Co‐localized neuropeptide Y and GABA have complementary presynaptic effects on sensory synaptic transmission

We have examined the morphological relationship of neuropeptide Y (NPY) and GABAergic neurons in the lamprey spinal cord, and the physiological effects of NPY and GABA_B receptor agonists on afferent synaptic transmission. NPY-containing fibres and cell bodies were identified in the dorsal root entry zone. NPY immunoreactive (–ir) fibres made close appositions with primary afferent axons. Co-localization of NPY and GABA-ir was found in the dorsal horn and dorsal column. Fifty-two per cent of NPY-ir profiles showed immunoreactivity to GABA at the ultrastructural level. Electron microscopic analysis showed that NPY-immunoreactivity was present throughout the axoplasm, including over dense core vesicles, whereas GABA-immunoreactivity was mainly found over small synaptic vesicles. Synthetic lamprey NPY, and the related peptide, peptide YY, reduced the amplitude of monosynaptic afferent EPSPs in spinobulbar neurons. NPY had no significant effect on the postsynaptic input resistance or membrane potential, the electrical component of the synaptic potential, or the response to glutamate, but it could reduce the duration of presynaptic action potentials, suggesting that it was acting presynaptically. NPY also reduced the excitability of the spinobulbar neurons, suggesting at least one postsynaptic effect. Because NPY and GABA colocalize, we compared the effects of NPY and the GABA_B agonist baclofen. Both presynaptically reduced EPSP amplitudes, baclofen having a larger effect and a faster onset and recovery than NPY. The GABA_B antagonist phaclofen reduced the effect of baclofen, but not that of NPY. We conclude that NPY and GABA are colocalized in terminals in the dorsal spinal cord of the lamprey, and that they have complementary actions in modulating sensory inputs.     

The termination pattern and postsynaptic targets of rubrospinal fibers in the rat spinal cord: a light and electron microscopic study.

The spinal course, termination pattern, and postsynaptic targets of the rubrospinal tract, which is known to contribute to the initiation and execution of movements, were studied in the rat at the light and electron microscopic levels by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) in combination with calbindin-D28k (CaBP), gamma-aminobutyric acid (GABA), and glycine immunocytochemistry. After injections of PHA-L unilaterally into the red nucleus, labelled fibers and terminals were detected at cervical, thoracic, and lumbar segments of the spinal cord. Most of the descending fibers were located in the dorsolateral funiculus contralateral to the injection site, but axons descending ipsilaterally were also revealed. Rubrospinal axon terminals were predominantly found in laminae V-VI and in the dorsal part of lamina VII at all levels and on both sides of the spinal cord, but stained collaterals were also seen in the ventrolateral aspect of Clark's column and in the ventral regions of lamina VII on both sides. The proportion of axonal varicosities revealed on the ipsilateral side varied at different segments and represented 10-28% of the total number of labelled boutons. Most of the labelled boutons were engaged in synaptic contacts with dendrites. Of the 137 rubrospinal boutons investigated, only 2 were found to establish axosomatic synaptic junctions in the lumbar spinal cord contralateral to the PHA-L injection. With the postembedding immunogold method, 80.8% of dendrites establishing synaptic contacts with rubrospinal terminals did not show immunoreactivity for either GABA or glycine, whereas 19.2% of them were immunoreactive for both amino acids. Rubrospinal axons made multiple contacts with CaBP-immunoreactive neurons in laminae V-VI. Synaptic contacts between rubrospinal terminals and CaBP-immunoreactive dendrites were identified at the electron microscopic level, and all CaBP-containing postsynaptic dendrites investigated were negative for both GABA and glycine. The results suggest that rubrospinal terminals establish synaptic contacts with both excitatory and inhibitory interneurons in the rat spinal cord, and a population of excitatory interneurons receiving monosynaptic rubrospinal input is located in laminae V-VI.     

