainly restricted to GABAergic neurons which do not use glycine as a co-transmitter.

Animals↗

PubMed1995

The relationship between glycine and gephyrin in synapses of the rat spinal cord.

In order to examine the relationship between gephyrin (the peripheral membrane protein associated with glycine receptors) and glycinergic boutons, we have carried out a post-embedding immunogold study of glycine-like immunoreactivity on sections of rat lumbar spinal cord which had previously been reacted with monoclonal antibody to gephyrin. In all three areas examined (laminae I and II, lamina III and lamina IX) the majority of profiles which were presynaptic at gephyrin-immunoreactive synapses were enriched with glycine-like immunoreactivity. It was estimated that at least 83% of profiles presynaptic to gephyrin-immunoreactive synapses in the superficial dorsal horn (laminae I and II) were glycine-immunoreactive, while for lamina III and the ventral horn (lamina IX) the proportions were at least 91% and 98% respectively. This provides strong evidence that glycine is a transmitter at those synapses where gephyrin- and glycine-like immunoreactivities are both present, but suggests that gephyrin may sometimes be expressed at non-glycinergic synapses and indicates the need for caution in using gephyrin-immunoreactivity as a marker for glycinergic synapses within the spinal cord. By reacting serial sections of dorsal horn with antisera to glycine and GABA, we have shown that many boutons in laminae I-III of the dorsal horn show both types of immunoreactivity and are therefore likely to use both amino acids as inhibitory transmitters. Many of the boutons which were presynaptic at axoaxonic synapses in the ventral part of lamina II and in lamina III were glycine- and GABA-immunoreactive and in many cases the postsynaptic element was the central axon of a type II synaptic glomerulus. Taken together with pharmacological evidence, this suggests that inhibitory interneurons in the dorsal horn which use both GABA and glycine may be important in controlling the flow of information from hair follicle afferents to other spinal neurons.

Afferent Pathways↗

PubMed1995

The types of neuron in spinal dorsal horn which possess neurokinin-1 receptors.

In order to provide further information about the types of spinal neuron which possess neurokinin-1 receptors, we have carried out pre-embedding immunocytochemistry on sections of rat lumbar spinal cord with an antiserum raised against a synthetic peptide corresponding to part of the sequence of the receptor, and combined this with post-embedding immunocytochemistry to detect GABA and glycine. Numerous neuronal cell bodies showing neurokinin-1 receptor-immunoreactivity were seen in lamina I, laminae III-VI, the lateral spinal nucleus and the area around the central canal. Most of the cells observed in lamina III were small and had relatively restricted dendritic trees which could often not be followed into lamina II, however some larger cells in laminae III and IV had dendrites which extended through lamina II and into lamina I. Cells of the latter type are likely to represent a major target of substance P released from small-diameter primary afferents in the superficial dorsal horn. The great majority (255 out of 283) of spinal neurons which possessed neurokinin-1 receptor-immunoreactivity, including all of those in lamina I, were not GABA- or glycine-immunoreactive, however a few cells in the deep part of the dorsal horn and the lateral spinal nucleus and several cells near the central canal were GABA-immunoreactive, and some of these were also glycine-immunoreactive. These results suggest that substance P acts through neurokinin-1 receptors mainly on excitatory neurons within the spinal cord.

Afferent Pathways↗

PubMed1994

Thyrotropin-releasing hormone- and GABA-like immunoreactivity coexist in neurons in the dorsal horn of the rat spinal cord.

In order to determine which types of neuron in spinal dorsal horn contain the peptide TRH, pre-embedding immunocytochemistry with antiserum to TRH was combined with post-embedding detection of GABA- and glycine-like immunoreactivity. The majority (88/101) of TRH-immunoreactive neurons were also GABA-immunoreactive, but none were glycine-immunoreactive. This suggests that TRH is mainly present in inhibitory interneurons which release GABA but not glycine, and provides further evidence that there are functional differences between those GABAergic neurons in the superficial dorsal horn that contain glycine, and those that do not.

Animals↗

PubMed1994

Subpopulations of GABAergic neurons in laminae I-III of rat spinal dorsal horn defined by coexistence with classical transmitters, peptides, nitric oxide synthase or parvalbumin.

GABAergic neurons in laminae I-III of the spinal dorsal horn may contain one or more of the following compounds: glycine, acetylcholine, neuropeptide Y, enkephalin, nitric oxide synthase or parvalbumin. Although the pattern of co-localization of some of these compounds is understood, it is not known which types of GABAergic neurons contain parvalbumin, or whether nitric oxide synthase coexists with peptides, acetylcholine or parvalbumin in any of these neurons, and in this study we have used immunocytochemistry and enzyme histochemistry to resolve these issues. Parvalbumin-immunoreactivity was restricted to those GABA-immunoreactive neurons that also showed glycine-immunoreactivity and was not co-localized with neuropeptide Y-immunoreactivity or NADPH diaphorase activity. By combining NADPH diaphorase histochemistry with immunocytochemistry with an antiserum to nitric oxide synthase, we were able to show that NADPH diaphorase activity was a reliable marker for nitric oxide synthase in the spinal cord. Neurons that possess GABA- but not glycine-immunoreactivity may contain neuropeptide Y, enkephalin, acetylcholine or NADPH diaphorase, and all of the cholinergic neurons appear to contain NADPH diaphorase. By combining immunofluorescent detection of neuropeptide Y or enkephalin with NADPH diaphorase histochemistry, we showed that peptide-immunoreactivity did not coexist with NADPH diaphorase. This suggests that neither of these peptides coexists with nitric oxide synthase or with acetylcholine in neurons in the superficial dorsal horn. Several phenotypically distinct groups of GABA-immunoreactive neuron can therefore be identified in laminae I-III of the dorsal horn, and these may represent different functional types of inhibitory neuron.

