GABAergic neurons in inferior colliculus of the GAD67-GFP knock-in mouse: electrophysiological and morphological properties

Utilizing slice preparations of GAD67-GFP knock-in mouse, in which GABAergic neurons are specifically labeled with GFP fluorescence, we studied electrophysiological characteristics of GABAergic neurons of IC by whole-cell patch clamp-recording combined with biocytin-intracellular-staining techniques. GABAergic neurons of IC fell into two distinct firing types; (1) tonic type neurons and (2) transient (phasic) type neurons. Tonic type neurons showed regularly repetitive discharge pattern in response to a long depolarizing current pulse (200 ms), and transient type neurons showed spike discharges just at the onset of current pulse. Most of neurons of both types showed depolarizing sag in response to hyperpolarizing current pulse, which were blocked by 0.1 mM ZD7288 (Ih blocker). All two types of tonic neurons showed an AHP, which was blocked by Cd2+ (0.1 mM) and high concentration of apamin (2 microM). One of tonic type neurons (BP) revealed a long delay in spike onset or a longer first spike interval when they were stimulated from hyperpolarized potentials. The remaining tonic neurons (RS) did not show this property. Tonic type neurons were distributed in all region of IC. Morphologically, they were not identical; heterogeneous in somatic diameter, dendritic field size and its orientation. One of transient type neurons (Th-) revealed an AHP after the spike. The other transient type neurons (Th+) showed a depolarization hump after the spike, which were blocked by 0.1-0.2 mM Ni2+. Th+ type neurons were found only in the dorsolateral region of IC, having small dendritic field. Th+ type neurons are likely to be a distinct, homogenous group of GABAergic neuron in IC.     

异丙酚诱导GAD_(67)-GFP基因敲入小鼠意识消失过程中GABA能神经元的活化

探讨在系统性给予谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠异丙酚后诱导的意识消失作用中的GA-BA能神经元的活化情况。GAD67-GFP阳性小鼠20只,腹腔注射异丙酚130mg/kg(对照组腹腔注射相同体积的生理盐水)。分别在注射后5min,30min,1h进行行为学评分,随后立即处死并取脑,应用免疫组织化学观察神经元活化标记物Fos在全脑的表达分布并用免疫荧光双标的方法观察Fos与GABA能神经元的共存情况。行为学结果表明在给予异丙酚后5min和30min均能达到适度或深度麻醉效果,而1h时则处于过渡状态,行为学评分为5min时13分,30min时14分,1h时为9分。与对照组相比,Fos在海马CA1区、杏仁核、丘脑室旁核、下丘脑室旁核、梨状核、下丘脑腹内侧核、中脑导水管周围灰质的表达在三个时间点都有明显增加(P<0.05),在嗅球外丛状层有表达但与对照组相比无明显差异(P>0.05)。注射后5min、30min和1h下丘脑腹内侧核有Fos与GABA共存,有共存的细胞占该区域Fos阳性神经元的80.3%、89.7%和91.6%。下丘脑腹内侧核GABA能神经元活化是异丙酚诱导意识消失的可能机制之一。     

GAD67-GFP精神分裂症小鼠行为学改变及海马齿状回颗粒细胞层GABA能神经元的表达

目的:探讨谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠制备精神分裂症模型后学习与记忆功能的改变及海马齿状回颗粒细胞层GABA能神经元的表达。方法:利用聚合酶链式反应(PCR)鉴定GAD67-GFP基因敲入小鼠,MK-801连续腹腔注射2周制备精神分裂症动物模型,通过悬尾实验、Morris水迷宫实验、免疫荧光标记技术等,观察GAD67-GFP基因敲入小鼠的学习与记忆功能的改变及GABA能神经元在海马齿状回颗粒细胞层的表达。结果:停药后实验组与对照组比较:(1)实验组体重增加明显低于对照组(P0.05);(2)行为学改变:1悬尾实验:实验组不动时间明显小于对照组(P0.05);2Morris水迷宫实验:定位航行实验中实验组逃避潜伏期,游泳总路程明显长于对照组(P0.05),而其平均游泳速度与对照组没有明显差异(P0.05);空间探查实验中实验组经过平台所在点的次数和在平台所在象限的时间明显小于对照组(P0.05);(3)在海马齿状回颗粒细胞层中实验组的GFP阳性细胞明显多于对照组(P0.05)。结论:通过对GAD67-GFP基因敲入小鼠进行腹腔注射MK-801制备精神分裂症模型后,其学习与记忆功能显著下降,且海马齿状回颗粒细胞层GABA能神经元明显增加。提示精神分裂症后学习记忆功能减退可能与GABA能神经元的表达有关。     