Colocalization of dopamine and GABA in spinal cord neurones in the sea lamprey

In this study, double immunofluorescence methods were used to investigate possible colocalization of the neurotransmitters dopamine [DA] and GABA in rostral spinal cord neurones in the upstream migrating adult sea lamprey (Petromyzon marinus). Double immunofluorescence revealed that all the DA-immunoreactive (ir) cerebrospinal fluid-contacting (CSF-c) cells, approximately 30% of the medioventral DA-ir cells, and most of the DA-ir cells located in the grey lateral to the central canal were also GABA-ir. The results also revealed some DA-ir cells located dorsally to the central canal, which increases the number of dopaminergic cell types known in lamprey. Double-labelled fibres were mainly distributed in the ventral column, and double-labelled boutons contacted some dorsal GABA-ir CSF-c cells, as well as some non-CSF-c GABA-ir cells and ventromedial dendrites of motoneurones. The findings reveal colocalization of dopamine and GABA in some cells and fibres, which suggests co-release of these substances in some synaptic terminals. Although dopaminergic/GABAergic CSF-c cells have been reported in some other vertebrates, the other double-labelled spinal populations appear exclusive to lampreys.     

Light and electron microscopic study of an avian pretectal nucleus, the lentiform nucleus of the mesencephalon, magnocellular division

The distribution of vasoactive intestinal polypeptide (VIP) was mapped by peroxidase immunocytochemistry in the spinal cords of seven Macaca fascicularis monkeys and two cats. The animals were perfusion fixed with different chemicals. Those that were perfused with either a Zamboni fixative or 5% acrolein had significantly greater immunoreactivity outside the sacral cords; those fixed with 4% paraformaldehyde had little in nonsacral regions. VIP-like immunoreactive (VIP) axons and terminals were found in the superficial dorsal horn, reticular nucleus of lamina V, intermediomedial nucleus, and lamina X at all levels from C2 to S4; a few axons and terminals were also seen in the ventral horn. Axons were found in Lissauer's tract at all levels, and axons appeared in the dorsolateral and ventrolateral white matter at midthoracic levels; in the lumbosacral cord the number and extent of axons in the lateral and ventral white matter increased progressively in a caudal direction. VIP neurons were identified in thoracic intermediate gray lateral to the central canal and in the intercalatus (IC) and intermediolateral (IML) nuclei. Electron microscopy of the VIP terminals in laminae I and II of the cervical cord revealed they contain small round vesicles and many large granular vesicles; some are glomerular terminals and most form asymmetrical synaptic contacts onto dendrites. These results indicate VIP is much more widely distributed in the spinal cord than previously thought; VIP may be associated with both visceral thoracic and lumbosacral afferents, and with other afferents in the cervical cord; VIP neurons are present in the thoracic intermediate gray; and VIP axons in the ventral and lateral white matter indicate that the spinal cord is supplied in part by VIP sources other than primary afferents.     

Zinc co-localizes with GABA and glycine in synapses in the lamprey spinal cord

The presence of zinc in synaptic terminals in the lamprey spinal cord was examined utilizing a modification of the Timm's sulfide silver method and with the fluorescent marker 6-methoxy-8-quinolyl-p-toluenesulfonamide (TSQ). Axons labeled with a Timm's staining method were predominantly located in the lateral region of the dorsal column. This correlated with a maximum of TSQ fluorescence in this region of the spinal cord. Single labeled terminals accumulating Timm reaction product were also found throughout the gray matter and fiber tracts. At the ultrastructural level, zinc was located in a population of synaptic terminals that co-localized gamma-aminobutyric acid (GABA) and glycine. Possible effects of Zn2+ on neuronal activity were examined. In spinobulbar interneurons, which receive GABAergic input in the dorsal column, zinc potentiated responses to GABA application, but it did not affect responses to GABA in motoneurons. Responses in motoneurons to pressure application of glycine were also not affected by Zn2+. Zinc, however, potentiated monosynaptic glycinergic inhibitory postsynaptic potentials (IPSPs) evoked in motoneurons by inhibitory locomotor network interneurons and increased frequency, but not amplitude of spontaneous miniature IPSPs recorded in the presence of tetrodotoxin (TTX), suggesting presynaptic effects. Glutamate responses and the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs) in motoneurons were reduced by zinc. These effects appeared to be mediated largely postsynaptically through an effect on the N-methyl-D-aspartate (NMDA) component of the glutamatergic input. Our results thus show that free zinc is present in inhibitory synaptic terminals in the lamprey spinal cord, and that it may function as a modulator of inhibitory synaptic transmission.     

