Electrically silent neurons in the substantia gelatinosa of the rat spinal cord.

Substantia gelatinosa (SG) neurons are usually categorized on three main types: tonic, adapting and delayed firing (DFNs), based on characteristic firing response evoked by sustained stimulation. Here, the existence of electrically silent neurons (ESNs, 9.3%) is reported by using patch-clamp recording and confocal microscopy in spinal cord slices from 3-5 weeks-old rats. Those neurons does not generate spikes at their resting membrane potential (approximately -69 mV) but only at preliminary depolarization to > -60 mV In the latter case, spikes appeared starting from the end of stimulation, which is characteristic feature of DFNs. With the exception of APs block, all other passive and active electrophysiological properties of ESNs and DFNs were similar. Their main morphological properties (vertical orientation of dendritic tree and axonal trajectory) were close too. A distinctive feature of ESNs was larger amplitude of outward A-type K+ current (K(A)). The results suggest that the latter might cause a block of APs in ESNs, while these cells likely are a functional (i.e., non-firing) subtype of DFNs. The role of DFNs in descending control of pain transmission via modulation of its K(A) is hypothesized.

Electrophysiological and morphological properties of neurons in the substantia gelatinosa of the mouse trigeminal subnucleus caudalis.

The excitability of the second order neurons within the trigeminal subnucleus caudalis underlies pain perception and processing in migraine and trigeminal neuralgia. These neurons were studied with whole-cell patch-clamp technique in slices from mouse brain stem. Electrical and morphological characteristics of 56 neurons were determined. Four categories were distinguished from electrophysiological properties: tonic (39%), phasic (34%), delayed (16%) and single spiking (11%). These categories did not show distinct morphological properties. Neurons had tetrodotoxin-sensitive sodium currents that activated and inactivated within milliseconds. They also showed a high voltage-activated, slowly inactivating calcium current: up to half of this current was blocked by omega-conotoxin GVIA (1microM) and omega-agatoxin IVA (100-300 nM), but it was not affected by nifedipine (10microM). Exogenously applied capsaicin (1microM) and alphabetamethylene-5'-adenosine triphosphate (100microM) elicited large amplitude, spontaneous excitatory postsynaptic currents that were blocked by capsazepine (10microM) and 5-[(3-phenoxybenzyl)-(1,2,3,4-tetrahydro-naphthalen-1-yl)-carbamoyl]-benzene-1,2,4-tricarboxylic acid (A-317491: 10microM), respectively. Thus, neurons of the mouse trigeminal subnucleus caudalis substantia gelatinosa exhibit N-type and P/Q-type voltage-gated calcium channels, and receive presynaptic afferents that express TRPV1 and P2X(2/3) receptors. These results suggest possible therapeutic interventions, and serve as a basis for the characterization of cellular changes that may underlie trigeminal neuropathic pain.

[Morphophysiologic properties of delayed firing neurons in substantia gelatinosa of the rat spinal cord.

Substantia gelatinosa (SG) neurons of the spinal cord are highly heterogeneous in their morphologic and physiologic properties. Based on characteristic firing response evoked by sustained depolarization, neurons can be categorized on three main types: tonic, adapting and delayed firing (DFNs). Here, properties of DFNs in spinal cord slices from 3-5 weeks-old rats were studied with the use of patch-clamp recording and confocal microscopy. Distinctive features of DFNs were increased rheobase (95.7 +/- 11.2 pA) and depolarized threshold to evoke action potential (-37.8 +/- 0.7 mV) than in neurons of other types. In voltage-clamp mode all DFNs expressed high amplitude outward A-type potassium current (K(A)), which started activation at approximately -70 mV, before Na+ current. Structurally, the majority of DFNs had vertically oriented dendritic tree, while their axons were not restricted to SG and projected predominantly to lamina I. Possible physiological functions of DFNs are discussed.

BDNF-mediated modulation of GABA and glycine release in dorsal horn lamina II from postnatal rats.

