Septal GABAergic neurons innervate inhibitory interneurons in the hippocampus of the macaque monkey.

The septohippocampal projection was visualized in three Macaca mulatta monkeys by anterograde transport of Phaseolus vulgaris leucoagglutinin. Following injections of the lectin into the medial septal nucleus, P. vulgaris leucoagglutinin-labelled fibres were found in the hippocampal complex, mainly in stratum oriens of the CA1 subfield, throughout the CA3 subfield, and in the hilus and stratum moleculare of the dentate gyrus. The majority of labelled axons were varicose, and formed multiple contacts with cell bodies and dendrites of calbindin D28k- and parvalbumin-immunoreactive non-pyramidal cells. GABA immunoreactivity of P. vulgaris leucoagglutinin-labelled axons and their postsynaptic targets was investigated by sectioning varicose axon segments for correlated light and electron microscopy, and processing alternate ultrathin sections for postembedding immunogold staining for GABA. All P. vulgaris leucoagglutinin-labelled boutons examined were GABA-immunoreactive and the majority of them formed symmetrical synapses with GABA-immunoreactive cell bodies and dendrites. The results demonstrate that a GABAergic septohippocampal pathway exists in the monkey, and, similar to the rat, terminates on different types of GABAergic neurons, including the parvalbumin- and calbindin D28k-containing non-pyramidal cells.     

Quantitative immunogold analysis reveals high glutamate levels in retinal and cortical synaptic terminals in the lateral geniculate nucleus of the macaque

An immunogold procedure has been used on ultrathin sections of the parvo- and magnocellular layers of the dorsal lateral geniculate of the rhesus monkey to estimate quantitatively at the electron microscopic level the intensity of immunoreactivity to an antibody against glutamate over profiles of retinal, cortical, GABAergic synaptic terminals and glial cells. GABAergic terminals were identified directly by immunogold reactivity to a GABA antibody or by ultrastructural features. The results showed that in both of the main subdivisions of the geniculate the densities of immunogold particles over cortical and retinal terminals were about two- to three-fold higher than those over GABAergic terminals or glial profiles. In addition, cortical and retinal terminals showed higher positive correlations of glutamate immunogold particle densities to synaptic vesicle densities than did GABAergic terminals. These differences suggest higher and lower concentrations of glutamate corresponding to transmitter and metabolic pools of this amino acid in axon terminals of retinal and cortical origins versus GABAergic terminals, respectively, in the dorsal lateral geniculate nucleus of the macaque.     

Changes in excitatory and inhibitory circuits of the rat hippocampus 12-14 months after complete forebrain ischemia.

Changes in interneuron distribution and excitatory connectivity have been investigated in animals which had survived 12-14 months after complete forebrain ischemia, induced by four-vessel occlusion. Anterograde tracing with Phaseolus vulgaris leucoagglutinin revealed massive Schaffer collateral input even to those regions of the CA1 subfield where hardly any surviving pyramidal cells were found. Boutons of these Schaffer collaterals formed conventional synaptic contacts on dendritic spines and shafts, many of which likely belong to interneurons. Mossy fibres survived the ischemic challenge, however, large mossy terminals showed altered morphology, namely, the number of filopodiae on these terminals decreased significantly. The entorhinal input to the hippocampus did not show any morphological alterations. The distribution of interneurons was investigated by neurochemical markers known to label functionally distinct GABAergic cell populations. In the hilus, spiny interneurons showed a profound decrease in number. This phenomenon was not as obvious in CA3, but the spiny metabotropic glutamate receptor 1alpha-positive non-pyramidal cells, some of which contain calretinin or substance P receptor, disappeared from stratum lucidum of this area. In the CA1 region, somatostatin immunoreactivity disappeared from stratum oriens/lacunosum-moleculare-associated cells, while in metabotropic glutamate receptor 1alpha-stained sections these cells seemed unaffected in number. Other interneurons did not show an obvious decrease in number. In stratum radiatum of the CA1 subfield, some interneuron types had altered morphology: the substance P receptor-positive dendrites lost their characteristic radial orientation, and the metabotropic glutamate receptor 1alpha-expressing cells became extremely spiny. The loss of inhibitory interneurons at the first two stages of the trisynaptic loop coupled with a well-preserved excitatory connectivity among the subfields suggests that hyperexcitability in the surviving dentate gyrus and CA3 may persist even a year after the ischemic impact. The dorsal CA1 region is lost; nevertheless hyperactivity, if it occurs, may have a route to leave the hippocampus via the longitudinally extensive axon collaterals of CA3 pyramidal cells, which may activate the subiculum and entorhinal cortex with a relay in the surviving ventral hippocampal CA1 region.     

Postsynaptic targets of somatostatin-immunoreactive interneurons in the rat hippocampus

Two characteristic interneuron types in the hippocampus, the so-called hilar perforant path-associated cells in the dentate gyrus and stratum oriens/lacunosum-moleculare neurons in the CA3 and CA1 regions, were suggested to be involved in feedback circuits. In the present study, interneurons identical to these cell populations were visualized by somatostatin-immunostaining, then reconstructed, and processed for double-immunostaining and electron microscopy to establish their postsynaptic target selectivity. A combination of somatostatin-immunostaining with immunostaining for GABA or other interneuron markers revealed a quasi-random termination pattern. The vast majority of postsynaptic targets were GABA-negative dendritic shafts and spines of principal cells (76%), whereas other target elements contained GABA (8%). All of the examined neurochemically defined interneuron types (parvalbumin-, calretinin-, vasoactive intestinal polypeptide-, cholecystokinin-, substance P receptor-immunoreactive neurons) received innervation from somatostatin-positive boutons. Recent anatomical and electrophysiological data showed that the main excitatory inputs of somatostatin-positive interneurons originate from local principal cells. The present data revealed a massive GABAergic innervation of distal dendrites of local principal cells by these feedback driven neurons, which are proposed to control the efficacy and plasticity of entorhinal synaptic input as a function of local principal cell activity and synchrony.     