Ultrastructural Relationships Between GABAergic Terminals and Cardiac Vagal Preganglionic Motoneurons and Vagal Afferents in the Cat: A Combined HRP Tracing and Immunogold Labelling Study

The ultrastructural relationships between gamma-aminobutyric acid-immunoreactive (GABA-ir) neurons and other neurons of the nucleus tractus solitarius (NTS) and motoneurons of the nucleus ambiguus (NA) and dorsal motor vagal nucleus (DMVN), were examined by electron microscopic (EM) immunogold labelling with an anti-GABA antiserum on brain stem sections in which vagal motoneurons and vagal afferent fibres were labelled with horseradish peroxidase (HRP). HRP was applied to the cervical vagus or the cardiac vagal branch of anaesthetized cats. After 24 - 48 h survival, brains were glutaraldehyde-fixed and a stable HRP-tetramethylbenzidine reaction product compatible with EM processing was revealed on 250 microm vibratome sections. Following osmium postfixation, dehydration and resin embedding, GABA-ir was localized on ultrathin sections by an immunogold technique. GABA-ir axon terminals, heavily and specifically labelled with gold particles, were very numerous within NTS, DMVN and NA. All terminals contained small, clear, pleomorphic vesicles and a few also contained larger dense cored vesicles. The density of gold particles over clear vesicles, dense cored vesicles and mitochondria was significantly greater than over the cytoplasm of these terminals. GABA-ir synapses were found on the soma and dendrites of neurons, but rarely on other axon terminals within NTS, where GABA-ir cell bodies and dendrites were also seen. These received synaptic contacts from both GABA-ir terminals and from HRP-labelled vagal afferents. In both the DMVN and NA, similar GABA-ir synapses were present on both the soma and dendrites of HRP-labelled motoneurons. GABA synapses were also present on other cell types in DMVN. These observations provide an anatomical basis for a GABAergic inhibition of neurons forming the central pathways of cardiovascular and other autonomic reflexes.     

Three types of GABA-immunoreactive cells in the lamprey spinal cord

Polyclonal antisera raised against conjugated GABA were used to study the distribution of GABAergic neurons in the spinal cords of lampreys (Lampetra fluviatilis and Ichtyomyzon unicuspis) using immunofluorescence and peroxidase-antiperoxidase techniques. Three morphologically distinct types of GABA-immunoreactive (GABA-ir) cell bodies were observed, multipolar neurons in the lateral grey cell column, apparently bipolar cells in the ventral aspect of the dorsal horn, and small liquor-contacting cells surrounding the central canal. A high density of immunoreactive fibers of spinal origin were present in the lateral and ventral funiculi, whereas the dorsal column had a relatively low density. Dense GABA-ir plexuses were situated in the lateral spinal margin, and in the dorsal part of the dorsal horn. A chronic lesion of the rostral spinal cord did not result in any observable loss of GABA-ir fibers below or above the lesion, suggesting that the 3 types of segmental GABA-ir neurons are the main sources of the GABAergic innervation of the lamprey spinal cord.     