Recent studies show that excitatory glutamatergic transmission is potentiated by BDNF in superficial dorsal horn, both at the pre- and the postsynaptic site. The role of BDNF in modulating GABA and glycine-mediated inhibitory transmission has not been fully investigated. To determine whether the neurotrophin is effective in regulating the spontaneous release of the two neurotransmitters, we have recorded miniature inhibitory postsynaptic currents (mIPSCs) in lamina II of post-natal rats. We show that application of BDNF enhanced the spontaneous release of GABA and glycine, in presence of tetrodotoxin. The effect was blocked by the trk-receptor inhibitor k-252a. Amplitude and kinetics of mIPSCs were not altered. Evoked GABA and glycine IPSCs (eIPSCs) were depressed by BDNF and the coefficient of variation of eIPSC amplitude was significantly increased. By recording glycine eIPSCs with the paired-pulse protocol, an increase of paired-pulse ratio during BDNF application was observed. We performed parallel ultrastructural studies to unveil the circuitry involved in the effects of BDNF. These studies show that synaptic interactions between full length functional trkB receptors and GABA-containing profiles only occur at non peptidergic synaptic glomeruli of types I and II. Expression of trkB in presynaptic vesicle-containing dendrites originating from GABAergic islet cells, indicates these profiles as key structures in the modulation of inhibitory neurotransmission by the neurotrophin. Our results thus describe a yet uncharacterized effect of BDNF in lamina II, giving further strength to the notion that the neurotrophin plays an important role in pain neurotransmission.

Transient loss of terminals from non-peptidergic nociceptive fibers in the substantia gelatinosa of spinal cord following chronic constriction injury of the sciatic nerve.

It is well known that following peripheral nerve injury, there are numerous changes in neurotransmitter and neuropeptide expression in the superficial dorsal horn, the dorsal root ganglion and the periphery. Of particular interest are the relative contributions of two sub-types of unmyelinated C-fibers in the initiation and maintenance of chronic pain, the peptidergic, and the non-peptidergic. Evidence gathered in recent years has led researchers to believe that the non-peptidergic nociceptive primary afferents are functionally distinct from their peptidergic counterpart. For our study, we used a well-established animal model of constriction neuropathy (the Kruger model) and studied Wistar rats at 5, 7, 10, 15 and 21 days after nerve lesion caused by the application of a fixed-diameter polyethylene cuff to the left sciatic nerve. Animals were assessed for the onset and evolution of mechanical allodynia using calibrated von Frey filaments and were additionally tested for thermal (heat and cold) hypersensitivity. Immunocytochemical detection of calcitonin gene-related peptide (CGRP) and isolectin B4 (IB4) binding was used to visualize the dorsal horn distribution of the boutons from the peptidergic and non-peptidergic fibers respectively. Using confocal microscopy and image analysis, we detected a significant decrease in the density of IB4-labeled boutons, ipsilateral to the lesion, at seven and 10 days following nerve injury. The density of IB4-labeled varicosities retuned to control levels by 15 days. There were no significant changes in the density of CGRP-labeled varicosities at all time points examined. Applying electron microscopy, we initially detected degenerative changes in the central elements of type I glomeruli and then a considerable reduction in their number followed by recovery at 15 days post-lesion. As the central boutons of type Ia represent varicosities from the fibers which bind IB4, the ultrastructural changes confirmed that there was a bona fide transient loss of varicosities, not simply a loss of IB4 binding. These data indicate that, in this animal model, morphological changes in the nociceptive C-fiber input of the rat dorsal horn are restricted to the non-peptidergic sub-population and are transient in nature. Furthermore, such changes do not correlate with the time-course of the allodynia.

Protein kinase C gamma-like immunoreactivity in the substantia gelatinosa of the medullary dorsal horn of the rat.

We examined protein kinase C gamma-immunoreactivity (PKCgamma-IR) in the substantia gelatinosa (SG) of the rat medullary dorsal horn (MDH). The density of PKCgamma-IR in the MDH was most intense in the SG. The number of neurons with PKCgamma-IR were also much larger in the SG than in the other layers of the MDH. Double-immunohistochemical studies indicated light and electron microscopically that substance P-containing fibers and I-B4 (isolectin from Bandeiraea simplicifolia)-labeled fibers made synapses on SG neurons with PKCgamma-IR, indicating that SG neurons with PKCgamma might receive nociceptive primary afferent fibers. The results support the notion that PKCgamma in the MDH may contribute to the regulation of the nociception.

The relationship between neurokinin-1 receptor and substance P in the medullary dorsal horn: a light and electron microscopic immunohistochemical study in the rat.