GAD67-GFP基因敲入小鼠5-羟色胺样和P物质样阳性终末与三叉神经中脑核神经元的联系

本研究应用免疫荧光组织化学三重标记技术,在激光共聚焦显微镜下观察了GAD67-GFP基因敲入小鼠三叉神经中脑核(Vme)内5-HT样和P物质(substanceP,SP)样阳性终末分别与小白蛋白(parvalbumin,PV)样和GFP阳性神经元之间的联系。结果显示:(1)Vme内有较多的PV样阳性神经元,这些神经元绝大多数为大、中型的假单极神经元,直径约为18～40μm;也可观察到少量的GFP阳性神经元,多为小的多极神经元,直径约为8～15μm;(2)5-HT样阳性终末广泛分布于Vme内,其中有部分阳性终末分别聚集在PV样或GFP阳性的Vme神经元的胞体周围,计数结果表明:大约有77.7%和22.3%的5-HT样阳性终末分别与PV样或GFP阳性胞体相接触;(3)SP样阳性终末也广泛分布于Vme内,并分别与PV样或GFP阳性神经元的胞体形成密切接触,其中有78.2%和21.8%的阳性终末分别与PV样或GFP阳性胞体相接触。以上结果提示,在口面部本体感觉信息向更高一级中枢传递过程中,5-HT能和SP能神经终末不仅对初级传入发挥直接的调控作用,还可能通过影响Vme内的GABA能神经元的活动,从而间接对该信息的传递发挥调控作用。     

GAD_(67)-GFP基因敲入小鼠脊髓背角GABA能神经元与不同5-HT受体亚型的共存研究

为初步探讨5-HT受体亚型对脊髓背角内抑制性GABA能神经元的介导作用,本研究以GAD67-GFP基因敲入小鼠为工具,利用免疫荧光双标记方法,检测了5-HT受体亚型与脊髓背角GABA能阳性神经元的共存情况。结果显示,GABA能阳性细胞与5-HT1A,5-HT2A和5-HT3等受体亚型共存,且共存率存在明显不同。以上结果提示5-HT受体各亚型在脊髓水平发挥不同的作用,从而参与完成5-HT复杂的生理效应。     

GAD67-GFP基因敲入小鼠黑质网状部神经元GABA与氯离子转运体KCC2和NKCC1的共存

为了观察谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠黑质网状部(SNr)内,表达GFP的GABA能神经元与一对功能相反的Cl-共转运体(K+-Cl-cotransporter2,KCC2;Na+-K+-Cl-cotransporter1,NKCC1)的共存情况,本研究分别运用原位分子杂交与免疫组织化学相结合以及GFP与KCC2或NKCC1免疫荧光染色相结合的双重标记方法,在光学显微镜和激光共聚焦显微镜下同时进行观察。结果显示:(1)SNr内95%以上的GFP阳性神经元同时表达KCC2 mRNA,而50%表达KCC2 mRNA的阳性神经元呈GFP阳性;(2)SNr内80%以上的GFP阳性神经元同时表达NKCC1 mRNA,约35%表达NKCC1 mRNA的阳性神经元呈GFP阳性;(3)SNr内90%以上的GFP阳性神经元同时表达KCC2,双标神经元约占KCC2阳性神经元的50.5%;(4)SNr内80.5%以上的GFP阳性神经元同时表达NKCC1,双标神经元约占NKCC1阳性神经元的42.5%。以上结果表明,SNr内表达GFP的GABA能神经元大部分与KCC2和NKCC1共存,提示氯离子共转运体可能对SNr内GABA能神经元起重要的调控作用。     

GAD67-GFP+ neurons in the Nucleus of Roller. II. Subthreshold and firing resonance properties

