Enrichment of Glutamate-like Immunoreactivity in Primary Afferent Terminals Throughout the Spinal Cord Dorsal Horn

Although several lines of evidence indicate that glutamate is a neurotransmitter in primary afferent terminals, controversies exist on the proportion and types of such terminals that release glutamate. In the present study quantitative analysis of immunogold labelling was used to assess the presence of glutamate-like immunoreactivity in primary afferent terminals in laminae I – V of the rat spinal cord dorsal horn. Anterograde transport of choleragenoid – horseradish peroxidase from a spinal ganglion and tetramethyl benzidine histochemistry were used to identify primary afferent terminals in laminae I and III – V. Presumed C-fibre terminals in lamina II were identified on morphological criteria (dense sinusoid axon terminals). Primary afferent terminals in all dorsal horn laminae displayed significantly higher levels of glutamate-like immunoreactivity than pleomorphic vesicle-containing profiles in laminae III – IV and large neuronal cell bodies in laminae III – V. The density of gold particles over primary afferent terminals also significantly exceeded the average density of gold particles over laminae II and III – IV. The highest densities of gold particles were present over dense sinusoid axon terminals in lamina II. These findings suggest that glutamate, alone or in combination with other neuroactive compounds, is involved in the transfer of all sensory modalities from primary afferent fibres to dorsal horn neurons.     

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.     

Expression of hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 in axon terminals of peptidergic nociceptive primary sensory neurons in the superficial spinal dorsal horn of rats

Hyperpolarization-activated cyclic nucleotide-gated cation channel proteins (HCN1-4), which are potentially able to modulate membrane excitability, are abundantly expressed by neurons in spinal dorsal root ganglia (DRG). In the present experiment, we investigated whether HCN2 protein is confined exclusively to the perikarya of DRG neurons or is transported from the somata to the central axons of DRG neurons that terminate in the spinal dorsal horn. Using immunohistochemical methods, we have demonstrated that laminae I-IIo of the superficial spinal dorsal horn of the adult rat spinal cord show a strong punctate immunoreactivity for HCN2. Dorsal rhizotomy resulted in a complete loss of immunostaining in the dorsal horn, suggesting that HCN2 is confined to axon terminals of primary afferents. In double labelling immunohistochemical studies, we have also shown that HCN2 widely co-localizes with calcitonin gene-related peptide, but is almost completely segregated from isolectin-B4 binding, indicating that HCN2 is primarily expressed in peptidergic nociceptive primary afferents. The expression of HCN2 in central terminals of peptidergic primary afferents was also verified with electron microscopy. Utilizing the pre-embedding nanogold method, we found that HCN2 is largely confined to axon terminals with dense-core vesicles. Within these terminals, some of the silver grains marking the accurate location of HCN2 molecules were associated with the cell membrane, and others were scattered in the axoplasm. Within the cell membrane, HCN2 was found almost exclusively in extrasynaptic locations. The results suggest that HCN2 may contribute to the modulation of membrane excitability of nociceptive primary afferent terminals in the spinal dorsal horn.     

Quantitative analysis of immunogold labeling indicates low levels and non-vesicular localization of L-aspartate in rat primary afferent terminals

The role of L-aspartate as an excitatory neurotransmitter in primary afferent synapses in the spinal cord dorsal horn is disputed. To further investigate this issue, we examined the presence of aspartate-like immunoreactivity in primary afferent nerve terminals and other tissue components of the dorsal horn. We also examined the relationship between aspartate and glutamate immunogold labeling density and the density of synaptic vesicles in primary afferent terminals and presumed inhibitory terminals forming symmetric synapses. Weak aspartate immunosignals, similar to or lower than those displayed by presumed inhibitory terminals, were detected in both C-fiber primary afferent terminals in lamina II (dense sinusoid axon terminals, identified by morphological criteria) and in A-fiber primary afferent terminals in laminae III-IV (identified with anterograde transport of choleragenoid-horseradish peroxidase conjugate). The aspartate immunogold signal in primary afferent terminals was only about one-fourth of that in deep dorsal horn neuronal cell bodies. Further, whereas significant positive correlations were evident between synaptic vesicle density and glutamate immunogold labeling density in both A- and C-fiber primary afferent terminals, none of the examined terminal populations displayed a significant correlation between synaptic vesicle density and aspartate immunogold labeling density. Thus, our results indicate relatively low levels and a non-vesicular localization of aspartate in primary afferent terminals. It is therefore suggested that aspartate, rather than being a primary afferent neurotransmitter, serves a role in the intermediary metabolism in primary afferent terminals.     

