Electron microscope study of Golgi-stained cells in lamina II of the rat spinal dorsal horn

Seven Golgi-stained cells in lamina II of the rat spinal dorsal horn were examined by electron microscopy. One of the cells studied was an islet cell, two were stalked cells, and the remaining four cells could not be classed into either group. The islet cell and three of the unclassified cells possessed presynaptic dendrites and formed synapses onto a variety of dendritic spines and shafts within lamina II. The axons of the islet cell and of one of the unclassified cells formed symmetric axodendritic synapses mainly onto dendritic shafts. The two stalked cells and the remaining unclassified cell did not possess vesicle-containing dendrites. This last cell bore some resemblance to a stalked cell and may have represented an atypical example of one. Most of the synapses involving the cells took place outside synaptic glomeruli, but all seven cells were postsynaptic to central axons within glomeruli and in most cases to both type I and type II central axons, suggesting a monosynaptic input from myelinated and unmyelinated primary afferent axons. In addition, most of the cells were postsynaptic to vesicle-containing dendrites. It is concluded that certain cells, which do not belong to the stalked or islet classes, possess presynaptic dendrites and function as presumed inhibitory interneurones within lamina II. The target of the cells with presynaptic dendrites includes other cells within lamina II and may also include cells in deeper laminae of the dorsal horn. Further evidence will be needed in order to determine whether all cells in lamina II that do not possess presynaptic dendrites form a single functional class.     

Ultrastructural and immunocytochemical study of lamina II islet cells in rat spinal dorsal horn.

In order to compare the ultrastructure of GABA-immunoreactive and nonimmunoreactive islet cells in lamina II of the rat dorsal horn, a combined ultrastructural and immunocytochemical study of nine Golgi-stained neurones was performed. Cell bodies of these neurones were tested with antiserum to GABA, and in most cases with antiserum to glycine, while parts of the cell body and dendritic tree were examined with the electron microscope. Four of the neurones had cell bodies that were immunoreactive with GABA antiserum, and 2 of these were also glycine-immunoreactive, while 2 were not. Cell bodies of the remaining five neurones were not immunoreactive with GABA antiserum, nor, in the 3 cases tested, with glycine antiserum. Three of the GABA-immunoreactive cells possessed vesicle-containing dendrites and were presynaptic at dendrodendritic synapses, whereas no vesicles were observed in the dendrites of any of the neurones that were not GABA-immunoreactive. The axon of one of the nonimmunoreactive cells was found with the electron microscope. It gave rise to boutons that contained round agranular vesicles and a few dense-cored vesicles. Three synapses formed by this axon were identified and all were asymmetric. No obvious differences were detected in the types of profile that were presynaptic to GABA-immunoreactive and nonimmunoreactive cells. These results suggest that GABAergic islet cells are a source of presynaptic dendrites in lamina II of the rat and that some presynaptic dendrites contain GABA and glycine, while others contain GABA without glycine. The nonimmunoreactive islet cells presumably represent a distinct functional class of neurones and some of these may release an excitatory amino acid transmitter, possibly in addition to one or more neuropeptides.     

Fine structure and synaptic architecture of HRP-labelled primary afferent terminations in lamina IIi of the rat dorsal horn