In the companion paper we show that GAD67-GFP+ (GFP+) inhibitory neurons located in the Nucleus of Roller of the mouse brain stem can be classified into two main groups (tonic and phasic) based on their firing patterns in responses to injected depolarizing current steps. In this study we examined the responses of GFP+ cells to fluctuating sinusoidal ("chirp") current stimuli. Membrane impedance profiles in response to chirp stimulation showed that nearly all phasic cells exhibited subthreshold resonance, whereas the majority of tonic GFP+ cells were nonresonant. In general, subthreshold resonance was associated with a relatively fast passive membrane time constant and low input resistance. In response to suprathreshold chirp current stimulation at a holding potential just below spike threshold the majority of tonic GFP+ cells fired multiple action potentials per cycle at low input frequencies (<5 Hz) and either stopped firing or were not entrained by the chirp at higher input frequencies (= tonic low-pass cells). A smaller group of phasic GFP+ cells did not fire at low input frequency but were able to phase-lock 1:1 at intermediate chirp frequencies (= band-pass cells). Spike timing reliability was tested with repeated chirp stimuli and our results show that phasic cells were able to reliably fire when they phase-locked 1:1 over a relatively broad range of input frequencies. Most tonic low-pass cells showed low reliability and poor phase-locking ability. Computer modeling suggested that these different firing resonance properties among GFP+ cells are due to differences in passive and active membrane properties and spiking mechanisms. This heterogeneity of resonance properties might serve to selectively activate subgroups of interneurons.     

Efferent connections of the ectostriatal core. An anterograde tracer study

In the present study the efferent connections of the ectostriatal core were investigated with biotinylated dextran-amine anterograde tracer in the chicken using light microscopy. The efferents of the ectostriatal core were labelled anterogradely, but retrograde labelling was also observed, which displayed the afferents of the same region. The ectostriatal belt received a few thin varicose fibres; retrogradely labelled cells also appeared. The most extended projection ended in the surrounding neostriatum, which turned out to be reciprocally connected to the ectostriatal core. On the basis of these connections, the neostriatum is said to be an important visual associative center. Efferent fibres reached the motor areas as well. A significant projection entered the paleostriatum augmentatum, especially the ventral part. The paleostriatum primitivum also received a few fibres. The other motor center, the medial part of the anterior archistriatum, was proved to be directly connected to the ectostriatal core as well. Considering that the archistriatum is also connected indirectly to the Wulst, the movements are able to be guided by well processed visual information.     

Intrinsic and commissural connections within the entorhinal cortex. An anterograde and retrograde tract-tracing study in the cat

Intrinsic and commissural connections within the entorhinal cortex (EC) were examined in the cat by the anterograde and retrograde tract-tracing methods with Phaseolus vulgaris leucoagglutinin and cholera toxin B subunit. Intrinsic axons to the superficial layers (layers I-III) arose mainly from layers II, III, Vd (deep part of layer V), and VI, were distributed more widely in the superficial layers than in the deep layers, and terminated progressively more densely in more superficial layers; most densely in layer I. In the medial entorhinal area (MEA) and the ventromedial and the ventrolateral divisions of the lateral entorhinal area (VMEA and VLEA), the longitudinal connections through the intrinsic fibers to the superficial layers is often more restricted in rostral direction than in caudal direction. In the dorsolateral division of the lateral EC (DLEA), the longitudinal connections through the intrinsic fibers to the superficial layers extended distantly in both rostral and caudal directions. Intrinsic fibers to the deep layers (layers IV-VI) originated mainly from layers IV and Vs (superficial part of layer V) and were distributed rather sparsely and diffusely; they were distributed more widely in the deep layers than in the superficial layers. Commissural axons to the homotopic EC regions originated from layers II and III of the MEA and DLEA and terminated in all EC layers, most densely in layer I.     