Immunolocalization and quantitation of a novel nerve terminal protein in spinal cord development

In the adult spinal cord, the neuron-specific protein NT75 is located in nerve terminals synapsing in the superficial laminae of the dorsal horn. The present study examines the occurrence of NT75 in the developing rat spinal cord. NT75 immunoreactivity is detectable in primary afferent axons at the dorsal root entry zone on embryonic day 15. Subsequently, staining of presumptive nerve terminals appears in the deeper laminae of the dorsal horn, expanding into the superficial laminae during the first postnatal week. NT75 staining also appears in developing corticospinal tract axons in the brainstem at birth, and at lumbosacral levels by postnatal day 5. As NT75-positive nerve terminals approach the adult distribution, staining of primary afferent and corticospinal axons decreases, becoming undetectable by postnatal day 30. Dense transient staining of presumed nerve terminals in the ventral horn is also apparent during early postnatal development. Quantitative analysis of developing spinal cord shows a low level of NT75 immunoreactivity at birth. NT75 activity then increases substantially, reaching values by the third and fourth postnatal weeks up to 2.5 times that seen in adults. The occurrence of NT75 immunoreactivity correlates with the reported time course of synaptic development in the spinal cord. In addition, the results suggest that NT75 immunoreactivity is maintained at high levels in the nerve terminals of certain neural pathways into adulthood, whereas in other systems NT75 immunoreactivity may be detectable only during development.     

A role for bombesin in sensory processing in the spinal cord

Bombesin (BN)-containing neuronal processes were demonstrated in laminae I and II of the dorsal horn of the cat, rat, and mouse spinal cord by immunocytochemistry and radioimmunoassay. Dorsal rhizotomy in the cat resulted in a marked decrease in BN immunoreactivity in the dorsal horn indicating that BN is contained in primary sensory afferents. BN-binding sites were also localized in superficial laminae of the dorsal horn. The presence of both BN and BN-binding sites in the dorsal horn suggested that BN may be involved in sensory processing in the spinal cord. Consistent with this hypothesis, it was demonstrated that an injection of BN into the spinal cord caused a biting and scratching response indicative of sensory stimulation. The effect was similar to that observed after injection of substance P into the cord with the exception that the BN effect lasted about 100 times longer than that induced by substance P. Taken together, these data indicate that BN may be a neurotransmitter of primary sensory afferents to the spinal cord.     

Distribution of protein kinase C-like immunoreactive neurons in rat brain

Distribution of protein kinase C in the CNS of rat is presented based on immunohistochemical analysis with monoclonal antibodies against this protein kinase. Protein kinase C-like immunoreactivity was discretely localized and associated with neurons. Most, if not all, glial cells were not significantly stained. The greatest density of the immunoreactive material was seen in the following regions: the olfactory bulb (external plexiform layer), olfactory tuberculum, anterior olfactory nucleus, cerebral cortex (layers I and IV), pyriform cortex, hippocampus (strata radiatum and oriens), amygdaloid complex (central and basolateral nuclei), cerebellar cortex (molecular layer), dorsal cochlear nucleus, nucleus spinal tract of the trigeminal nerve, and dorsal horn of the spinal cord (substantia gelatinosa). Image analysis revealed that the regional distribution of the protein kinase C-like immunoreaction generally agreed with that of phorbol ester-binding sites. Immunoreactive perikarya were found in the following areas: the cerebral cortex (layers V and VI), caudate putamen, hippocampus, thalamus, amygdaloid complex, medial and lateral geniculate nucleus, superior colliculus, cerebellar cortex, nucleus spinal tract of the trigeminal nerve, dorsal cochlear nucleus, and dorsal horn of the spinal cord. Intense protein kinase C-like immunoreactivity in the neuron was observed both in the membrane and cytoplasm of the perikarya, dendrites, axons, and axon terminals, while weak immunoreaction was seen in the nuclei but almost never in the nucleoles. A map of protein kinase C-containing neurons was constructed. Such an uneven distribution in the brain suggests that this enzyme may play roles in controlling neuronal function in the areas noted.     

