QUANTITATIVE HISTOCHEMISTRY OF γ-AMINOBUTYRIC ACID IN CAT SPINAL CORD WITH SPECIAL REFERENCE TO PRESYNAPTIC INHIBITION

Abstract— A method is described for quantifying the GABA distribution in cat spinal cord at 200–500 μn resolution. Isolated spinal cord (L5–S1) was frozen and sectioned at about 150 μm thickness. The frozen tissue section was cut into 200 or 500 μm square blocks. The GABA content of each square tissue block was determined by enzymic micromethods and GABA distribution was mapped quantitatively. Average GABA concentrations were: 0·4 mmol/l. in white matter, 1·2 mmol/l. in ventral horn and 1·7 mmol/l. in dorsal horn. The highest concentrations of GABA (2–3 mmol/l.) were found in the dorsolateral part of dorsal horn. In order to destroy the interneurons of dorsal horn, the blood vessels supplying the dorsal horn of the lumbar enlargement were unilaterally cauterized. Seven to 30 days after operation, both the size of dorsal root potential and the GABA level in the dorsal horn were markedly reduced on the cauterized side. These results suggest that GABA is highly concentrated in the interneurons of dorsal horn and functions as a transmitter of presynaptic inhibition.     

Immunohistochemical studies on the spinal dorsal horn of the turtle Chrysemys d'orbigny.

Immunohistochemical methods were used to characterize some of the systems of nerve fibers occurring in the spinal dorsal horns of the turtle Chrysemys d'orbigny. Substance P (SP), calcitonin gene-related peptide (CGRP) and leuenkephalin (Enk) immunoreactive fibers were found concentrated in the superficial horn region, termed here synaptic field Ia. From this zone the immunoreactive fibers project to deeper dorsal horn regions. Comparison with histological images obtained after HRP labeling of dorsal root axons indicates that SP-, CGRP- and Enk-immunoreactive fibers are small-diameter primary sensory fibers entering the cord via synaptic field Ia. It is posulated here that these three substances may coexist in the same fibers. Enk-positive fibers also occur randomly scattered in the lateral funiculi, showing a conspicuous increase in density at the perimedullary plexus level. Tyrosine hydroxylase (TH) immunoreactive fibers were found in the more compact dorsal horn neuropil (synaptic field II) and also forming bilateral conspicuous bundles in the lateral funiculi. TH-immunoreactive cell bodies were found in the epithelium lining the central canal. Taking into account data derived from Golgi impregnated material it is proposed that they represent epithelial cells undergoing neural differentiation.     

Anti-idiotypic antibodies as a tool for cytochemical identification of substance P receptors in the central nervous system

Anti-idiotypic antibodies may serve as valuable probes for cytological identification of peptide receptors in the CNS. We have previously described the preparation of anti-substance P (SP) anti-idiotypic antibodies (anti-Id Ab) and have shown that they recognize SP receptors. These anti-Id Ab can be used in cytology to label SP receptors in CNS. We chose rat cervical spinal cord as a model because SP is present in large amounts in the dorsal and ventral horns, where it is implicated in pain and in motor function, respectively. After application of an indirect immunoperoxidase technique to tissue sections from perfused animals, immunolabeling was seen in the two superficial layers of the dorsal horn, the area surrounding the central canal, extending along the white matter in lamina VII, and in part of the ventral horn. This localization is in accordance with the classical distribution of SP receptors as seen by autoradiography with labeled SP. In the light of control experiments, as well as of biochemical and pharmacological arguments, we discuss the specificity of the immunolabeling. We conclude that anti-Id Ab recognize NK-P receptors, although crossreaction with NK-A or NK-B receptors cannot be totally ruled out.     