GAD67-GFP+ neurons in the Nucleus of Roller: a possible source of inhibitory input to hypoglossal motoneurons. I. Morphology and firing properties

In this study we examined the electrophysiological and morphological properties of inhibitory neurons located just ventrolateral to the hypoglossal motor (XII) nucleus in the Nucleus of Roller (NR). In vitro experiments were performed on medullary slices derived from postnatal day 5 (P5) to P15 GAD67-GFP knock-in mouse pups. on cell recordings from GFP+ cells in NR in rhythmic slices revealed that these neurons are spontaneously active, although their spiking activity does not exhibit inspiratory phase modulation. Morphologically, GFP+ cells were bi- or multipolar cells with small- to medium-sized cell bodies and small dendritic trees that were often oriented parallel to the border of the XII nucleus. GFP+ cells were classified as either tonic or phasic based on their firing responses to depolarizing step current stimulation in whole cell current clamp. Tonic GFP+ cells fired a regular train of action potentials (APs) throughout the duration of the pulse and often showed rebound spikes after a hyperpolarizing step. In contrast, phasic GFP+ neurons did not fire throughout the depolarizing current step but instead fired fewer than four APs at the onset of the pulse or fired multiple APs, but only after a marked delay. Phasic cells had a significantly smaller input resistance and shorter membrane time constant than tonic GFP+ cells. In addition, phasic GFP+ cells differed from tonic cells in the shape and time course of their spike afterpotentials, the minimum firing frequency at threshold current amplitude, and the slope of their current-frequency relationship. These results suggest that GABAergic neurons in the NR are morphologically and electrophysiologically heterogeneous cells that could provide tonic inhibitory synaptic input to HMs.     

Early development of GABAergic cells of the retina in sharks: an immunohistochemical study with GABA and GAD antibodies

We studied the ontogeny and organization of GABAergic cells in the retina of two elasmobranches, the lesser-spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus) by using immunohistochemistry for gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). Both antibodies revealed the same pattern of immunoreactivity and both species showed similar organization of GABAergic cells. GABAergic cells were first detected in neural retina of embryos at stage 26, which showed a neuroepithelial appearance without any layering. In stages 27-29 the retina showed similar organization but the number of neuroblastic GABAergic cells increased. When layering became apparent in the central retina (stage-30 embryos), GABAergic cells mainly appeared organized in the outer and inner retina, and GABAergic processes and fibres were seen in the primordial inner plexiform layer (IPL), optic fibre layer and optic nerve stalk. In stage-32 embryos, layering was completed in the central retina, where immunoreactivity appeared in perikarya of the horizontal cell layer, inner nuclear layer and ganglion cell layer, and in numerous processes coursing in the IPL, optic fibre layer and optic nerve. From stage 32 to hatching (stage 34), the layered retina extends from centre-to-periphery, recapitulating that observed in the central retina at earlier stages. In adults, GABA/GAD immunoreactivity disappears from the horizontal cell layer except in the marginal retina. Our results indicate that the source of GABA in the shark retina can be explained by its synthesis by GAD. Such synthesis precedes layering and synaptogenesis, thus supporting a developmental role for GABA in addition to act as neurotransmitter and neuromodulator.     

Synaptic connections between nonspiking afferent neurons and motor neurons underlying phase-dependent reflexes in crayfish.

1. This paper analyzes the synaptic connections made by nonspiking afferent neurons of the thoracocoxal muscle receptor organ (TCMRO) with basal limb motor neurons in the crayfish. The T fiber, a dynamically sensitive afferent, monosynaptically excites promotor motor neurons. Evidence suggests that both tonic graded chemical transmission and electrical synaptic transmission may be involved, depending on the motor neuron under consideration. 2. In preparations in the active state (spontaneously producing reciprocal motor patterns), the T fiber also inhibits promotor motor neurons in a phase-dependent manner. This inhibitory pathway is probably indirect, because it involves additional synaptic delay. 3. The statically sensitive S fiber also excites promotor motor neurons, but phase-dependent inhibition of promotor motor neurons by the S fiber was not seen. 4. The T fiber excites a subclass of remotor motor neurons (group 1) by a combination of direct chemical input and electrical input. This connection underlies the positive feedback reflex that excites these remotor motor neurons, in a phase-dependent manner, on stretch of the TCMRO during the active state. In inactive preparations, this connection remains subthreshold. 5. Central synaptic outputs of group 1 remotor motor neurons can also inhibit promotor motor neurons. This pathway may contribute to the phase-dependent reflex inhibition of promotor motor neurons that occurs in the active state.     