Glycine-immunoreactive neurons in the developing spinal cord of the sea lamprey: comparison with the gamma-aminobutyric acidergic system

The development and cellular distribution of the inhibitory neurotransmitter glycine in the spinal cord of the sea lamprey were studied by immunocytochemistry and double immunofluorescence and compared with the distribution of gamma-aminobutyric acid (GABA). Results in lamprey embryos and prolarvae reveal that the appearance of glycine-immunoreactive (-ir) spinal neurons precedes that of GABA-ir neurons. Throughout development, glycine-ir cells in the lateral and dorsomedial gray matter of the spinal cord are more numerous than the GABA-ir cells. Only a subset of these neurons shows colocalization of GABA and glycine, suggesting that they are primarily disparate neuronal populations. In contrast, most cerebrospinal fluid (CSF)-contacting neurons of the central canal walls are strongly GABA-ir, and only a portion of them are faintly glycine-ir. Some edge cells (lamprey intraspinal mechanoreceptors) were glycine-ir in larvae and adults. The glycine-ir and GABA-ir neuronal populations observed in the adult spinal cord were similar to those found in larvae. Comparison of glycine-ir and GABA-ir fibers coursing longitudinally in the spinal cord of adult lamprey revealed large differences in diameter between these two types of fiber. Commissural glycine-ir fibers appear in prolarvae and become numerous at larval stages, whereas crossed GABA-ir are scarce. Taken together, results in this primitive vertebrate indicate that the spinal glycinergic cells do not arise by biochemical shift of preexisting GABAergic cells but instead suggest that glycine is present in the earliest circuitry of the developing lamprey spinal cord, where it might act transiently as an excitatory transmitter.     

GABA and glycine-like immunoreactivity at axoaxonic synapses on 1a muscle afferent terminals in the spinal cord of the rat

The object of this study was to analyze the synaptic interactions of identified muscle spindle afferent axon terminals in the spinal cord of the rat. Group 1a muscle afferents supplying the gastrocnemius muscle were impaled with microelectrodes in the dorsal white matter of the spinal cord and stained by intracellular injection with Neurobiotin. Postembedding immunogold techniques were used to reveal GABA- and glycine-like immunoreactivity in boutons presynaptic to afferent terminals in the ventral horn and the deep layers of the dorsal horn. Serial-section reconstruction was used to reveal the distribution of synaptic contacts of different types on the afferent terminals. The majority of afferent boutons received axoaxonic and made axodendritic or axosomatic synaptic contacts. In the ventral horn, 91% of boutons presynaptic to the afferent terminals were immunoreactive for GABA alone and 9% were immunoreactive for both GABA and glycine. The mean number of axo-axonic contacts received per terminal was 2.7, and the mean number of synaptic contacts at which the terminal was the presynaptic element was 1.4. In the deep layers of the dorsal horn, 58% of boutons presynaptic to afferent terminals were immunoreactive for GABA alone, 31% were immunoreactive for GABA and glycine, and 11% for glycine alone. The mean number of axoaxonic contacts received per afferent terminal in this region was 1.6 and the mean number of synaptic contacts at which the terminal was the presynaptic element was 0.86. This clearly establishes the principle that activity in 1a afferents is modulated by several neurochemically distinct populations of presynaptic neuron.     

The GABAergic neurons and axon terminals in the lateralis medialis-suprageniculate nuclear complex of the cat: GABA-immunocytochemical and WGA-HRP studies by light and electron microscopy

In order to get more detailed information on the neural circuit of the lateralis medialis-suprageniculate nuclear (LM-Sg) complex of the cat, the GABAergic innervation of this complex was studied by GABA immunohistochemical techniques. Small immunoreactive cells were found throughout the LM-Sg complex. On the basis of their ultrastructural features, these GABAergic cells were identified as Golgi type II interneurons. The neuropil of this nucleus displayed a conspicuous granular immunoreactivity. Ultrastructurally, the immunoreactive neural profiles in the neuropil were identified as the presynaptic dendrites of interneurons, myelinated axons, or axon terminals. The GABAergic dendritic profiles, containing pleomorphic synaptic vesicles, were involved in synaptic glomeruli. Additionally, GABAergic axon terminals containing pleomorphic synaptic vesicles formed symmetric axodendritic synaptic contacts mainly in the extraglomerular neuropil. They appeared to correspond to either axon terminals from the thalamic reticular nucleus (TRN) or the axon terminals of interneurons. The projections from the TRN to the LM-Sg complex were studied by using wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Following injection of WGA-HRP into the LM-Sg complex, a number of retrogradely labeled cells were observed in the TRN. The connections between the TRN and the LM-Sg complex appeared to be topographically organized, the dorsal TRN being connected mainly with the dorsomedial portion of the LM-Sg complex, and the ventral TRN being connected chiefly with the ventrolateral portion of the LM-Sg complex. Following injection of the tracer into the TRN, ultrastructural examination of anterograde labeling in the LM-Sg complex revealed that labeled terminals contain pleomorphic vesicles and make symmetric synaptic contacts mainly with small to medium-sized dendrites. The labeled terminals were not involved in synaptic glomeruli. The present results provide anatomic support for the contention that the projection cells of the LM-Sg complex may be inhibited by both the TRN axons and interneurons, probably through the mediation of GABA.     