The fine structure and synaptic architecture of the afferent terminations in dorsal horn lamina II are studied using a combined light and electron microscopic procedure after anterograde labelling with horseradish peroxidase. Vibratome parasagittal sections, stained with heavy metal intensified diaminobenzidine after tracer application to the dorsal roots, were flat-embedded in Epon. The five types of labelled terminal arbors occurring in lamina IIi (Cruz et al., '87: J. Comp. Neurol. 261:221-236) were drawn and relocated in 5-microns sections cut serially from the thick sections. Ultrathin sections were then cut from the 5-microns sections so that the terminal fibers and swellings observed in the light microscope could be traced in the electron microscope. The flame-shaped arbors arose from fine myelinated stem fibers. Terminal strands generated large oval central terminals of type II synaptic glomeruli (CII), which established frequent axoaxonal contacts. Similar terminals have been labelled in the cat after tracer injections into hair-follicle fibers (Réthelyi et al., '82: J. Comp. Neurol. 207:381-393). The other four plexuses arose from unmyelinated stem fibers. The swarms of ultrafine boutons consisted of extremely thin terminal fibers generating very small, round, or polygonal glomerular terminals containing tightly packed agranular synaptic vesicles of variable size and one mitochondrion at best. The terminal strands of the bouquet plexus bore long and scalloped central varicosities of type I synaptic glomeruli (CI) with pleomorphic agranular vesicles and a relative abundance of dendroaxonal contacts. These features, together with the location in dorsal lamina IIi, suggest their belonging to the fluoride resistant acid phosphatase (FRAP)-reactive population. The boutons of the undulating fibers and those of the lateral plexus were, like those of the bouquets, scalloped and elongated rostrocaudally (CI), but contained a few large granular vesicles. The occurrence of the swarm, undulating, and lateral plexuses in ventral lamina IIi, which seems to lack FRAP or peptidergic terminals, suggests an origin from other, still unidentified neurochemical populations of fine primary afferents.     

The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord

Transganglionic transport of wheatgerm agglutinin conjugated horse-radish peroxidase (WGA-HRP) was used to reveal the central distribution of terminals of primary afferent fibers from peripheral nerves innervating the hind leg of the rat. In separate experiments the sizes and locations of cutaneous peripheral receptive fields were determined by electrophysiological recording techniques for each of the nerves that had been labeled with WGA-HRP. By using digital image analysis, the sizes and positions of the peripheral receptive fields were correlated with the areas of superficial dorsal horn occupied by terminals of primary afferents from each of these receptive fields. Data were obtained from the posterior cutaneous nerve of the thigh, lateral sural, sural, saphenous, superficial peroneal, and tibial nerves. The subdivisions of the sciatic nerve, the sural, lateral sural, superficial peroneal, and tibial nerves each projected to a separate and distinct region of the superficial dorsal horn and collectively formed a "U"-shaped zone of terminal labeling extending from lumbar spinal segments L2 to the caudal portions of L5. The gap in the "U" extended from L2 to the L3-4 boundary and was occupied by terminals from the saphenous nerve. Collectively, all primary afferents supplying the hindlimb occupied the medial 3/4 of the superficial dorsal horn with terminals from the tibial nerve lying most medially and occupying the largest of all the terminal fields. Afferents from the superficial peroneal lay in a zone between the medially situated tibial zone and the more laterally placed sural zone. Afferents from the posterior cutaneous nerve were located most caudally and laterally. Terminal fields from the posterior cutaneous and saphenous nerves differed from the others in having split representations caused presumably by their proximity to the mid-axial line of the limb. Comparisons between the peripheral and the central representations of each nerve revealed that 1 mm2 of surface area of the superficial dorsal horn serves approximately 600-900 mm2 of hairy skin and roughly 300 mm2 of glabrous skin. The vast majority of terminal labeling observed in the dorsal horn was found in the marginal layer and substantia gelatinosa, suggesting that small diameter afferents have an orderly somatotopic arrangement in which each portion of the skin surface is innervated by afferent fibers that terminate in preferred localities within the dorsal horn.     