Localization of Endomorphin-2-Like Immunoreactivity in the Rat Medulla and Spinal Cord

Martin-Schild, S., J. E. Zadina, A. A. Gerall, S. Vigh and A. J. Kastin. Localization of endomorphin-2-like immunoreactivity in the rat medulla and spinal cord. Peptides 18(10) 1641–1649, 1997.—Endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH₂) are endogenous ligands that have greater affinity and selectivity for the μ-opiate receptor than any other known mammalian peptide. A polyclonal antiserum, screened for specificity to endomorphin-2 by immunodot-blot assay and preabsorption controls, was used for localization of this peptide. Immunocytochemistry performed on the brainstem, spinal cord, and sensory ganglia of rats by the avidin–biotin–peroxidase method revealed a continuous dense aggregation of endomorphin-2-like immunoreactive varicose fibers in the superficial laminae of the dorsal horn of the medulla and spinal cord. Immunoreactive fibers were detected in the dorsal root as well as within the dorsal root ganglia. The results suggest that endomorphin-2 is synthesized in primary sensory neurons in ganglia, transported to the superficial dorsal horn, and released near neurons expressing μ receptors. Its distribution appears to represent a functional unit likely to be associated with modulation of nociceptive stimuli.     

Alteration of sensorineural circuits in spinal cord by chronic contact dermatitis

In the present study, eczema-induced alteration of sensorineural circuits of the spinal dorsal horn was investigated. Eczematous lesions resembling atopic dermatitis were induced by repeated application of diphenylcyclopropenone (DCP) onto murine right hind paws. Immunohistochemical labeling of calcitonin gene-related peptide and substance P was increased in the dorsal horn on the DCP-treated side. Expression of calcium binding proteins, calretinin and calbindin-D28K, normally widely seen in dorsal horn interneurons, was up-regulated on the DCP-treated side. E-Cadherin and alpha-N-catenin, synapse-related molecules, were intensely expressed in the spinal dorsal horn of the DCP-treated side. Interestingly, c-Fos positive cells were also significantly increased in laminae I and III of the DCP-treated side. These results suggest an enhanced release of neuropeptides from peripheral afferents and alterations in the sensorineural circuitry of the dorsal horn. These changes may account for the enhanced sensory sensitivity recognized in patients with chronic eczema and atopic dermatitis.     

Transganglionic transport of the lectin Soybean agglutinin (Glycine max) following injection into the sciatic nerve of the adult rat

The lectin soybean agglutinin (SBA) from Glycine max binds to small-sized dorsal root ganglion cells and their central terminals in the superficial dorsal horn of the spinal cord. Here we investigated the ability of SBA and SBA conjugated to horseradish peroxidase(SBA-HRP) to trace thin calibre afferents into the spinal cord from a peripheral nerve. Following injection into the sciatic nerve, labelled cells in the dorsal root ganglion were predominantly small-sized but some medium-sized cells were also labelled. Colocalisation studies of transported SBA with various neuronal markers showed that all neurons that transported SBA-HRP showed SBA binding, indicating high uptake specificity for the conjugate. 15% were immunoreactive for RT97 indicating that some axons were myelinated, and 54% also expressed binding sites for isolectin B4 from Griffonia simplicifolia, a selective marker for a subpopulation of unmyelinated afferents. With regard to neurotransmitter content, 43% of the SBA cells contained calcitonin gene-related peptide, 33% contained substance P and 2.5% somatostatin. In addition, 3% contained carbonic anhydrase. Centrally, injection of SBA in the sciatic nerve resulted in labelled terminals in somatotopically appropriate regions of laminae I–II of the dorsal horn, and in the gracile nucleus. A few neurons in the dorsal horn were labelled indicating that some transneuronal transport of SBA had occurred. The results show that SBA can be used as a transganglionic tracer to label fine calibre primary afferents that project to laminae I–II of the spinal cord and the gracile nucleus. It appears to label more afferents than isolectin B4, including also a subpopulation of myelinated afferents.     

Effects of systemic administration of strychnine, L-allylglycine, bicuculline and picrotoxin on the transsynaptic neural destruction in the medullary dorsal horn following transection of the rat inferior alveolar nerve

Adult rats underwent unilateral transection of the inferior alveolar nerve and subsequent intraperitoneal injection of strychnine (1 mg/kg, 3-23 days) at various posttransectional intervals. When they were sacrificed at 18-30 days posttransectionally, many pyknotic neuronal cell bodies were observed in plastic embedded toluidine blue-stained 1 micron-thick sections of the medullary dorsal horn. They were mostly found in the dorsal part of the dorsal horn ipsilateral to the neurotomy and were more abundant in laminae I/II than in laminae III/IV. Similar pyknotic neurons were found after 1 or 2 days of L-allylglycine administration (55.7 mg/kg/day) at a posttransectional interval of 20 days. Unlike those observed after strychnine treatment, the pyknotic neurons after L-allylglycine treatment were evenly distributed throughout laminae I-IV of the dorsal half of the medullary dorsal horn. Twenty-three days of bicuculline (2 mg/kg/day) or picrotoxin (0.5 mg/kg/day) treatment at an interval of 7 days did not yield pyknotic neurons. The results are discussed in the light of intrinsic synaptic circuitry of the dorsal horn.     