Synaptic relationship of the neurons containing a metabotropic glutamate receptor, MGluR5, with nociceptive primary afferent and GABAergic terminals in rat spinal superficial laminae

Recent pharmacological evidence showed that metabotropic glutamate receptors (mGluRs), particularly mGluRs1/5, had a potential role in spinal nociceptive processing. However, previous morphological studies on mGluRs have been limited mainly to their distribution in the spinal cord. In the present study, electron microscopic immunocytochemistry was employed to identify the synaptic relationship of the neurons containing mGluR5, with nociceptive primary afferent and gamma-aminobutyric acid-ergic (GABAergic) terminals in the superficial dorsal horn of the spinal cord. Nociceptive C- and A(delta)-primary afferent terminals selectively labeled with horseradish peroxidase conjugated to wheat-germ agglutinin were in asymmetric synaptic contacts with or in direct apposition to mGluR5 positive dendritic profiles. The double-labeling studies revealed that mGluR5 immunoreactive dendrites also received symmetric synaptic contacts from axon terminals labeled with immunogold particles indicating GABA. The present demonstration of mGluR5 neurons receiving inputs from both nociceptive primary afferents and GABAergic terminals of presumed interneurons further supports the involvement of mGluR5 in the transmission and modulation of nociceptive information in the spinal cord.     

Rostral ventrolateral medulla: a source of the glutamatergic innervation of the sympathetic intermediolateral nucleus

To determine whether the sympathoexcitatory projection from the rostral ventrolateral medulla (RVL) to the sympathetic intermediolateral nucleus (IML) of the spinal cord might use glutamate as an excitatory transmitter, we performed a dual-label, transport and immunocytochemical ultrastructural study. Axon terminals within the IML were examined to determine whether anterogradely transported Phaseolus vulgaris-leucoagglutinin (PHA-L) following injections into the RVL, was colocalized with glutamate immunoreactivity using an antibody to hemocyanin-conjugated L-glutamate (Hepler et al., J. Histochem. Cytochem., 36 (1988) 13-22). Transported PHA-L was visualized with the peroxidase-antiperoxidase technique while glutamate-like immunoreactivity was localized within the same section of the thoracic spinal cord with immunoautoradiography. By light microscopy, PHA-L immunoreactivity was found within a plexus of fine fibers and varicose processes localized to the IML. Silver grains indicative of glutamate immunoreactivity were concentrated over the IML and also over the superficial layers of the dorsal horn. Electron microscopic analysis revealed PHA-L immunoreactivity in axons and axon terminals within the IML. They ranged in diameter from 0.5 to 2.0 microns, contained numerous small clear and 0-3 large, dense-core vesicles, and formed primarily asymmetric synaptic contacts on small dendrites of IML neurons. Some of the PHA-L immunoreactive terminals making asymmetric (excitatory) synaptic contacts on the small dendrites of IML neurons also contained glutamate-like immunoreactivity. We conclude that at least a portion of the input to the IML from the RVL uses glutamate as its transmitter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and ultrastructure of primary afferent axons in Lissauer's tract in the northern leopard frog (Rana pipiens)