Chronic peripheral nerve section diminishes the primary afferent A-fibre mediated inhibition of rat dorsal horn neurones

The inhibitory effect of A-primary afferent activity on A- and C-evoked activity in dorsal horn convergent neurones has been investigated in the decerebrate spinal rat. A-afferent conditioning stimuli produce a powerful inhibition of the C-evoked activity in the majority of units recorded in lamina 5 but were almost without effect on the C-evoked activity in units recorded within the substantia gelatinosa (laminae 1 and 2). The ability of an A-volley to inhibit the response to a C-volley begins immediately after the arrival of the A-volley and lasts for 50–70 ms. Conditioning A-stimuli also inhibit the A-evoked activity of dorsal horn neurones, the inhibition lasting up to 125 ms. Unlike the effect of A-conditioning stimuli on C-responses, which was restricted to units in lamina 5, the A-volleys inhibited the response of both substantia gelatinosa and lamina 5 units. In rats with chronically sectioned sciatic nerves (7–14 days) both the A on A and A on C inhibitions were significantly diminished in spite of intact afferent volleys and postsynaptic activity. In neurones activated by stimulation of the sectioned nerve, the A-conditioning stimuli either failed to produce an inhibition or produced a weak and shorter effect. These results are discussed in terms of the possible functional significance of A-afferent mediated inhibition.     

The properties of neurones recorded in the superficial dorsal horn of the rat spinal cord

The physiological properties of neurones in the superficial laminae of the dorsal horn of the fourth and fifth lumbar segments of the rat spinal cord have been investigated in decerebrate spinal animals. Both extracellular recordings with platinum-plated tungsten microelectrodes (n = 72) and intracellular recordings with glass microelectrodes (N = 79) were made. Attempts were made to fill cells intracellularly with horseradish peroxidase or Lucifer Yellow. Thirty-seven percent of the intracellularly injected neurones were recovered after histological processing and their cell bodies found to be in lamina 1 or 2 and in the dorsal white matter overlying lamina 1. The dendritic spread of the stained neurones was maximal in the rostrocaudal plane with a restricted mediolateral spread. The physiological properties of the extracellularly recorded units, the intracellularly unidentified units, and the intracellularly stained units were the same. The neurones were characterized by low background activity and all had excitatory receptive fields on the lower limb. Some neurones responded only to low-threshold mechanical stimulation of the skin or only to noxious skin stimulation but the majority of units (58%) were wide-dynamic-range cells responding to both types of stimuli. Receptive field classification was made questionable, however, by the existence of cells (9%) that exhibited a spontaneous shift in the size of their receptive fields and in the type of stimulus that elicited a response. The neurones in the superficial dorsal horn commonly showed a marked inhibition to repeated cutaneous stimuli (27%) or a prolonged afterdischarge followed a single stimulus (20%). Afferent input from the sural nerve was found to be from A and C fibres in both extra- and intracellular recordings. Aδ- and C-mediated excitations were most common although convergent inputs from Ab?-fibres occurred in 40% of units. No correlation was found between cell structure or distribution of dendritic fields and physiological properties in our small sample of intracellularly stained cells. The morphology of the cells was highly diverse, as were the different receptive fields. There was, however, some correlation between the location of cell bodies and their responses. Neurones responding only to low-threshold stimuli were distributed either in the dorsal white matter or in inner lamina 2. Wide-dynamic-range cells were distributed throughout the superficial dorsal horn. These results suggest that neurones of different shapes and positions may subserve the same function and, conversely, that neurones of the same shape and position may subserve different functions.     

Neuronal uptake of [3H]GABA and [3H]glycine in laminae I-III (substantia gelatinosa Rolandi) of the rat spinal cord. An autoradiographic study

[3H]GABA or [3H]glycine were injected into the subarachnoidal space of adult rats at C4-C5 level. After 10-60 min, the animals were perfused with 4% paraformaldehyde-0.5% glutaraldehyde and thick sections of the cervical spinal cord were postosmicated and Epon embedded. Light microscope autoradiographs of transverse cord sections showed numerous silver grains over the dorsal column and laminae I-III, higher grain densities occurring over lamina I for GABA and lamina III for glycine. In [3H]GABA-injected animals nerve cell bodies in lamina I or at the transition to lamina II appeared strongly labeled in light and electron microscope autoradiographs. These cells were smaller and less rich in RER than marginal cells and poor in axosomatic synaptic contacts. High grain densities appeared over axon terminals synapsing with dendrites in laminae I-II and over the light peripheral axon endings of synaptic glomeruli of laminae II-III. After [3H]glycine treatment, a number of nerve cell bodies were labeled in lamina III. It is suggested that two types of inhibitory interneurons occur in the rat gelatinosa, one GABAergic with cell body in lamina I, and another glycinergic in lamina III.     

