D.J. Maxwell

The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid‐lumbar spinal cord of the rat. The great majority (94–100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2‐immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin‐ and enkephalin‐containing boutons (including somatostatin‐immunoreactive boutons that lacked calcitonin gene‐related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present.

Selective loss of spinal GABAergic or glycinergic neurons is not necessary for development of thermal hyperalgesia in the chronic constriction injury model of neuropathic pain.

&NA; GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABAA and glycine receptor antagonists produces behavioural signs of allodynia, suggesting that these transmitters have an important role in spinal pain mechanisms. Several studies have described a substantial loss of GABA‐immunoreactive neurons from the dorsal horn in nerve injury models, and it has been suggested that this may be associated with a loss of inhibition, which contributes to the behavioural signs of neuropathic pain. We have carried out a quantitative stereological analysis of the proportions of neurons in laminae I, II and III of the rat dorsal horn that show GABA‐ and/or glycine‐immunoreactivity 2 weeks after nerve ligation in the chronic constriction injury (CCI) model, as well as in sham‐operated and naïve animals. At this time, rats that had undergone CCI showed a significant reduction in the latency of withdrawal of the ipsilateral hindpaw to a radiant heat stimulus, suggesting that thermal hyperalgesia had developed. However, we did not observe any change in the proportion of neurons in laminae I–III of the ipsilateral dorsal horn that showed GABA‐ or glycine‐immunoreactivity compared to the contralateral side in these animals, and these proportions did not differ significantly from those seen in sham‐operated or naïve animals. In addition, we did not see any evidence for alterations of GABA‐ or glycine‐immunostaining in the neuropil of laminae I–III in the animals that had undergone CCI. Our results suggest that significant loss of GABAergic or glycinergic neurons is not necessary for the development of thermal hyperalgesia in the CCI model of neuropathic pain.

Conditional Rhythmicity of Ventral Spinal Interneurons Defined by Expression of the Hb9 Homeodomain Protein

The properties of mammalian spinal interneurons that underlie rhythmic locomotor networks remain poorly described. Using postnatal transgenic mice in which expression of green fluorescent protein is driven by the promoter for the homeodomain transcription factor Hb9, as well as Hb9-lacZ knock-in mice, we describe a novel population of glutamatergic interneurons located adjacent to the ventral commissure from cervical to midlumbar spinal cord levels. Hb9+ interneurons exhibit strong postinhibitory rebound and demonstrate pronounced membrane potential oscillations in response to chemical stimuli that induce locomotor activity. These data provide a molecular and physiological delineation of a small population of ventral spinal interneurons that exhibit homogeneous electrophysiological features, the properties of which suggest that they are candidate locomotor rhythm-generating interneurons.

Ultrastructure and synaptic connections of cutaneous afferent fibres in the spinal cord

Abstract The combination of intracellular recording and staining techniques with electron microscopy offers a unique opportunity for correlating physiological and ultrastructural properties of single neurones. The spinal terminations of cutaneous primary afferent fibres have been studied by this method, and examination of the ultrastructural synaptology of their boutons sheds light on how sensory information may be processed at the first synapse in the spinal cord. The combination of intracellular staining techniques with other neuroanatomical tract-tracing methods can reveal some of the neuronal networks of the spinal cord and, particularly, the contributions made by primary afferent fibres to these networks.

Distribution and colocalisation of glutamate decarboxylase isoforms in the rat spinal cord.