Vasoactive intestinal polypeptide and substance P in primary afferent pathways to the sacral spinal cord of the cat

An analysis of vasoactive intestinal polypeptide immunoreactivity (VIP-IR) and substance P-IR in the cat spinal cord has revealed marked differences in the distribution of the two peptides. While substance P-IR was located at all levels of the cord, VIP-IR was most prominent in the sacral segments in Lissauer's tract and lamina I on the lateral edge of the dorsal horn. VIP-IR was also present in the sacral cord in (1) laminae V, VII, and X, (2) a thin band on the medial side of the dorsal horn, (3) the dorsal commissure, (4) the lateral band of the sacral parasympathetic nucleus, and (5) in a few animals in Onuf's nucleus. In other segments of the spinal cord VIP-IR was much less prominent but was present in Lissauer's tract and laminae I, II, and X. Substance P-IR was more uniformly distributed at all segmental levels in laminae I-III, V, VII, and X and in the dorsal commissure. In ventrolateral lamina I of the sacral spinal cord both VIP-IR and substance P-IR exhibited a distinctive periodic pattern in the rostrocaudal axis. The peptides were associated with bundles of dorsoventrally oriented axons and varicosities spaced at approximately 210-micron intervals center to center along the length of the spinal cord. The bundles in lamina I continued into lamina V where they further divided into smaller bundles that extended medially through laminae V and VII. The most prominent bundles of VIP axons passed ventrally from lateral laminae V and VII to enter lamina X and the ventral part of the dorsal gray commissure. On the other hand the majority of substance P axons in lamina V turned dorsally to join with axons on the medial side of the dorsal horn and to pass into the dorsal part of the dorsal gray commissure. Rostrocaudal VIP axons were present not only in Lissauer's tract but also in dorsolateral lamina I, in the lateral funiculus and in the ependymal cell layer of the central canal. Following unilateral transection of the sacral dorsal roots (2 weeks-22 months) the density of VIP axons and terminals was markedly reduced in ipsilateral Lissauer's tract and lateral laminae I and V; however, no change was detected in lamina X. Sacral deafferentation reduced substance P-IR in the dorsal gray commissure and in lateral laminae I and V.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dual ultrastructural immunocytochemical labeling of μ and δ opioid receptors in the superficial layers of the rat cervical spinal cord

The δ opioid receptor (DOR) and μ opioid receptor (MOR) are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. To determine the cellular basis for these functional associations, we examined the electron microscopic immunocytochemical localization of DOR and MOR in single sections through the superficial layers of the dorsal horn in the adult rat spinal cord (C2–C4). From a total of 270 DOR-labeled profiles, 49% were soma and dendrites, 46% were axon terminals and small unmyelinated axons, and 5% were glial processes. 6% of the DOR-labeled soma and dendrites, and <1% of the glial processes also showed MOR-like immunoreactivity (MOR-LI). Of 339 MOR-labeled profiles, 87% were axon terminals and small unmyelinated axons, 12% were soma and dendrites, and 2% were glial processes. 21% of the MOR-labeled soma and dendrites, but none of the axon terminals also contain DOR-LI. The subcellular distributions of MOR and DOR were distinct in axon terminals. In axon terminals, both DOR-LI and MOR-LI were detected along the plasmalemma, but only DOR-LI was associated with large dense core vesicles. DOR-labeled terminals formed synapses with dendrites containing MOR and conversely, MOR-labeled terminals formed synapses with DOR-labeled dendrites. These results suggest that the synergistic actions of selective MOR- and DOR-agonists may be attributed to dual modulation of the same or synaptically linked neurons in the superficial layers of the spinal cord.     