The synaptic relationship between substance P (SP) and its receptor, i.e. neurokinin-1 receptor (NK1R), was examined in the superficial laminae of the caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn; MDH) of the rat. For confocal laser-scanning microscopy, double-immunofluorescence histochemistry for NK1 and SP was performed. In electron microscopic double-immunolabeling study, immunoreactivity for NK1R was detected with the silver-intensified gold method, while immunoreactivity for SP was detected with peroxidase immunohistochemistry. SP-immunoreactive axon terminals were observed to be in synaptic (mostly asymmetric) contact with NK1R-immunoreactive neuronal profiles in lamina I and lamina IIo. Although some SP-immunoreactive axon terminals were in synaptic contact with NK1R-immunoreactive sites of plasma membranes, NK1R-immunoreactivity was observed at both synaptic and non-synaptic sites of plasma membrane. Thus, SP released from axon terminals might not only act on NK1Rs facing the SP-containing axon terminals, but also diffuse in the extracellular fluid for distances larger than the synaptic cleft to act on NK1Rs at some distances from the synaptic sites.

FRAP-positive and capsaicin-sensitive terminals in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis.

FRAP (fluoride-resistant acid phosphatase)-reactivity in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis (STNC) was examined by light and electron microscopy. Degenerated figures of terminals caused by capsaicin were compared with the FRAP-positive terminals. Scalloped (fan-like) or indented, sinuous, slender, and cap-like figures with closely packed agranular synaptic vesicles of various sizes were common to both FRAP-positive and capsaicin-sensitive terminals. These terminals had glomerular or nonglomerular endings. Sometimes FRAP-positive and capsaicin-sensitive glomerular terminals made presynapses with surrounding dendrites. Frequently, both nonglomerular terminals were in direct contact with the neuronal soma. The terminal features of FRAP-positive and capsaicin-sensitive ones in the mouse STNC are the same as those seen in the superficial dorsal horn of the spinal cord. These findings suggest that some of the FRAP-positive terminals are capsaicin-sensitive, thereby indicating their nociceptive primary afferent.

Nitric oxide-producing islet cells modulate the release of sensory neuropeptides in the rat substantia gelatinosa.

The substantia gelatinosa of the spinal cord (lamina II) is the major site of integration for nociceptive information. Activation of NMDA glutamate receptor, production of nitric oxide (NO), and enhanced release of substance P and calcitonin gene-related peptide (CGRP) from primary afferents are key events in pain perception and central hyperexcitability. By combining reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry for NO-producing neurons with immunogold labeling for substance P, CGRP, and glutamate, we show that (1) NO-producing neurons in lamina IIi are islet cells; (2) these neurons rarely form synapses onto peptide-immunoreactive profiles; and (3) NADPH diaphorase-positive dendrites are often in close spatial relationship with peptide-containing terminals and are observed at the periphery of type II glomeruli showing glutamate-immunoreactive central endings. By means of confocal fluorescent microscopy in acute spinal cord slices loaded with the Ca2+ indicator Indo-1, we also demonstrate that (1) NMDA evokes a substantial [Ca2+]i increase in a subpopulation of neurons in laminae I-II, with morphological features similar to those of islet cells; (2) a different neuronal population in laminae I-IIo, unresponsive to NMDA, displays a significant [Ca2+]i increase after slice perfusion with either substance P and the NO donor 3morpholinosydnonimine (SIN-1); and (3) the responses to both substance P and SIN-1 are either abolished or significantly inhibited by the NK1 receptor antagonist sendide. These results provide compelling evidence that glutamate released at type II glomeruli triggers the production of NO in islet cells within lamina IIi after NMDA receptor activation. The release of substance P from primary afferents triggered by newly synthesized NO may play a crucial role in the cellular mechanism leading to spinal hyperexcitability and increased pain perception.

AMPA receptors at primary afferent synapses in substantia gelatinosa after sciatic nerve section.