Substance pin spinal cord dorsal horn decreases following peripheral nerve injury

Substance P was located in the spinal cord of rats by immunocytochemistry.Section and ligation of the sciatic nerve produced a depleted area low in substance P in the medial two-thirds of laminae 1 and 2 of segments L4 and 5.The time of depletion began about 5 days after the nerve had been cut and substance P reached a steady minimum by about 9 days and remained depleted for the entire period examined, 31 days.Crush lesions of the sciatic nerve failed to produce the marked and rapid changes of spinal cord substance P observed after section and ligation.     

Organization of cutaneous ventrodorsal and rostrocaudal axial lines in the rat hindlimb and trunk in the dorsal horn of the spinal cord

The somatotopic organization of cutaneous primary afferents projecting to the dorsal horn of the rat spinal cord was investigated. The fluorescent neurotracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was applied to cutaneous incisions made along ventrodorsal axial lines (VDALs) or rostrocaudal axial lines (RCALs) of the trunk and hindlimb. DiI-induced fluorescent zones appeared in laminae I-III of the dorsal horn in the transverse section. Several fluorescent zones appeared at different mediolateral portions after tracer application to VDALs. After tracer was applied to RCALs, a single zone of fluorescence was observed. Serial transverse sections were used to reconstruct fluorescent zones in lamina II and to illustrate the rostrocaudally elongated band-like projection fields in a horizontal plane. In the horizontal plane, the fluorescent zones of VDALs were reconstructed to band-like projection fields. These fields were arranged mediolaterally and extended rostrocaudally for approximately the length of one spinal cord segment or less. The fluorescent zones of RCALs were reconstructed to one band-like projection field. This field extended rostrocaudally over several spinal cord segments. Cutaneous afferents from the ventral median line of the trunk, tail, hindlimb, sole, and ventral side of the digits projected to the medial margin of the dorsal horn. Cutaneous afferents from the dorsal median lines projected to the lateral margin of the dorsal horn. By analyzing the pattern of the body surface regions and the VDALs and RCALs, the central projection fields of body surface regions could be hypothesized, based on the central projection fields of the individual VDAL and RCAL afferents. Thus, we established a detailed dorsal view map of the central projection fields of cutaneous primary afferents.     

Choline acetyltransferase-immunoreactive profiles are presynaptic to primary sensory fibers in the rat superficial dorsal horn

The specific aim of this study was to search for morphological counterparts to the known antinociceptive effects of cholinomimetic drugs at the spinal cord level. For this, the light microscopic and ultrastructural distribution of choline acetyltransferase immunoreactivity was studied in laminae I-III of the rat cervical spinal cord. Immunoreactivity was present in cell bodies in lamina III, and in dendrites and axons of all three laminae. Immunoreactive axonal varicosities were often presynaptic to the central varicosities of type II synaptic glomeruli in lamina II and lamina III, less often presynaptic to the central elements of type I glomeruli in lamina II, and often presynaptic to dendrites in both type I and type II glomeruli. In addition, immunoreactive dendrites were often postsynaptic to the central varicosities of glomeruli of all morphological types. These results indicate that 1) primary sensory fibers excite cholinergic interneurons; 2) the acetylcholine released by the axon terminals of these interneurons modulates both nociceptive and non-nociceptive sensory information at the spinal cord level through both pre- and postsynaptic mechanisms. Furthermore, our results reinforce current ideas on reciprocal sensory interaction between thick and fine afferent fibers.     