Dorsal roots were transected and filled with horseradish peroxidase to identify the primary afferent axons in Lissauer's tract (LT) of the spinal cord in a ranid frog with light and electron microscopy. Axons in LT could be traced at least 2-3 segments rostral and caudal to the level of the filled root. Some axons in LT entered a nucleus in the dorsal lateral funiculus at brachial and lumbar levels, while other axons terminated in widespread regions of the dorsal horn. Electron microscopy revealed unlabelled terminals with numerous large, dense-cored vesicles in LT. However, large vesicles were rarely observed and were never abundant in labelled primary afferent axons. S type synaptic contacts between labelled axons and unlabelled profiles were observed. The presynaptic axons in these synapses contained many small, spherical vesicles. The cross-sectional area of LT was related to the spinal cord level, with the largest area at the brachial level, and intermediate area at the lumbar level, and the smallest area at the thoracic level. No difference in number of labelled axons was observed in the medial and lateral parts of LT.     

Developmental changes of the GABA-immunoreactive fibers in the lamprey spinal cord

The changes in distribution and number of GABA immunoreactive (GABA-ir) fibers from postembryonic stages to adulthood in the lamprey spinal cord white matter were studied by using immunocytochemical techniques. From prolarvae to adult spawning animals there was an increase of the number of GABA-ir fibers. Three phases can be distinguished: (a) from prolarvae to middle size larvae (around 50 mm in body length) an increase in the number of GABA-ir fibers per section is observed. Furthermore, an adult-like pattern of GABA-ir fibers distribution is established during this phase. (b) Then, the number of GABA-ir fibers remains stable until metamorphosis, the end of the larval period. (c) Finally, in young postmetamorphic and adult animals the number of GABA-ir fibers is higher than in larvae. These observations, joined to the changes previously reported in the GABA-ir neurons, indicate that at least parts of the GABA inhibitory component of the spinal locomotor network is reorganized during the lamprey life cycle and it may indicate different inhibitory requirements in the locomotor network.     

Organization of glutamate-like immunoreactivity in the rat superficial dorsal horn: light and electron microscopic observations

Glutamate has been shown to be a neurotransmitter in the central nervous system of vertebrates, and it has been hypothesized that glutamate is functional as a neurotransmitter in the spinal cord dorsal horn. A monoclonal antibody to fixative-modified glutamate was used in this study to examine the light microscopic and ultrastructural profiles of glutamate-like immunoreactivity in the superficial dorsal horn of the rat spinal cord. Glutamate-like immunoreactivity was observed in neurons, fibers, and terminals of both laminae I and II. Marginal zone immunoreactive neurons ranged from 10 to 30 micron in diameter and received many nonimmunoreactive somatic synapses. In substantia gelatinosa, immunoreactive neurons were observed in both inner and outer layers, ranged 5 to 10 micron in diameter, and received few nonimmunoreactive somatic synapses. Glutamate-like immunoreactive dendrites were observed in both laminae and were contacted primarily by nonimmunoreactive synaptic terminals that generally contained small clear vesicles. Both myelinated and unmyelinated immunoreactive axons were observed in Lissauer's tract. Immunoreactive terminals contained small (40 nm) clear vesicles and generally formed simple synaptic contacts with nonimmunoreactive dendrites in laminae I and II. The results of this study corroborate the importance of glutamate as a neurotransmitter in spinal sensory mechanisms.     

Electron microscopy of immunoreactivity patterns for glutamate and γ-aminobutyric acid in synaptic glomeruli of the feline spinal trigeminal nucleus (subnucleus caudalis)