Increased excitability of superficial laminae of the spinal cord may contribute to the pathological pain consequent to peripheral nerve injury. Among several mechanisms that may be responsible for this occurrence is upregulation of receptors for glutamate in the spinal cord. To explore this possibility, we investigated changes in AMPA receptors in substantia gelatinosa of rats after section of the sciatic nerve. Immunofluorescence was performed on sections from the fourth lumbar segment. Quantitative analysis of digitally captured images suggested that staining for an antibody to a sequence shared by GluR2 and GluR3 (GluR2/3) was increased on the side ipsilateral to the lesion. To determine whether antigen accumulation was at synaptic sites and to probe whether it was selective for primary afferent terminals, we performed electron microscopy on immunogold-labelled material. Gold particles coding for GluR2/3 subunits were counted from synaptic active zones of glomerular terminals in substantia gelatinosa that originate from small calibre afferent fibres, and from active zones of terminals of probable intrinsic origin. Counts were significantly increased on the side ipsilateral to the lesion only at synapses of primary afferent terminals. These results document selective upregulation of receptor protein at the synapse. This upregulation may contribute to the increased sensitivity of dorsal horn neurons following peripheral nerve injury.

Fluoride-resistant acid phosphatase (FRAP)-positive afferent terminals make synaptic contact with interneuronal soma in the substantia gelatinosa of the mouse spinal dorsal horn.

Fluoride-resistant acid phosphatase (FRAP)-reactive terminals making contact with interneuronal soma are found in the substantia gelatinosa of the mouse spinal dorsal horn. About one half of the interneuronal somata receive FRAP-positive boutons. By electron microscopy, these FRAP-positive terminals appear small, dark, slender, roundish, cap-like, ellipsoid or sinuous and electron-dense, scalloped (fan-like) contours with clear spherical synaptic vesicles of variable size, some large dense-core vesicles and mitochondria. All these features are very similar to those of capsaicin-sensitive terminals. Thus they are considered to be nociceptive primary afferent endings. Therefore, some of the FRAP-positive terminals are suggested to have a modulatory role in the nociceptive circuit in the substantia gelatinosa.

[Experimental and ultrastructural study of synapses in the substantia gelatinosa after electrical acupuncture].

Ultrastructural changes of synapses in the rat substantia gelationsa of the spinal caudal trigeminal nucleus were quantitatively examined following electrical acupuncture at "Sibai" for 30 minutes. Various structural changes were observed: Spherical synaptic vesicles were clustered toward the presynaptic membrane, the number of the vesicles tended to decrease, the width of the postsyiincreased and mitochondria were also increased in the bouton. These various types of synaptic alterations related to boutons are interpreted as the morphological changes induced by the acupuncture in the region corresponding to "Sibai".

Alz-50 recognizes epitopes in primary sensory fibres and in neurons of the substantia gelatinosa of the spinal cord. An ultrastructural study in the rat.

The monoclonal antibody Alz-50 has been proposed as a marker for cellular pathological changes in Alzheimer's disease. However, it has been reported that this antibody also reacts with specific epitopes in normal individuals. Furthermore, intense Alz-50 immunoreactivity has been recently described in the hypothalamus and spinal cord of rat and monkey. In the present study, we analysed the distribution pattern of Alz-50 immunostaining in the spinal cord of the adult rat. Using light microscopy, immunostained fibres and varicosities were detected mainly in laminae I-II, although some immunostaining lamina I and the outer two thirds of lamina II. The varicosities appeared either scalloped or dome-shaped and contained numerous agranular synaptic vesicles and a few dense-core vesicles. Most varicosities were presynaptic to dendrites. A few immunostained cell bodies and dendrites were also observed, but glial cells were never immunostained. Some ultrathin sections were processed for postembedding immunogold detection of calcitonin gene-related peptide and GABA immunoreactivities. Most of the varicosities which were immunoreactive for Alz-50 also showed calcitonin gene-related peptide immunoreactivity. In contrast, GABA immunoreactivity was never co-localized with Alz-50 immunoreactivity. These results indicate that, in the superficial dorsal horn, the epitope recognized by the Alz-50 antibody is located mainly, but not exclusively, in primary sensory fibres.

The effects of cholinoceptor agonists and antagonists on C-fibre evoked responses in the substantia gelatinosa of neonatal rat spinal cord slices.

1. The effects of cholinoceptor agonists and antagonists were studied on neurones in the substantia gelatinosa (SG) of an in vitro spinal cord slice and nerve preparation from neonatal rats. 2. Bath application of carbachol (1-50 microM) reduced, in a dose-related manner, the amplitude and duration of the excitatory postsynaptic potentials (e.p.s.ps) evoked in response to nerve stimulation. 3. The latencies and stimulation thresholds required to evoke these e.p.s.ps suggested that the majority were due to C-fibre activation. 4. The reduction in e.p.s.p. amplitude and duration produced by carbachol was reversed by the muscarinic antagonists, atropine (in 8 out of 11 cells), pirenzepine (in 7 out of 9 cells) and methoctramine (in 8 out of 9 cells) and by the nicotinic antagonist mecamylamine (in 3 out of 7 cells). 5. Injection of small hyperpolarizing or depolarizing pulses was associated with no change in conductance in 19 out of 26 (73%) of cells tested, suggesting that an action at a site presynaptic to the neurone studied could account for part of the effect of carbachol. 6. It is proposed that some of the cholinoceptors associated with the e.p.s.p. depression are located on C-fibres.