Identification and immunohistochemical characterization of colospinal afferent neurons in the rat

The classification, morphology and function of enteric neurons have been extensively studied in the small and large intestine. However, little is known about enteric neurons that directly project to the CNS. Previous studies have identified these unique neurons in the rectum, rectospinal neurons, but little was done to characterize them. Therefore, the aim of this study was to identify and characterize enteric neurons in the rat colon that directly project to the CNS by using retrograde neuronal tracing and immunohistochemistry. By applying the retrograde tracers 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) and Fluorogold (FG) to the L6/S1 segments of the spinal cord, we identified these neurons in both the myenteric and submucosal plexuses of the colon. These neurons were immunoreactive for neurofilament (NF) a marker for Aδ-fibers and isolectin-B4 (IB₄) a marker for C-fibers. These neurons expressed the enzyme neuronal nitric oxide synthase (nNOS) as well as peptides associated with sensory neurons such as substance P (SP) and vasoactive intestinal polypeptide (VIP) but did not express calcitonin gene-related peptide (CGRP). The N-methyl-d-aspartate (NMDA) receptor subunits NR1 and NR2D and proteinase-activated receptor-2 (PAR2) were also found in these neurons. However they did not express the transient receptor potential receptor V1 (TRPV1) or neurokinin 1 receptor (NK1). The expression of the peptides and receptors suggests that there are at least two separate populations of neurons projecting from the colon to the CNS. The data suggest that these colospinal afferent neurons (CANs) might be involved in nociception. Whether sensory information from CANs is perceived by the animal or is part of the parasympathetic reflex is currently not known.     

Tyrosine hydroxylase-positive GABAergic juxtaglomerular neurons are the main source of the interglomerular connections in the mouse main olfactory bulb

The interglomerular connections in the mouse olfactory bulb were examined with the retrograde-tracer experiments using Fluorogold. When the injections were restricted to the glomerular layer, we encountered tracer-labeled cells in the glomerular layer and the superficial part of the external plexiform layer, not only near the injection sites but also more than 500 microm distant from the injection sites. Almost of those tracer-labeled neurons distant from the injection sites were large tyrosine hydroxylase-positive juxtaglomerular neurons, some of which were confirmed to have intraglomerular dendrites. Thus, the long interglomerular connections were mainly made by a particular type of dopaminergic-GABAergic juxtaglomerular neurons.     

Projections from the hippocampal and parahippocampal regions to the entorhinal cortex. An anterograde and retrograde tract-tracing study in the cat

Projections from the hippocampal and parahippocampal regions to the entorhinal cortex (EC) were examined in the cat by anterograde and retrograde tract-tracing with Phaseolus vulgaris leucoagglutinin and cholera toxin B subunit. CA1 fibers to EC were distributed more densely in the medial EC than in the lateral EC; these were seen in all EC layers, but most densely in layers II and III. The septotemporal axis of the area of origin of CA1-EC fibers corresponded to a caudal-to-rostral axis of the area of their termination in the EC. CA2 and CA4 also sent a small number of fibers to the EC. The subiculum sent fibers mainly to the lateral EC; more densely to layers IV-VI than to layers I-III. The septotemporal axis of the area of origin of subiculum-EC fibers corresponded to a caudolateral-to-rostromedial axis of their termination in the EC. Distribution pattern of fibers from the prosubiculum regions close to CA1 or from prosubiculum regions close to the subiculum was similar to that of CA1 fibers or subiculum fibers, respectively. The presubiculum sent fibers mainly to the medial EC; most densely to layers I and III. The parasubiculum sent fibers mainly to the medial EC; most densely to layer II. Fibers to the contralateral EC were detected only from the presubiculum; they originated from the superficial layers and terminated in layer III of the medial entorhinal area.     

Trigeminal nociceptive neurons in the subnucleus reticularis ventralis. I. Response properties and afferent connections.