Calcitonin gene-related peptide 8-37 inhibits the evoked discharge frequency of wide dynamic range neurons in dorsal horn of the spinal cord in rats.

The present study was performed to explore the effect of calcitonin gene-related peptide 8-37 (CGRP8-37) on the electrical stimulation-evoked discharge frequency of wide dynamic range (WDR) neurons in the dorsal horn of the spinal cord in rats. The discharge frequencies of WDR neurons were evoked by transdermic electrical stimulation applied on the ipsilateral hindpaw. CGRP8-37 was applied directly on the dorsal surface of the L3 to L5 spinal cord. After the administration of 3 nmol of CGRP8-37, the evoked discharge frequency of WDR neurons decreased significantly, an effect lasting more than 30 min. The results indicate that CGRP receptors play an important role in the transmission of presumed nociceptive information in the dorsal horn of the spinal cord.     

Calcitonin gene-related peptide-receptor component protein expression in the uterine cervix, lumbosacral spinal cord, and dorsal root ganglia

The neuropeptide calcitonin gene-related peptide (CGRP) may play a role in neurogenic inflammation, tissue remodeling of the uterine cervix, promoting vasodilation, parturition, and processing of sensory information in the spinal cord. CGRP-immunoreactive nerves of the cervix and spinal cord have been studied but cellular identification of the CGRP receptor has received little attention. CGRP-receptor component protein (CGRP-RCP) is a small protein associated with the CGRP receptor; thus, immunostaining for the CGRP-RCP can be used to identify sites of the CGRP receptor. We determined sites of CGRP-RCP immunoreactivity relative to the presence of CGRP-ir nerve fibers in the female rat uterine cervix, spinal cord, and dorsal root ganglia. CGRP-RCP immunoreactivity was expressed in the dorsal horn of the spinal cord, venules of the uterine cervix, and perikarya of sensory neurons in dorsal root ganglia. CGRP-immunoreactive fibers were adjacent to CGRP-RCP-immunoreactive vessels in the cervix and among CGRP-RCP-immunoreactive structures in the dorsal horn of the spinal cord. This suggests CGRP-RCP is associated with structures innervated by CGRP nerves and these interactions may be changed in tissues in response to an appropriate stimulus.     

Abnormal physiology of the dorsal horn as related to the deafferentation syndrome

The spinal cord dorsal horn has been implicated in the generation of pain and dysesthesias following nerve and nerve root damage and/or avulsion, as well as following damage in adjacent spinal cord regions. Alterations in the functional properties of dorsal horn neurons occur after deafferentation and may underlie the occurrence of abnormal sensations referred to the denervated body part. Abnormal activity following deafferentation has also been noted at thalamic and cortical levels. Some of these post-denervation functional changes, determined anatomically and/or electrophysiologically, are reviewed as well as the results of behavioral studies of the deafferentation syndrome in the rat.     

Somatotopy of digital nerve projections to the dorsal horn in the monkey

The dorsal horn projection patterns of finger nerves were investigated in four Macaca mulatta monkeys. Proper digital branches of the median nerves, serving the radial aspect of a digit on each hand, were loaded with wheatgerm agglutinin-horseradish peroxidase complex (WGA:HRP). The distribution of the lectin-enzyme complex was mapped in the right and left dorsal horns. The dorsal horn projections of the digital nerves were localized in segments C6-C8 in laminae I-VI, primarily in laminae I-IV. The wedge-shaped termination zones were somatotopically organized, in agreement with the projections of the digits in cats. The fingers are represented medially, as they are in the cat. This similarity suggests that there is a mediolateral gradient of dorsal horn organization similar to that of the cat, with distal skin represented medially and proximal skin represented laterally. The rostrocaudal trajectory of finger representation, with digit 1 most rostral and digit 5 most caudal, is also in agreement with the organization of hindlimb toe projections in the cat. There was a high degree of bilateral symmetry for homologous nerves, and little overlap of projections from nerves innervating adjacent fingers. The sample size was too small to permit us to assess interanimal variation. These results suggest a similar somatotopy of projections, and presumably of dorsal horn cell somatotopy, in monkey and cat.     