Spinal cord afferent systems containing the nerve terminal protein NT75

In the adult spinal cord, immunocytochemical staining for NT75 is concentrated in nerve terminals in the superficial laminae of the dorsal horn. Deeper laminae of the dorsal horn contain moderate immunocytochemical labeling, but the ventral horn is only sparsely stained. The origin of spinal nerve terminals containing NT75 was investigated with lesion techniques, colchicine treatment, and retrograde tracing in combination with immunocytochemical staining. Primary afferent neurons express NT75 immunoreactivity and account for most of the dense staining in the superficial dorsal horn and part of the labeling in the deeper laminae. It was found that corticospinal and virtually all brainstem neurons with descending projections to the spinal cord also express NT75 immunoreactivity, including those terminating in the ventral horn. Colchicine treatment of the spinal cord also resulted in NT75 staining in most, if not all, spinal neurons. It appears that neurons in all three major sources of spinal afferents (primary sensory, descending, and intrinsic systems) can express NT75 immunoreactivity, but that some neurons normally contain higher levels of the protein in their nerve terminals. Previous analysis of developing spinal cord has shown widespread, dense NT75 labeling throughout the spinal gray in the early postnatal period, which later becomes restricted to the adult pattern. These studies support the hypothesis that many spinal pathways express high levels of NT75 immunoreactivity during development, but that only certain pathways maintain high levels in the adult. © 1993 Wiley-Liss, Inc.     

Taurine-like immunoreactivity in the brain of the honeybee

Taurine (2-aminoethanesulfonic acid) is one of the most abundant free amino acids in the insect central nervous system. We have investigated the distribution of taurine-like immunoreactivity in the brain of the honeybee with an antiserum recognizing fixed taurine. Taurine-like immunoreactivity appeared within neuronal perikarya, neurites, and terminals, whereas glial cells were unlabelled. All photoreceptor cells of the compound eyes and the ocelli were stained. So were the fibers of the anterior superior optic tract, which connects the optic lobes to the mushroom bodies in the median protocerebrum. In the mushroom bodies the majority of intrinsic Kenyon cells showed high levels of taurine-like immunoreactivity. The lateral antennoglomerular tract, which interconnects the mushroom bodies with the antennal lobes, was also intensely stained. In the antennal lobes, strong labelling was observed within a few fibers that invade a set of posterior glomeruli from the posterior margin. Sensory projections from the antennal nerve into the antennal lobes showed only intermediate levels of staining. Sensory projections into the dorsal lobe were devoid of taurine-like immunoreactivity. Labral, mandibular, maxillary, and labial nerves, which innervate the various parts of the feeding apparatus, contain a set of five to eight heavily stained fibers. A comparison of taurine-like immunoreactivity with glutamate- and GABA-like immunoreactivities in the brain of the honeybee indicates that the three amino acids are enriched in distinct neuronal populations.     

Ultrastructural localization of μ-opioid receptors in the superficial layers of the rat cervical spinal cord: extrasynaptic localization and proximity to Leu-enkephalin