Trigeminal nociceptive neurons within the subnucleus reticularis ventralis medullae oblongatae (SRV), which lies ventral to the trigeminal subnucleus caudalis and subnucleus reticularis dorsalis medullae oblongatae, were studied in urethane/chloralose-anesthetized cats and monkeys. These neurons were called 'SRV neurons'. They were almost regularly excited by pressure to the ipsilateral cornea or to both corneas at a strength well above the human corneal pain threshold. Most of them were activated by noxious mechanical stimulation of the pinna, face and/or tongue. A significant fraction of SRV units was responsive to tapping of the ipsilateral dorsum of the nose and/or electrical stimulation of tooth pulp afferents. Evidence was obtained that responses to tapping of the dorsum of the nose were due to mechanical distortion of the nasal mucosa. Intracellular injection of HRP into SRV neurons demonstrated that injected neurons were large neurons characteristic of the SRV. Trigeminal tractotomy just rostral to the obex did not eliminate responses of SRV units to trigeminal inputs. Neurons relaying trigeminal inputs to SRV neurons were electrophysiologically identified in the nucleus reticularis parvocellularis which is ventromedially adjacent to the subnuclei oralis and interpolaris of the trigeminal spinal tract nucleus. These findings were supported by HRP injection into the SRV. Units having receptive fields similar to those of SRV neurons were found in lamina VII of the first cervical cord, suggesting that SRV neurons may be trigeminal lamina VII neurons.     

Quantitative and immunohistochemical analysis of neuronal types in the mouse caudal nucleus tractus solitarius: focus on GABAergic neurons

γ-Aminobutyric acid-ergic (GABAergic) neurons are major inhibitory interneurons that are widely distributed in the central nervous system. The caudal nucleus tractus solitarius (cNTS), which plays a key role in respiratory, cardiovascular, and gastrointestinal function, contains GABAergic neurons for regulation of neuronal firing. In the present study, GABAergic neuronal organization was analyzed in relation to the location of subnuclei in the mouse cNTS. According to the differential expression of glutamate decarboxylase 67 (GAD67), vesicular glutamate transporter 2 (VGLUT2), calbindin, and tyrosine hydroxylase (TH) mRNAs, the cNTS was divided into four subnuclei: the subpostrema, dorsomedial, commissural, and medial subnuclei. The numerical density and size of soma in the four subnuclei were then quantified by an unbiased disector analysis. Calbindin-positive cells constituted subpopulations of small non-GABAergic neurons preferentially localized in the subpostrema subnucleus. TH-positive cells constituted large neurons preferentially localized in the medial subnucleus. GABAergic neurons constituted a subpopulation of small neurons, preferentially localized in the commissural and medial subnuclei, which represented ≥50% of small cells in these subnuclei. Thus, the GABAergic small neurons were located around TH-positive large cells in the ventrolateral portion of the cNTS. This finding, in combination with results of previous studies in the rat cNTS showing that large cells originate efferents from the cNTS, suggests that GABAergic small neurons in the commissural and medial subnuclei might regulate output from the cNTS.     

Fluoro-Gold tracing of zinc-containing afferent connections in the mouse visual cortices

Summary To identify zinc-containing projections to the visual areas, we injected Fluoro-Gold into the occipital cortex of the mouse. Five days later, the mice underwent an intravital selenium-labeling procedure to demonstrate the somata of neurons that give rise to zinc-containing boutons. Numerous double-labeled cells were seen in the ipsi- and contralateral primary (layers II/III and VI), and secondary visual cortices (layers II/III and VI). A few double-labeled cells were apparent in other cortical areas concerned with visual processing: the orbital cortex (layers II and III), the posterior portion of the medial agranular frontal cortex (layer V/VI border), and the temporal cortex (layer VI). The cingulate, retrosplenial, perirhinal, and lateral entorhinal cortices had lamina projecting to the visual cortex and separate lamina harboring zinc-containing cells. A spatial segregation of fluorescent and zinc-containing neurons was also seen in the claustrum. This integration or segregation of projecting and zinc-containing neurons may reflect the function of the cortical areas. N-methyl-d-aspartate receptor function is antagonized by physiological concentrations of zinc in vitro. It is proposed that zinc-positive projections from areas that perform basic visual functions are less likely to be modified by N-methyl-d-aspartate receptor-mediated processes than the zinc-negative connections from associational areas.     

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.