We studied the ultrastructure of the synaptic organization in the feline spinal trigeminal nucleus, emphasizing specific neurotransmitter patterns within lamina II of the pars caudalis/medullary dorsal horn. Normal adults were perfused, and Vibratome sections from pars caudalis were processed for electron microscopy. Ultrathin sections were reacted with antibodies for the excitatory neurotransmitter glutamate (Glu) and for the inhibitory neurotransmitter γ-aminobutyric acid (GABA) by using postembedding immunogold techniques. Both single- and double-labeled preparations were examined. Results with single labeling show that Glu-immunoreactive terminals have round synaptic vesicles and form asymmetric synaptic contacts onto dendrites. GABA-immunoreactive axon terminals and vesicle-containing dendrites have pleomorphic vesicles, and the axon terminals form symmetric contacts onto dendrites and other axons. Double labeling on a single section shows glomeruli with central Glu-immunoreactive terminals that are presynaptic to dendrites, including GABA⁺ vesicle-containing dendrites. These Glu⁺ terminals are also postsynaptic to GABA⁺ axon terminals, and these GABA-immunoreactive terminals may also be presynaptic to the GABA⁺ vesicle-containing dendrites. Quantitative analyses confirm the specificity of the Glu and GABA immunoreactivities seen in the various glomerular profiles. The results suggest that a subpopulation of Glu-immunoreactive primary afferents (excitatory) may be under the direct synaptic influence of a GABA-immunoreactive intrinsic pathway (inhibitory) by both presynaptic and postsynaptic mechanisms. © 1996 Wiley-Liss, Inc.     

Group III mGluR-mediated depression of sensory synaptic transmission

The effect of L-AP4, a group III mGluR agonist, on sensory synaptic transmission in the lamprey spinal cord has been analyzed. Paired recordings were made between cutaneous mechanosensory neurons (dorsal cells) and postsynaptic spinobulbar giant interneurons. L-AP4 reduced the monosynaptic dorsal cell-evoked EPSP, but at concentrations higher (200-500 microM) than those necessary to depress reticulospinal axon-evoked EPSPs. Stimulation of the dorsal column, which contains dorsal cell axons and the axons of putative nociceptive and theromosensory axons, elicited compound EPSPs that were consistently depressed by L-AP4. Sensory inputs in the lamprey are thus inhibited by group III mGluRs.     

Synaptic interactions between GABA-immunoreactive profiles and the terminals of functionally defined myelinated nociceptors in the monkey and cat spinal cord.

This study analyzes the synaptic interactions between the central terminals of A delta high threshold mechanoreceptors (A delta HTMs) and GABA-immunoreactive profiles. A delta HTM primary afferents from three monkeys and one cat were electrophysiologically identified and intracellularly labeled with HRP, and their terminal arborizations in laminae I and II of the sacrocaudal spinal cord were studied at the ultrastructural level. GABA-immunoreactive profiles in relation to A delta HTM terminals were demonstrated using postembedding colloidal gold techniques. Monkey A delta HTM terminals (n = 131) usually constituted the central element of synaptic glomeruli; they established large asymmetric synaptic contacts with 1-13 dendrites (modal value 2-4) and were surrounded by 0-6 peripheral axon terminals (modal value 2-3). The large majority (around 85%) of the peripheral axon terminals were GABA immunoreactive. They were found presynaptic to the A delta HTM terminal and/or to dendrites postsynaptic to the primary afferent terminal. Furthermore, all peripheral axon terminals found presynaptic to the A delta HTM terminals showed GABA immunoreactivity. Within a single A delta HTM fiber, this synaptic arrangement was found in 20-60% of its boutons. In addition, 28% of the postsynaptic dendritic profiles displayed weak GABA immunoreactivity. Some of them contained vesicles; however, only in a few cases did we observe synapses between a GABA-immunoreactive vesicle-containing dendrite and a dendritic profile postsynaptic to an A delta HTM terminal. Similar synaptology and interactions with GABA-immunoreactive profiles were displayed by the terminals of the single cat A delta HTM fiber studied. Our data support the hypothesis that GABA-containing neurons use both presynaptic and/or postsynaptic mechanisms to exert a powerful control, presumably inhibitory, over the transmission of nociceptive information between A delta HTM afferents and second-order neurons in monkey and cat spinal cord. Our results also imply that GABA may be released within the synaptic glomeruli formed by A delta HTM terminals either by local dendrites or by axon terminals. We discuss the possibility that these GABAergic synapses can be driven by inputs from both primary afferents and/or descending systems to modulate the transmission of nociceptive sensory information.     