Many of the analgesic effects of opiate drugs and of endogenous opioid ligands, such as Leu⁵-enkephalin (LE) are thought to be mediated in part by μ-opioid receptors (MOR) in the dorsal horn of the spinal cord. To establish the cellular sites for the spinally mediated analgesic effects of MOR activation and potential anatomical substrates for interactions with LE, we examined the ultrastructural localization of MOR and LE immunoreactivities in the adult rat cervical spinal cord (C3-C5). Anti-MOR sera recognizing the car☐yl terminal domain of MOR was localized using immunoperoxidase and immunogold-silver methods. μ-opioid receptor-like immunoreactivity (MOR-LI) was observed mainly in the superficial layers of the dorsal horn. Electron microscopy of this region revealed that small unmyelinated axons and axon terminals constituted 48% 91/189 and 15% (28/189), respectively, while dendrites comprised 36% (68/189) of the total population of neuronal profiles containing MOR. MOR-LI was localized mainly along extrasynaptic portions of the plasma membrane in both axons and dendrites. In sections dually labeled for MOR and LE, 21% (14/68) of the dendrites containing MOR-LI closely apposed or received synaptic contact from axon terminals exhibiting LE reaction product. The results provide the first ultrastructural evidence that within the dorsal horn of the spinal cord, LE, as well as exogenous opiates may alter both axonal release of neurotransmitters and postsynaptic responsiveness of target neurons to afferent input through activation of extrasynaptic MOR.     

Hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 ion channels modulate synaptic transmission from nociceptive primary afferents containing substance P to secondary sensory neurons in laminae I-IIo of the rodent spinal dorsal horn

We have previously demonstrated that hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 (HCN2) is expressed by terminals of peptidergic nociceptive primary afferents in laminae I-IIo of the rat spinal dorsal horn. In this study, we investigated the possible neurotransmitters and postsynaptic targets of these HCN2-expressing primary afferent terminals in the superficial spinal dorsal horn by using immunocytochemical methods. We demonstrated that HCN2 widely colocalizes with substance P (SP), and that HCN2-positive terminals that are also immunoreactive for SP form serial close appositions with dendrites and perikarya of neurokinin 1 receptor-immunoreactive neurons. It was also found that HCN2-immunoreactive terminals are frequently apposed to neurons that are immunoreactive for calbindin, micro-opioid receptor and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunit GluR2, markers for excitatory interneurons. Investigating HCN2 immunoreactivity in glutamic acid decarboxylase 65-green fluorescent protein transgenic mice, we found that HCN2-positive terminals occasionally also contact cells that contain an isoform of glutamic acid decarboxylase (glutamic acid decarboxylase 65), a marker for GABAergic inhibitory neurons. Application of ZD7288, an antagonist of HCN channels, onto neurons that were recorded in spinal cord slices with whole-cell patch-clamp electrodes reduced the number of monosynaptic excitatory postsynaptic potentials evoked by electrical stimulation of primary afferents at nociceptive intensities. The results suggest that HCN2 may contribute to the modulation of membrane excitability of SP-containing nociceptive primary afferent terminals, may increase the reliability of synaptic transmission from primary afferents to secondary sensory neurons and thus may play a role in the fine-tuning of pain transmission from nociceptive primary afferents to neurons in the spinal dorsal horn.     

Commissural propriospinal connections between the lateral aspects of laminae III-IV in the lumbar spinal cord of rats

It has been established that there is a strong functional link between sensory neural circuits on the two sides of the spinal cord. In one of our recent studies we provided a morphological confirmation of this functional phenomenon, presenting evidence for the presence of a direct commissural connection between the lateral aspects of the dorsal horn on the two sides of the lumbar spinal cord. By using a combination of neural tracing and immunocytochemical detection of neural markers like vesicular glutamate transporters, glutamic acid decarboxylase, glycine transporter, and met-enkephalin (which are characteristic of various subsets of excitatory and inhibitory neurons), we investigated here the distribution, synaptic relations, and neurochemical characteristics of the commissural axon terminals. We found that the cells of origin of commissural fibers in the lateral aspect of the dorsal horn were confined to laminae III-IV and projected to the corresponding area of the contralateral gray matter. Most of the commissural axon terminals established synaptic contacts with dendrites. Axospinous or axosomatic synaptic contacts were found in limited numbers. We demonstrated that interactions among commissural neurons also exist. More than three-fourths of the labeled axon terminals were immunostained for glutamic acid decarboxylase and/or glycine transporter, but none of them showed positive immunoreaction for met-enkephalin and vesicular glutamate transporters. The results indicate that there is a substantial reciprocal commissural synaptic interaction between the lateral aspects of laminae III-IV on the two sides of the lumbar spinal cord and that this pathway may transmit both inhibitory and excitatory signals to their postsynaptic targets.     