Acetylcholine↗

PubMed1994

Some inhibitory neurons in the spinal cord develop c-fos-immunoreactivity after noxious stimulation.

In order to determine which types of spinal neuron produce c-fos in response to noxious stimulation, we have combined pre-embedding detection of c-fos-like immunoreactivity with post-embedding immunocytochemistry using antibodies against GABA and glycine, 2 h after subcutaneous injection of formalin into a hindpaw of anaesthetized rats. Throughout the spinal cord, the majority of c-fos-immunoreactive neurons (72-81%) did not possess GABA- or glycine-like immunoreactivity, while the remaining cells contained one or both types of immunoreactivity. In the superficial dorsal horn (laminae I and II) and dorsal white matter, between 14 and 20% of c-fos-immunoreactive neurons were GABA-immunoreactive, and some of these were also glycine-immunoreactive. A single neuron in lamina I in one animal was glycine- but not GABA-immunoreactive. In the remainder of the spinal cord, between 21 and 35% of the c-fos-immunoreactive cells were GABA- or glycine-immunoreactive, and the majority of these neurons contained both types of immunoreactivity. These results suggest that some inhibitory neurons in both the superficial and deep parts of the dorsal horn are activated by noxious stimuli. It is known that some of the cells which produce c-fos in response to noxious stimulation are projection neurons, with axons ascending to the brainstem or thalamus, however, because of the large number of c-fos-immunoreactive cells in the dorsal horn, it is likely that many are interneurons, and some of these are probably excitatory cells which use glutamate as a transmitter. It therefore appears that after noxious stimulation c-fos is produced in several types of spinal neuron, including projection cells and both excitatory and inhibitory interneurons.

Animals↗

PubMed1994

Immunocytochemical evidence that neurotensin is present in glutamatergic neurons in the superficial dorsal horn of the rat.

In order to determine whether glutamate is enriched in neurotensin-containing axons in the superficial dorsal horn of the rat spinal cord, we have carried out preembedding immunocytochemistry with an antiserum to neurotensin and then used a postembedding immunogold method with antiserum to glutamate. The immunogold label (corresponding to glutamate-like immunoreactivity) over 40 neurotensin-immunoreactive boutons in laminae I and II of the lumbar dorsal horn was compared with that over nearby axons that formed asymmetrical or symmetrical synapses. In addition, for 20 of these boutons, the labeling was compared with that over mossy and parallel fiber terminals (both of which are thought to use glutamate as a transmitter) from sections of cerebellum that had been processed together with those of spinal cord. Glutamate-like immunoreactivity was consistently high over neurotensin-immunoreactive boutons relative to most surrounding profiles. Immunostaining over these boutons was slightly (11%) lower than that over matched terminals that formed asymmetrical synapses, but considerably higher than that over the terminals that formed symmetrical synapses. The level of glutamate immunoreactivity in neurotensin-immunoreactive boutons in dorsal horn was similar to that in cerebellar parallel fiber terminals, but significantly lower than that in mossy fiber terminals. These results suggest that glutamate is a transmitter used by neurotensin-immunoreactive axons in the dorsal horn, and since these axons are thought to be largely or entirely derived from neurotensin-containing neurons in laminae I-III, they provide immunocytochemical evidence for a population of excitatory glutamatergic neurons in this region.

Animals↗

PubMed1993

Coexistence of NADPH diaphorase with GABA, glycine, and acetylcholine in rat spinal cord.

The enzyme NADPH diaphorase is present in many spinal neurons, and is thought to correspond to nitric oxide synthase. In order to determine which types of neuron in the spinal cord contain this enzyme, we have carried out a combined enzyme histochemical and immunocytochemical study with antibodies to GABA, glycine, and choline acetyltransferase. Two hundred rats were tested for GABA- and glycine-like immunoreactivity. The majority of these neurons (207/224) were GABA-immunoreactive and 139 were also glycine-immunoreactive. NADPH diaphorase-positive neurons in laminae I and II generally showed both types of immunoreactivity, while those in deeper laminae of the dorsal horn and around the central canal either showed both types or else were only GABA-immunoreactive. Since GABA and acetylcholine are thought to coexist in spinal neurons, NADPH diaphorase staining was combined with immunostaining for choline acetyltransferase. Immunoreactive neurons in laminae III and IV were all NADPH diaphorase-positive, while only some of those around the central canal and in the deeper laminae of the dorsal horn were positive. Choline acetyltransferase-immunoreactive neurons in the intermediolateral cell column (presumed sympathetic preganglionic neurons) were often NADPH diaphorase-positive, whereas those in the ventral horn (presumed motoneurons) were not. NADPH diaphorase-positive cells in the intermediolateral cell column were not immunoreactive with GABA or glycine antibodies.

Acetylcholine↗