The inhibitory neurotransmitter GABA is synthesized by glutamic acid decarboxylase (GAD), and two isoforms of this enzyme exist: GAD65 and GAD67. Immunocytochemical studies of the spinal cord have shown that whilst both are present in the dorsal horn, GAD67 is the predominant form in the ventral horn. The present study was carried out to determine the pattern of coexistence of the two GAD isoforms in axonal boutons in different laminae of the cord, and also to examine the relation of the GADs to the glycine transporter GLYT2 (a marker for glycinergic axons), since many spinal neurons are thought to use GABA and glycine as co-transmitters. Virtually all GAD-immunoreactive boutons throughout the spinal grey matter were labelled by both GAD65 and GAD67 antibodies; however, the relative intensity of staining with the two antibodies varied considerably. In the ventral horn, most immunoreactive boutons showed much stronger labelling with the GAD67 antibody, and many of these were also GLYT2 immunoreactive. However, clusters of boutons with high levels of GAD65 immunoreactivity were observed in the motor nuclei, and these were not labelled with the GLYT2 antibody. In the dorsal horn, some GAD-immunoreactive boutons had relatively high levels of labelling with either GAD65 or GAD67 antibody, whilst others showed a similar degree of labelling with both antibodies. GLYT2 immunoreactivity was associated with many GAD-immunoreactive boutons; however, this did not appear to be related to the pattern of GAD expression. It has recently been reported that there is selective depletion of GAD65, accompanied by a loss of GABAergic inhibition, in the ipsilateral dorsal horn in rats that have undergone peripheral nerve injuries [J Neurosci 22 (2002) 6724]. Our finding that some boutons in the superficial laminae showed relatively high levels of GAD65 and low levels of GAD67 immunoreactivity is therefore significant, since a reduction in GABA synthesis in these axons may contribute to neuropathic pain.

Networks of inhibitory and excitatory commissural interneurons mediating crossed reticulospinal actions

Axonal projections and neurotransmitters used by commissural interneurons mediating crossed actions of reticulospinal neurons were investigated in adult cats. Eighteen interneurons, located in or close to lamina VIII in midlumbar segments, that were monosynaptically excited by reticulospinal tract fibres and projected to contralateral motor nuclei were labelled by intracellular injection of tetramethylrhodamine‐dextran and Neurobiotin. The nine most completely labelled interneurons were analysed with combined confocal and light microscopy. None of the stem axons gave off ipsilateral axon collaterals. Seven cells had axon collaterals that arborized in the contralateral grey matter in the ventral horn of the same segments. Transmitters were identified by using antibodies raised against vesicular glutamate transporters 1 and 2, glutamic acid decarboxylase and the glycine transporter 2. The axons of two cells were immunoreactive for the glycine transporter 2 and hence were glycinergic. Three cells were immunoreactive for the vesicular glutamate transporter 2 and hence were glutamatergic. None of the axons displayed immunoreactivity for glutamic acid decarboxylase. Electron microscopy of two cells revealed direct synaptic connections with motoneurons and other neurons. Axonal swellings of one neuron formed synapses with profiles in motor nuclei whereas those of the other formed synapses with other structures, including cell bodies in lamina VII. The results show that this population of commissural interneurons includes both excitatory and inhibitory cells that may excite or inhibit contralateral motoneurons directly. They may also influence the activity of motoneurons indirectly by acting through interneurons located outside motor nuclei in the contralateral grey matter but are unlikely to have direct actions on interneurons in the ipsilateral grey matter.

Fine structure of serotonin-containing axons in the marginal zone of the rat spinal cord

Descending serotoninergic systems in the spinal cord may be involved in antinociception. In this study 6 rat spinal cords were prepared for serotonin immunostaining and marginal zones (lamina I of Rexed) from the dorsal horn were examined with electron microscopy. Stained terminals were often seen to be closely associated with the somata of lamina I neurones or other unstained axon terminals, but seldom formed conventional synaptic junctions. It is concluded that serotoninergic systems in this region may influence the activity of these associated structures in a diffuse non-synaptic manner.