Growth-associated protein 43 immunoreactivity in the superficial dorsal horn of the rat spinal cord is localized in atrophic C-fiber,and not in sprouted A-fiber,central terminals after peripheral nerve injury

Peripheral nerve injury induces the up-regulation in dorsal root ganglion cells of growth-associated protein 43 (GAP-43) and its transport to the superficial laminae of the dorsal horn of the spinal cord, where it is located primarily in unmyelinated axons and growth-cone like structures. Peripheral nerve injury also induces the central terminals of axotomized myelinated axons to sprout and form novel synaptic contacts in lamina II of the dorsal horn. To investigate whether the sprouting of A-fiber central terminals into lamina II is the consequence of GAP-43 incorporation into their terminal membranes, we have used an ultrastructural analysis with double labelling to identify the localization of GAP-43 immunoreactivity. Transganglionic transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was used to identify C-fiber terminals. Transganglionic transport of the B fragment of cholera toxin conjugated to horseradish peroxidase (B-HRP) was used to label A-fiber sciatic nerve central terminals in combination with GAP-43 immunocytochemistry. GAP-43 was found to colocalize only with WGA-HRP- and not with B-HRP-labelled synapses or axons. In addition, many single-labelled GAP-43 synapses were observed. Many of the WGA-HRP-labelled terminals that were characterized by degenerative changes were GAP-43 immunoreactive. Our results indicate that peripheral nerve injury induces novel synapse formation of A fibers in lamina II but that up-regulated levels of GAP-43 are present mainly in other axon projections to the superficial dorsal horn. J. Comp. Neurol. 386:111–118, 1997. © 1997 Wiley-Liss, Inc.     

Immunocytochemical approach of GABAergic innervation of the mouse spinal cord using antibodies to GABA

The distribution of gamma-amino-butyric acid containing neurons in the Mouse spinal cord has been studied at both the light and electron microscope levels using antibodies against GABA and revelation by the Fab-peroxidase technique. At the light microscope level immunoreactive profiles of perikarya and neuronal processes were particularly abundant in the superficial laminae (I-IV) of the dorsal horn. Scattered soma profiles were found in the other layers and more particularly in the lamina X where Liquor contacting immuno-reactive neurons could be detected. GABAergic cell bodies were very sparse in the ventral horn. Electron microscopic observations confirmed the light microscope results: terminals constituted synaptic symmetrical contacts that provide a morphological basis for inhibition in the dorsal horn and for post-synaptic inhibition of motoneurons in the ventral horn.     

Somatostatin immunoreactive structures in the developing rat spinal cord

Somatostatin immunoreactive (SOM-I) perikarya were first observed in the ventral horn at E12, in the presumptive intermediate gray area at E14, and in the alar plate of the rostral spinal cord at E14. In general, after their initial appearance, their density increased and then decreased during development. A moderate density of SOM-I varicosities became obvious in the superficial laminae of the E20 dorsal horn. By E12 a few SOM-I perikarya, interpreted to be dorsal root ganglia, were observed lateral to the spinal cord, and by E13, SOM immunoreactivity was visualized within the central and peripheral processes of dorsal root ganglion axons. In the marginal zone, SOM-I fibers were first demonstrable in the ventral funiculus at E14, and in the lateral funiculus at E15. After their initial appearance, their density increased and then decreased with age, with the exception of the dorsal part of the lateral funiculus where it increased at the early stages of development to an apparently stable level. The early detection of SOM immunoreactivity in specific spinal regions corresponds well with the birth dates of cells in those regions. This indicates that the SOM-I cells are capable of synthesizing the substance at least as early as they have entered their final cell division.     

Different populations of parvalbumin- and calbindin-D28k-immunoreactive neurons contain GABA and accumulate 3H-D-aspartate in the dorsal horn of the rat spinal cord.