Neuropeptide Y-immunoreactive terminals form axo-axonic synaptic arrangements in the substantia gelatinosa (lamina II) of the cat spinal dorsal horn.

The ultrastructural organization of nerve terminals containing neuropeptide Y-immunoreactivity was studied in the substantia gelatinosa of the cat spinal dorsal horn. Seventy immunoreactive boutons were examined through serial sections and 67 of them were found to form between one and five synaptic junctions with dendrites (59.5% of synapses), somata (3% of synapses) and other axon terminals (37.5% of synapses). The postsynaptic axon terminals were often the central boutons of glomeruli. These findings suggest that neuropeptide Y regulates spinal sensory transmission through both a postsynaptic action upon dorsal horn neurons and a presynaptic action upon primary afferent terminals.

Immunocytochemical staining of neuropeptides in terminal arborization of primary afferent fibers anterogradely labeled and identified at light and electron microscopic levels.

A method is described to combine, at the ultrastructural level, horseradish peroxidase (HRP) anterograde tracing of primary afferents and peptide immunocytochemistry, using the lateral plexus of primary afferent fibers in laminae I-IIo of the rat dorsal horn as a model system. Free HRP was crushed against the dorsal roots. After a 14-h survival, animals were perfused, and the spinal cord was sliced at 50 microns with a Vibratome in a parasagittal plane. From these thick sections, camera lucida drawings of HRP-labeled fibers were obtained. Following osmication and Epon flat embedding, thick sections were re-cut at 5 microns and the labeled arbors matched with those previously drawn from the 50-microns sections. Ultrathin sections were cut from the 5-microns semithin sections and directly stained on grids using a post-embedding immunogold labeling procedure. Single and/or double immunocytochemical staining was performed using a rat monoclonal antibody against substance P and a rabbit polyclonal antiserum against calcitonin gene-related peptide (CGRP). Immunocytochemical reactions were visualized using appropriate immunoglobulin G-gold conjugates and the double-labeled synaptic boutons were matched with the varicosities previously visualized at the light level in the thick and semi-thin sections. The major advantages of this method are: (i) correlative studies at light and electron microscope level are made possible; (ii) tissue ultrastructure and antigenicity are adequately preserved so that a reliable subcellular localization of antigens under study is obtained; (iii) the markers used for tracing and immunocytochemistry are clearly distinguishable, even when present in the same nerve profile; and (iv) anterograde tracing can easily be combined with multiple immunolabeling.

GABAergic neurons in the mouse superficial dorsal horn with special emphasis on their relation to primary afferent central terminals.

Immunocytochemical studies were carried out on the morphological relation between primary afferent central terminals (C-terminals) and GABAergic neurons in the mouse superficial dorsal horn. The superficial dorsal horn is composed of many synaptic glomeruli comprising two types: Type I with centrally located CI-terminals surrounded by several dendrites and few axonal endings, and Type II with centrally located CII-terminals surrounded by several dendrites and a few axonal endings. The CI-terminals are sinuous or scalloped with densely packed agranular synaptic vesicles, a few granular synaptic vesicles and mitochondria, and show an electron dense axoplasm, whereas the CII-terminals are large and round or rectangular with evenly distributed agranular synaptic vesicles, a number of granular synaptic vesicles and mitochondria, and show an electron opaque axoplasm. The immunoreaction of GABA was remarkable in the superficial laminae of the dorsal horn. Many interneuronal somata in the substantia gelatinosa showed GABAergic immunoreactivity. The immunoreaction was seen in the entire GABAergic neuroplasm, but not in the nucleus and its envelope. Most GABAergic features appeared as dendrites making postsynaptic contact with CI- or CII-terminals; i.e., numerous C-terminals made presynaptic contact with GABAergic dendrites. GABA immunoreactivity was seen over round synaptic vesicles and mitochondrial membranes. A few CII-terminals made presynaptic contact with GABAergic interneuronal somata. Previous physiological and anatomical studies have suggested that not only the cutaneous nociceptive primary afferent C-terminals but also mechanoreceptive primary afferent C-terminals make presynaptic contact with the GABAergic dendrites, boutons and soma. The presynaptic relation of these primary afferents with GABAergic neurons seems to provide morphological support for the essential feature of the gate control theory: primary afferent fibers may play a part in the modulation of nociceptive information via GABAergic neurons in the superficial dorsal horn. Small GABAergic terminals were found to make contact with blood capillaries suggesting the release of GABA into circulation.