Evidence for Ascending and Descending Intraspinal as Well as Primary Sensory Somatostatin Projections in the Rat Spinal Cord

Somatostatin distribution was measured quantitatively in the rat spinal cord by radioimmunoassay. Rostro-caudally, somatostatin content was about 50% higher in lumbar-sacral cord than in cervical or thoracic levels. The dorso-ventral distribution is more uneven: somatostatin is highest in the dorsal horn, where the peptide is 15 times as concentrated as it is in the ventral white matter, the region of lowest concentration. However, measurable amounts of the peptide were found in all regions studied. Dorsal root ganglionectomy decreased somatostatin levels in the dorsal cord, supporting the previously proposed role for this peptide as a primary sensory neurotransmitter or modulator; but somatostatin content also was decreased both rostral and caudal to spinal transection, indicating the presence of ascending and descending somatostatin pathways within the spinal cord. Brain levels did not change. Met-enkephalin and substance P were also measured after the above surgical manipulations. Met-enkephalin content was not altered and substance P content was lowered significantly only after ganglionectomy. Although this study confirms the primary sensory neuron as the origin of a part of spinal cord somatostatin, it further indicates the presence of ascending and descending somatostatin pathways within the rat spinal cord.     

The modulation of sensory transmission through the medullary dorsal horn during cortically driven mastication

During mastication, reflexes are modulated and sensory transmission is altered in interneurons and ascending pathways of the rostral trigeminal sensory complex. The current experiment examines the modulation of sensory transmission through the most caudal part of the trigeminal sensory system, the medullary dorsal horn, during fictive mastication produced by cortical stimulation. Extracellular single unit activity was recorded from the medullary dorsal horn, and multiple unit activity was recorded from the trigeminal motor nucleus in anesthetized, paralyzed rabbits. The masticatory area of sensorimotor cortex was stimulated to produce rhythmic activity in the trigeminal motor nucleus (fictive mastication). Activity in the dorsal horn was compared in the presence and absence of cortical stimulation. Fifty-two percent of neurons classified as low threshold and 83% of neurons receiving noxious inputs were influenced by cortical stimulation. The cortical effects were mainly inhibitory, but 21% of wide dynamic range and 6% of low threshold cells were excited by cortical stimulation. The modulation produced by cortical stimulation, whether inhibitory or excitatory, was not phasically related to the masticatory cycle. It is likely that, when masticatory movements are commanded by the sensorimotor cortex, the program includes tonic changes in sensory transmission through the medullary dorsal horn.     

Intrathecally delivered glial cell line-derived neurotrophic factor produces electrically evoked release of somatostatin in the dorsal horn of the spinal cord

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor with an established role in sensory neuron development. More recently it has also been shown to support adult sensory neuron survival and exert a neuroprotective effect on damaged sensory neurons. Some adult small-sized dorsal root ganglion (DRG) cells that are GDNF-sensitive sensory neurons express the inhibitory peptide somatostatin (SOM). Thus, we tested the hypothesis that prolonged GDNF administration would regulate SOM expression in sensory neuron cell bodies in the dorsal root ganglia (DRG) and activity-induced release of SOM from axon terminals in the dorsal horn. Continuous intrathecal delivery of GDNF for 11-13 days significantly increased the number of small DRG cells that expressed SOM. Furthermore, GDNF treatment evoked SOM release in the isolated dorsal horn following electrical stimulation of the dorsal roots that was otherwise undetectable in control rats. Conversely capsaicin-induced release of SOM (EC(50) 50 nM) was not modified by GDNF treatment. These results show that GDNF can regulate central synaptic function in SOM-containing sensory neurons.     