The colocalization of parvalbumin (PV), calbindin-D28k (CaBP), GABA immunoreactivities, and the ability to accumulate 3H-D-aspartate selectively were investigated in neurons of laminae I-IV of the dorsal horn of the rat spinal cord. Following injection of 3H-D-aspartate into the basal dorsal horn (laminae IV-VI), perikarya selectively accumulating 3H-D-aspartate were detected in araldite embedded semithin sections by autoradiography, and consecutive semithin sections were treated to reveal PV, CaBP and GABA by postembedding immunocytochemistry. Perikarya accumulating 3H-D-aspartate were found exclusively in laminae I-III, and no labelled somata were found in deeper layers or in the intermediolateral column although the labelled amino acid clearly spread to these regions. More than half of the labelled cells were localized in lamina II. In this layer, 16.4% of 3H-D-aspartate-labelled perikarya were also stained for CaBP. In contrast to CaBP, PV or GABA was never detected in neurons accumulating 3H-D-aspartate. A high proportion of PV-immunoreactive perikarya were also stained for GABA in laminae II and III (70.0% and 61.2% respectively). However, the majority of CaBP-immunoreactive perikarya were GABA-negative. GABA-immunoreactivity was found in less than 2% of the total population of cells stained for CaBP in laminae I-IV. A significant proportion of the GABA-negative but PV-immunoreactive neurons also showed CaBP-immunoreactivity in laminae II and IV. These results show that out of the two calcium-binding proteins, CaBP is a characteristic protein of a small subpopulation of neurons using excitatory amino acids and PV is a characteristic protein of a subpopulation of neurons utilizing GABA as a transmitter. However, both proteins are present in additional subgroups of neurons, and neuronal populations using inhibitory or excitatory amino acid transmitters are heterogeneous with regard to their content of calcium-binding proteins in the dorsal horn of the rat spinal cord.     

Ultrastructural description of taurine-like immunoreactive cells and processes in the rat hippocampus

A monoclonal antibody against taurine conjugated to KLH was used to identify and describe taurine-like immunoreactive processes in the rat hippocampus. Tissue from perfused rats was processed for immunohistochemical visualization of taurine and embedded for electron microscopy. Representative tissue samples from three regions, the dentate gyrus, CA3, and CA1, were sectioned, examined, and photographed. In the dentate gyrus, both granule cells and pyramidal basket cells were taurine-like immunoreactive. Some axon terminals in the dentate gyrus molecular layer as well as some mossy fiber boutons in the hilus were also taurine-like immunoreactive. In the CA3 region both pyramidal neurons and glial cells were taurine-like immunoreactive A few small-diameter axon terminals in stratum radiatum and some mossy fiber boutons in stratum lucidum were taurine-like immunoreactive. In CA1, pyramidal neurons and some glia were intensely taurine-like immunoreactive. A few immunoreactive axon terminals were seen in stratum radiatum and stratum oriens. In all regions, dendritic staining predominated. Our results support the hypothesis that while taurine may act as a neurotransmitter in a small portion of hippocampal terminals, its main function is probably as a neuromodulator or ionic regulator.     

Calcineurin and synaptophysin in the human spinal cord of normal individuals and patients with familial dysautonomia

Summary This report concerns the immunohistochemical demonstration of two neuronal Ca²⁺-binding proteins, calcineurin and synaptophysin, in the spinal cord of normal controls and from patients with familial dysautonomia. In controls, calcineurin immunoreactivity was highly concentrated in small nerve cells and fibers of the substantia gelatinosa. Synaptophysin immunoreactivity was normally distributed throughout the spinal cord gray matter, being highly concentrated in the substantia gelatinosa, the dorsal nucleus of Clarke and the anterior horn. In patients with familial dysautonomia, no apparent changes in calcineurin immunoreactivity were found in the substantia gelatinosa. By contrast, there was a significant depletion of synaptophysin-positive axon terminals in the substantia gelatinosa and in the dorsal nucleus of Clarke of patients with familial dysautonomia.