The action of capsaicin on primary afferent central terminals in the superficial dorsal horn of newborn mice.

Capsaicin was injected subcutaneously (50 mg/kg) into 10 mice on days 2 or 3 after birth, and 12 h, 3 and 5 days later the distribution and structure of degenerated primary afferent central axons or terminals (C-terminals) in the lumbar spinal dorsal horn were examined by electron microscopy. Degenerated terminal axons with dense or lamellar bodies or higher electron density were conspicuous 12 h after treatment with capsaicin. Severely degenerated unmyelinated axons, including dense or lamellar bodies engulfed by microglial cells, were numerous in the most superficial (marginal) layer, but rarely seen in the substantia gelatinosa. Two types of primary afferent central terminals in the substantia gelatinosa showed various extents of degeneration: small dark C-terminals (CI-terminals) with densely packed agranular synaptic vesicles, and large light ones (CII-terminals) with less dense agranular synaptic vesicles and a few granular synaptic vesicles. Thus, many central axon terminals of dorsal root ganglion (DRG) neurons that are sensitive to capsaicin enter the marginal layer and substantia gelatinosa. Degenerated primary afferent central axons or terminals markedly decreased in the superficial dorsal horn 3 and 5 days after capsaicin treatment, still, there were many degenerating DRG neurons at this time as shown by our previous study. Previously we also reported that fewer slightly degenerating unmyelinated dorsal root axons and small DRG neurons appear at 12 h and larger DRG neurons degenerate later than smaller ones after treatment with capsaicin. As a result, the discovery of many severely degenerated terminal axons in the superficial dorsal horn soon after treatment supports the idea that capsaicin first acts on the central terminals and that this is followed by damage to larger DRG neurons.

Ultrastructural changes of the central scalloped (C1) primary afferent endings of synaptic glomeruli in the substantia gelatinosa Rolandi of the rat after peripheral neurotomy.

Fine structural changes were observed in the dark scalloped central C1 terminals of type I synaptic glomeruli in spinal cord segments C6-C7 of the rat 3 days after cutting the three main forelimb nerves. Twenty-six per cent of the C1 terminals occurring on the ipsilateral side showed a lighter appearance due to a decrease in the number of synaptic vesicles. The number of synaptic vesicles per unit section area was only 42% of that present in normal C1 terminals on the contralateral side. The number of synaptic contacts of C1 terminals with the profiles surrounding them in each glomerulus was diminished and glial envelopment was increased to 15% of C1 terminal contour. Up to day 12, vesicle and synaptic losses were gradually aggravated and glial apposition was increased, but no obvious signs of glial engulfment were observed. From day 3 to day 12, altered C1 terminals increased in number, while those that appeared normal decreased. The latter had disappeared at day 12 and the altered ones at day 15, and from this stage type I glomeruli were no longer present on the treated side. The lack of electron-dense degenerative bouton changes characteristic of Wallerian degeneration offers an explanation for the lack of or minimal amount of argyrophilic structures which has been found consistently in the substantia gelatinosa during transganglionic degeneration. The gradual decay of the C1 terminals raises the question of their fate. Future studies with the use of a stable marker might provide an answer.

GABA-like immunoreactivity in type I glomeruli of rat substantia gelatinosa.

A postembedding immunogold study of type I synaptic glomeruli in lamina II of rat dorsal horn was carried out using antiserum to gamma-aminobutyric acid (GABA). Gold particles were concentrated over some peripheral axons and vesicle-containing dendrites within these glomeruli and both types of profile were presynaptic to central axons. These results suggest that GABA is involved in presynaptic inhibition of unmyelinated primary afferents and is released by some presynaptic dendrites within lamina II.