Morphology of inhibitory and excitatory interneurons in superficial laminae of the rat dorsal horn

If we are to stand any chance of understanding the circuitry of the superficial dorsal horn, it is imperative that we can identify which classes of interneuron are excitatory and which are inhibitory. Our aim was to test the hypothesis that there is a correlation between the morphology of an interneuron and its postsynaptic action. We used in vitro slice preparations of the rat spinal cord to characterize and label interneurons in laminae I–III with Neurobiotin. Labelled cells (n= 19) were reconstructed in 3D with Neurolucida and classified according to the scheme proposed by Grudt & Perl (2002) . We determined if cells were inhibitory or excitatory by reacting their axon terminals with antibodies to reveal glutamate decrboxylase (for GABAergic cells) or the vesicular glutamate transporter 2 (for glutamatergic cells). All five islet cells retrieved were inhibitory. Of the six vertical (stalked) cells analysed, four were excitatory and, surprisingly, two were inhibitory. It was noted that these inhibitory cells had axonal projections confined to lamina II whereas excitatory vertical cells projected to lamina I and II. Of the remaining neurons, three were radial cells (2 inhibitory, 1 excitatory), two were antennae cells (1 inhibitory, 1 excitatory), one was an inhibitory central cell and the remaining two were unclassifiable excitatory cells. Our hypothesis appears to be correct only for islet cells. Other classes of cells have mixed actions, and in the case of vertical cells, the axonal projection appears to be a more important determinant of postsynaptic action.

Direct observations of synapses between GABA-immunoreactive boutons and muscle afferent terminals in lamina VI of the cat's spinal cord.

Single group Ia muscle afferent fibres in the lumbar spinal cord of the cat were impaled with microelectrodes and labelled with horseradish peroxidase. Two collateral axons were prepared for combined light and electron microscopy. Arbors selected from lamina VI were processed by the postembedding immunogold technique with antiserum which specifically recognizes GABA in glutaraldehyde-fixed tissue. Twelve Ia boutons were examined through series of thin sections with the electron microscope and all of them were associated with presynaptic axon terminals which were positively labelled for GABA. Some Ia boutons received synaptic contacts from several GABAergic terminals. The present study establishes that a GABA-like substance is present in axon terminals presynaptic to Ia afferent boutons in lamina VI of the spinal cord. This evidence provides a morphological basis for presynaptic inhibition of Ia afferent input into lamina VI.

Central boutons of glomeruli in the spinal cord of the cat are enriched withl-glutamate-like immunoreactivity

Several lines of evidence indicate that L-glutamate may be a neurotransmitter of fine myelinated and unmyelinated primary afferent fibres in the spinal cord. The aim of the present study was to determine if L-glutamate was enriched in the terminals of these fibres. We performed the post-embedding immunogold technique on sections taken from the superficial regions of the lumbar cord in two cats. An antiserum, raised against protein-conjugated L-glutamate, was employed. Several tests on tissue and on a model system indicated that the antiserum recognized a glutaraldehyde-fixed L-glutamate-like substance. Terminals of fine afferent fibres were identified in the substantia gelatinosa as central boutons of synaptic glomeruli. Central boutons were examined through serial sections following immunogold reactions and were found to be heavily labelled with gold particles in consecutive sections. Quantitative analysis indicated that central boutons were more than two and a half times as densely labelled with gold particles than the tissue average. It was concluded that this represents a genuine enrichment of L-glutamate in these structures. Comparisons were made between L-glutamate-immunoreactive properties of central terminals and immunoreactivity for GABA, aspartate and glutamine. Statistical analysis revealed that central boutons in sections incubated in GABA antiserum and glutamine antiserum were associated with significantly lower densities of gold particle labelling than the average for the same tissue. Particle densities of central boutons in sections incubated in aspartate antiserum were not significantly different from average tissue densities. It was concluded that central boutons were not enriched with these three amino acids. Central boutons of synaptic glomeruli were classified into three groups on morphological criteria: (1) dense sinusoidal boutons; (2) large dense-core vesicle-containing boutons; and (3) regular synaptic vesicle-containing boutons. Quantitative analysis revealed that all of these groups were enriched in glutamate immunoreactivity, however, there were differences between the groups; large dense-core vesicle-containing boutons were associated with significantly lower densities of particles than regular synaptic vesicle-containing and dense sinusoidal terminals. The evidence indicates that central boutons, which most probably originate from fine myelinated and unmyelinated primary afferent fibres, are enriched with L-glutamate which may serve as a neurotransmitter in such fibres.