Exogenous tumor necrosis factor-alpha rapidly alters synaptic and sensory transmission in the adult rat spinal cord dorsal horn

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is involved in the generation of inflammatory and neuropathic pain. This study investigated if TNF-alpha has any effect on spinal synaptic and/or sensory transmission by using whole-cell recordings of substantia gelatinosa (SG) neurons in transverse lumbar spinal cord slices of adult rats and by using behavioral tests. After intrathecal administration of TNF-alpha in adult rats, spontaneous hind paw withdrawal behavior and thermal hyperalgesia were rapidly induced (approximately 30 min), while mechanical allodynia slowly developed. Bath application of TNF-alpha (0.1-1 nM, 8 min) depressed peak amplitude of monosynaptic Adelta and C fiber-evoked excitatory postsynaptic currents (EPSCs) without changing in holding currents and input resistances, whereas this application generally potentiated polysynaptic Adelta fiber-evoked EPSCs. Moreover, the frequencies, but not the amplitudes, of spontaneous and miniature EPSCs and spontaneous inhibitory postsynaptic currents were significantly increased by bath-applied TNF-alpha in most of the SG neurons. The effects of TNF-alpha on Adelta/C fiber-evoked monosynaptic and polysynaptic or spontaneous EPSCs were significantly blocked by 5 microM TNF-alpha antagonist that inhibits TNF-alpha binding to its type 1 receptor (TNFR1). Because this study also found high protein expression of TNFR1 in the adult dorsal root ganglion and no change of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) induced whole-cell currents by TNF-alpha, we conclude that presynaptic TNFR1 at Adelta/C primary afferent terminals contributes to the rapid alteration of synaptic transmission in the spinal SG, and the development of abnormal pain hypersensitivity by exogenous TNF-alpha.     

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

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

Effects of iontophoresis of noradrenaline and stimulation of the periaqueductal gray on single-unit activity in the rat superficial dorsal horn

Recordings were made with a new form of low-noise carbon fibre microelectrode from 75 units in the superficial laminae of the lumbar dorsal horn of the anaesthetized rat. The response of each unit to adequate stimulation of its peripheral receptive field, to noradrenaline (NA) applied iontophoretically, and to electrical stimulation of the periaqueductal gray (PAG) was investigated. Only units that could be excited by iontophoresis of glutamate (10–100 nA) were analyzed. Recording sites in the spinal cord and stimulation sites in the brainstem were identified histologically at the end of each experiment. Forty-six units with low-threshold receptive fields and small spike amplitudes were found, mainly located in laminae II and III. Both stimulation of the PAG and NA iontophoresis excited the majority (32/46) of these units. The rest were unaffected. Eight high-threshold (HT) units were located in the region of lamina I. Twenty-one wide-dynamic-range (WDR) units were found mainly in deeper laminae. Both WDR and HT units were inhibited by NA and PAG stimulation. This inhibition affected both glutamate-evoked activity and responses to nociceptive stimuli. We suggest that the small LT units are inhibitory interneurones of the substantia gelatinosa (SG), which act on the WDR and HT units to produce nociceptive-specific inhibition. The inhibition can be modality-specific without necessarily being presynaptic because of the laminar arrangement of the dorsal horn. The PAG could thus exert its known antinociceptive effects at least partly via descending noradrenergic axons that excite these SG cells.     

Morphology and axonal arborization of rat spinal inner lamina II neurons hyperpolarized by mu-opioid-selective agonists