Synaptic and nonsynaptic monoaminergic neuron systems in the lamprey spinal cord

In the lamprey spinal cord, dopamine- (DA) and 5-hydroxytryptamine- (5-HT) containing cells appear to play an important role in controlling the firing properties of motoneurons and interneurons and, thereby, in modulating the efferent motor pattern. To determine the detailed morphology and synaptic connectivity of the intraspinal DA and 5-HT systems in Lampetra fluviatilis and Ichthyomyzon unicuspis, DA and 5-HT antisera were used in light and electron microscopic immunocytochemical experiments. Two main groups of labeled cells were distinguished: DA-containing liquor-contacting (LC) cells distributed along the central canal, and 5-HT + DA-containing multipolar cells located near the midline ventral to the central canal. Both types were synaptically connected with other neuronal elements. The DA-immunoreactive LC cells, which extended a ciliated process into the central canal, received symmetrical synapses from unlabeled terminals containing small synaptic vesicles. The distal process of the LC cells could be traced to the lateral cell column, to the ventral aspect of the dorsal column, or to the ventromedial area. Ultrastructural analysis of DA fibers in these regions showed the presence of labeled terminals containing numerous small synaptic vesicles and a few dense-core vesicles. These terminals formed symmetrical synapses with unlabeled cell bodies and dendrites, with GABA-immunopositive LC cells, and with the multipolar DA+5-HT cells. The multipolar DA+5-HT cells also received input from unlabeled synapses. Intracellular recording from these cells showed that they received excitatory postsynaptic potentials in response to stimulation of fibers in the ventromedial tracts and dorsal roots. The terminals of the multipolar DA+5-HT neurons in the ventromedial spinal cord contained numerous dense-core vesicles and small synaptic vesicles, but no synaptic specializations could be detected. In addition, a small number of larger DA-immunoreactive cells were observed in the lateral cell column at rostral levels. The lamprey spinal cord thus contains distinct populations of synaptically interconnected monoaminergic neurons. Dopamine-containing LC cells synapse onto DA+5-HT-containing multipolar cells, in addition to GABAergic LC cells and unidentified spinal neurons. In contrast, the multipolar cells appear to exert their influence by nonsynaptic mechanisms. © 1996 Wiley-Liss, Inc.     

Zinc-enriched GABAergic terminals in mouse spinal cord

Electrophysiological experiments have shown that zinc ions modulate glutamate and GABA receptors in brain slices. All the zinc-enriched neuronal pathways in the brain analyzed up until now have been found to be glutaminergic. Many years ago, zinc-enriched terminals with flat vesicles and symmetric synapses were found to be present in rat spinal cord by Henrik Daa Schr?der, and recently these findings have been supported by immunohistochemical and electron microscopical data in lamprey, mouse and rat. In the present study we expanded these observations by revealing a colocalization of zinc ions, zinc transporter-3 (ZnT3) and glutamic acid decarboxylase (GAD) in synaptic vesicles of zinc-enriched terminals throughout the mouse spinal cord. Confocal analysis of ZnT3 and GAD immunofluorescence was used at light microscopical levels, and a combination of zinc selenium autometallography and GAD immunocytochemistry at electron microscopic levels. Zinc-enriched/GABAergic terminals were observed in all laminae of the spinal gray matter, but most densely populated were laminae I and III in the dorsal horn. In the lateral and ventral funiculi of the white matter, rows of inhibitory zinc-enriched boutons were seen radiating from the gray matter. Ultrastructurally, colocalization of zinc ions and GAD immunoreactivity was seen in a pool of presynaptic terminals in the above locations. Some zinc-enriched terminals were not GAD-positive and some GAD-positive terminals were void of zinc ions. The majority of the zinc-enriched, not GABAergic terminals could be classified as excitatory based on their morphology, i.e. round clear vesicles and symmetric synapses. We conclude that a majority of the spinal cord zinc-enriched terminals are GABAergic. The zinc-enriched terminals with excitatory morphology are most likely glutaminergic, a few have an inhibitory morphology but are not GABAergic. These are most likely glycinergic.