Neuronal nitric oxide synthase in the rabbit spinal cord visualised by histochemical NADPH-diaphorase and immunohistochemical NOS methods

The NADPH-diaphorase (NADPH-d) staining method is widely used in the investigation of both the central and peripheral nervous systems. Neuronal nitric oxide synthase (nNOS) has previously been shown to be responsible for the NADPH-d activity in neurons. However, NADPH-d activity does not always fully represent the enzyme nNOS. We investigated the distribution of NADPH-d activity and nNOS protein in the rabbit spinal cord for all groups of neurons and Rexed's laminae. In most laminae the distribution of NADPH-d activity was identical to nNOS immunoreactivity. Both were present in the dorsal horn and in pericentral areas of the spinal cord, but some differences existed. The superficial part of the dorsal horn (laminae I-III) stained more intensely for NADPH-d than for nNOS. However, the most prominent difference was seen in the lateral part of the dorsal horn--the lateral collateral pathway (LCP). The LCP stained strongly for NADPH-d activity, while nNOS staining was absent. Although there is an excellent correlation between NADPH-d staining and nNOS immunohistochemical staining in the spinal cord in general, the presence of staining differences necessitates the use of immunohistochemistry for some specialized applications.     

Neurokinin A, calcitonin gene-related peptide, and dynorphin A (1-8) in spinal dorsal horn contribute to descending inhibition evoked by nociceptive afferent pathways: an immunocytochemical study

Immunocytochemical technique was used to compare the contents of neurokinin A (NKA), calcitonin gene-related peptide (CGRP), and dynorphin A (1-8) (DynA) on two sides of the lumbar dorsal horn of rats in which the unilateral thoracic dorsalateral funiculus (DLF) was transected while formalin (0.2 ml, 0.5%) was injected equally into two hindpaws. The results showed that all the NKA-like, CGRP-like, and DynA (1-8)-like immunoreactivities were significantly lower in the superficial laminae of the dorsal horn on the side ipsilateral to the lesioned DLF than that on the side with intact DLF. This implies that peripheral noxious inputs activate the supraspinal descending inhibitory systems which in turn modulate the transmission of noxious message at the spinal level by changing the release of related neuropeptides.     

Speaking out of turn: a role for silent synapses in pain

Severe tissue or nerve injury can result in a chronic and inappropriate sensation of pain, mediated in part by the sensitization of spinal dorsal horn neurons to input from primary afferent fibers. Synaptic transmission at primary afferent synapses is mainly glutamatergic. Although a functioning excitatory synapse contains both alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the postsynaptic membrane, recent evidence suggests that dorsal horn neurons contain some "silent" synapses, which exhibit purely NMDA receptor-mediated evoked postsynaptic currents and do not conduct signals at resting membrane potential. Serotonin, which is released onto dorsal horn neurons by descending fibers from the rostroventral medulla, potentiates sensory transmission by activating silent synapses on those neurons, i.e., by recruiting functional AMPA receptors to the postsynaptic membrane. This phenomenon may contribute to the hyperexcitability of dorsal horn neurons seen in chronic pain conditions.     

Somatotopy of Digital Nerve Projections to the Dorsal Horn in the Monkey

The dorsal horn projection patterns of finger nerves were investigated in four Macaca mulatto monkeys. Proper digital branches of the median nerves, serving the radial aspect of a digit on each hand, were loaded with wheatgerm agglutinin—horseradish peroxidase complex (WGA:HRP). The distribution of the lectin—enzyme complex was mapped in the right and left dorsal horns.

The dorsal horn projections of the digital nerves were localized in segments C₆-C₈ in laminae I-VI, primarily in laminae I-IV. The wedge-shaped termination zones were somatotopically organized, in agreement with the projections of the digits in cats. The fingers are represented medially, as they are in the cat. This similarity suggests that there is a mediolateral gradient of dorsal horn organization similar to that of the cat, with distal skin represented medially and proximal skin represented laterally. The rostrocaudal trajectory of finger representation, with digit 1 most rostral and digit 5 most caudal, is also in agreement with the organization of hindlimb toe projections in the cat. There was a high degree of bilateral symmetry for homologous nerves, and little overlap of projections from nerves innervating adjacent fingers. The sample size was too small to permit us to assess interanimal variation. These results suggest a similar somatotopy of projections, and presumably of dorsal horn cell somatotopy, in monkey and cat.