The ventral or inner region of spinal substantia gelatinosa (SG; lamina II(i)) is a heterogeneous sublamina important for the generation and maintenance of hyperalgesia and neuropathic pain. To test whether II(i) neurons can be hyperpolarized by the mu-opioid agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO; 500 nM) and to address possible downstream consequences of mu-opioid-evoked inhibition of II(i) neurons, we combined in vitro whole-cell, tight-seal recording methods with fluorescent labeling of the intracellular tracer biocytin and confocal microscopy. Twenty-one of 23 neurons studied had identifiable axons. Nine possessed axons that projected ventrally into laminae III-V; six of these were hyperpolarized by DAMGO. Three of four neurons with identifiable axons that projected to lamina I were hyperpolarized by DAMGO. Most neurons could be classified as either islet cells or stalked cells. Five of nine labeled islet cells and only two of seven stalked cells were hyperpolarized by DAMGO. Three were stellate cells: one resembled a spiny cell and three could not be classified. DAMGO hyperpolarized each of the stellate cells, the spiny cell, and 1 of the unclassified cells. Our data support the hypothesis that part of the action of mu-opioid agonists involves the inhibition of interneurons that are part of a polysynaptic excitatory pathway from primary afferents to neurons in the deep and/or superficial dorsal horn.     

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

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

Numbers of synapses in laminae I-IV of the rat dorsal horn

The present study determines numerical densities (NVsyn) and total numbers of synaptic discs in laminae I-IV of the rat S2 dorsal horn. Previous methods for NVsyn have the advantage of being relatively simple, but these assume that the discs are round, flat, and of uniform size. In our material, serial reconstructions indicate that these assumptions are not met. Accordingly we use a stereological method that is not as dependent on these assumptions. This method is to divide the surface density of the discs by the mean surface area of a disc (NVsyn = SVsyn/Ssyn). We refer to this as a reconstruction method because synaptic discs are reconstructed from serial sections. We also calculate numerical densities by several previously used standard methods, and the findings are similar but not identical. We find that numerical density and total synaptic numbers are smallest in lamina I, and densities and total numbers are not significantly different when lamina II is compared to laminae III and IV. Thus the intense labeling of terminals with certain compounds that characterize lamina I and II does not imply an increase in total synaptic numbers or in synaptic density. In addition there is a general increase in synaptic densities and numbers as one proceeds from lamina I to lamina IV. Another point is that the numerical density of synapses in the dorsal horn is approximately that of the cerebral cortex. These data will serve as a basis from which to judge the effects of denervations and other manipulations that purportedly change synaptic numbers.     

Somatotopic organization of cutaneous afferent terminals and dorsal horn neuronal receptive fields in the superficial and deep laminae of the rat lumbar spinal cord

The somatotopic organization of A- and C-afferent fibre terminals in the dorsal horn of the rat lumbar spinal cord was compared with the spatial location of second-order dorsal horn neuronal mechanoreceptive fields. The central terminal fields of the sural, saphenous, and tibial nerve were mapped by labelling the nerves with horseradish peroxidase (HRP). A previous study used the transganglionic transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) to produce a somatotopic map of high-threshold C-fibre terminal fields in lamina II (Swett and Woolf: J. Comp. Neurol. 231:66-77, '85). In the present study the terminal fields of low-threshold A beta afferents that terminate in laminae III and IV were mapped by using unconjugated HRP at prolonged survival times (72 hours). Unfixed tissue was used to increase the sensitivity of the tetramethylbenzidine reaction, thus allowing these afferent terminals to be clearly seen. The general spatial arrangement of the terminal fields in laminae III/IV closely resembled that found in lamina II in the mediolateral and rostrocaudal planes but because of a dorsoventral obliquity of the afferent terminals, the superficial and deeper fields are not in strict vertical register. The input to laminae II-IV of the dorsal horn may therefore be viewed as two horizontally arranged sheets of afferent terminals both accurately representing the skin surface, the more superficial sheet representing the high-threshold C-afferents and the deeper sheet, low-threshold A-beta afferents. The spatial organization of high-threshold A-delta afferents in laminae I and V appears to be quite different, with a transverse rather than a longitudinal orientation. To study dorsal horn cell receptive field organization two single units with mechanoreceptive fields were recorded extracellularly in each of 87 vertical tracks in the lumbar spinal cord, one unit in the superficial dorsal horn and the second in the deep dorsal horn. In general the somatotopic organization of the receptive fields of both sets of units followed that of the afferent terminal fields but there were cells with receptive fields that were anomalous relative to the recording site. No evidence of any vertical relation or columnar arrangement in receptive field size, threshold, or location on the body surface was found when comparing the two units in a pair. Furthermore, no laminar functional specialization was found, the majority of neurones having both low- and high-threshold inputs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphological characterization of substance P-like immunoreactive glomeruli in the superficial dorsal horn of the rat spinal cord and trigeminal subnucleus caudalis: a quantitative study

The aim of this work was to study the ultrastructural distribution of substance P-like immunoreactivity in laminae I and II of rat spinal cord and trigeminal subnucleus caudalis in relation to synaptic glomeruli. A bispecific monoclonal antibody directed against substance P and horseradish peroxidase was used, combining sensitive immunocytochemistry with preservation of fine ultrastructural detail. Some of the quantitative observations were carried out with an automated image analysis system. The study revealed that in lamina I of the spinal cord, almost all immunoreactive profiles counted were nonglomerular, and a considerable number of them contacted medium-size or large dendrites or were in direct contact with other vesicle-containing profiles. In ventral lamina II, 9.4% of the labeled axonal varicosities were central boutons of type I glomeruli (CI). They could be identified by their scalloped contour, number and types of peripheral profiles, reduced density of mitochondria, and localization in the dorsal horn. However, these immunoreactive glomerular CI boutons (14.1% of the total number of CI) differed statistically from the prevailing population of nonimmunoreactive CI, by being surrounded by less peripheral neuronal profiles, which established fewer synapses. In addition, they contained more than three dense-core vesicles per central profile. In the trigeminal subnucleus caudalis laminae I and II, the substance P fibers and varicosities had a plexiform orientation at the light microscopic level, which contrasted with the mainly rostrocaudal orientation of the spinal cord's lamina II plexus. However, the main ultrastructural findings were similar. These results demonstrate that substance P-like immunoreactivity occurs in a large number of type I synaptic glomeruli with specific morphological features and reinforce the current concept that the substantia gelatinosa of the spinal cord and trigeminal subnucleus caudalis are homologous structures.     

BDNF sensitizes the response of lamina II neurons to high threshold primary afferent inputs

Brain-derived neurotrophic factor (BDNF) is up-regulated and released in the dorsal horn following peripheral inflammation and has therefore been implicated in spinal mechanisms of sensitization. Despite these observations, the mechanisms associated with such a role for BDNF are not yet fully determined. Here, we investigate the effect of BDNF on dorsal root-evoked synaptic transmission in lamina II neurons. In a transverse spinal cord slice preparation from neonatal rats (P1-15), the whole cell patch-clamp technique was used to record from these neurons. Brief application of BDNF (50-200 ng/mL) facilitated the evoked synaptic currents; they remained enhanced even after BDNF was washed out. A significant minority of cells was minimally affected by BDNF and consistent with this, not all neurons in lamina II were immunoreactive for the tyrosine kinase (trk) B receptor. No facilitation was elicited when N-methyl-d-aspartate (NMDA) receptors were blocked with D-APV, when the postsynaptic NMDA receptors were selectively blocked with intracellular MK-801, or when postsynaptic neurons were loaded with BAPTA. Additionally, inhibiting phospholipase C (PLC) or protein kinase C (PKC) prior to BDNF application completely blocked facilitation. However, once synaptic current underwent BDNF-induced facilitation, the PKC inhibitors failed to reverse the effect, suggesting that PKC is needed for initiation, but not maintenance of BDNF-induced facilitation. These results demonstrate that BDNF functions at the spinal level to enhance synaptic efficacy in an NMDA receptor-dependent manner and requires the action of the PLC/PKC pathway. This action of BDNF may contribute to central sensitization and exaggerated pain states.