Nitric oxide-containing pyramidal neurons of the subiculum innervate the CA1 area

Neurons and axon terminals containing neuron-specific nitric oxide synthase (nNOS) were examined in the rat subiculum and CA1 area of Ammon's horn. In the subiculum, a large subpopulation of the pyramidal neurons and non-pyramidal cells are immunoreactive for nNOS, whereas in the neighbouring CA1 area of Ammon's horn only non-pyramidal neurons are labelled with the antibody against nNOS. In the pyramidal layer of the subiculum, nNOS-positive axon terminals form both asymmetric and symmetric synapses. In the adjacent CA1 area the nNOS-positive terminals that form symmetric synapses are found in all layers, whereas those terminals that form asymmetric synapses are only in strata radiatum and oriens, but not in stratum lacunosum-moleculare. In both the subiculum and CA1 area, labelled terminals make symmetric synapses only on dendritic shafts, whereas asymmetric synapses are exclusively on dendritic spines. Previous observations demonstrated that all nNOS-positive non-pyramidal cells are GABAergic local circuit neurons, which form exclusively symmetric synapses. We suggest that nNOS-immunoreactive pyramidal cells of the subiculum may innervate neighbouring subicular pyramidal cells and, to a smaller extent, pyramidal cells of the adjacent CA1 area, forming a backward projection between the subicular and hippocampal principal neurons. Electronic Publication     

Immunoelectron microscopic study of γ-aminobutyric acid inputs to identified thalamocortical projection neurons in the anterior thalamus of the rat

 We have carried out a semi-quantitative ultrastructural study to determine the characteristics and distribution of γ-aminobutyric acid (GABA)-containing constituents of the anterodorsal (AD) and anteroventral (AV) thalamic nuclei in adult rats. We used a polyclonal antibody to GABA and a postembedding immunogold detection method in animals in which the cortical projection neurons of these nuclei had been labelled by retrograde transport of cholera toxin/horseradish peroxidase (HRP) injected into the retrosplenial granular cortex. Two types of GABA-immunopositive structures were identified, with gold particle densities 4–40 times higher than the highest densities over blood-vessel lumens and areas of empty resin: (1) an apparently homogeneous population of axon terminals with Gray type-2 (symmetric) synaptic contacts corresponding to F-axon terminals; and (2) small–medium sized myelinated axons scattered individually or in small groups within the neuropil which may be their parent axons. These axons and terminals may originate from the ipsilateral thalamic reticular nucleus; others may arise from the basal forebrain or brainstem. The GABA-immunopositive terminals comprised approximately 16% of all axon terminal profiles in AD and 12% in AV, a significant difference. However, because the immunoreactive axon terminals in AD were significantly larger than those in AV (1.09±0.47 μm² vs 0.90±0.43 μm²) and would therefore be encountered more frequently, it is not possible to conclude that the GABAergic innervation of AD is heavier than that of AV. The GABA-positive terminals established synaptic contacts with cell bodies and dendrites of all sizes (some of which were HRP-labelled) with the following frequency distribution (AD/AV, no significant difference): somata 5%/7%; large dendrites (≥1.5 μm) 14%/9%; medium dendrites (1.00–1.49 μm) 35%/45% and small dendrites (<1 μm) 46%/40%. Despite evidence from previous studies, we found no evidence in this study for the presence of GABAergic interneurons or for GABA-containing projection neurons in AD or AV. Received: 4 March 1998 / Accepted: 15 January 1999     

Glutamic acid decarboxylase isoforms are differentially distributed in the septal region of the rat

The septal region of the brain consists of a heterogeneous population of GABAergic neurons that play an important role in the generation of hippocampal theta rhythms. While GABAergic neurons employ two isoforms of the enzyme glutamic acid decarboxylase (GAD) for the synthesis of GABA, distribution of GAD isoforms has not been investigated in the septum. Immunohistochemical techniques were used to investigate the expression of GAD enzymes in medial and lateral septum. GAD65 and GAD67 immunohistochemistry revealed dense fibers and punctuated immunoreactivity in septal regions. While few GAD65-positive neuronal somas were detected in medial septum, a significantly higher number of immunoreactive neurons were detected in lateral septum. GAD65- and GAD67-positive neurons in the lateral septum exhibit higher complexity of dendritic arborizations than in the medial septum where staining was mainly restricted to the soma. Presumptive axon terminals (puncta) showed abundant immunoreactivity predominantly for GAD65 isoforms in all septal regions. This suggests that septal GABAergic neurons differentially express GAD enzymes thereby potentially reflecting functional differences. Differences found between medial and lateral septal GABAergic neuronal populations are in agreement with the concept that medial and lateral septum are brain structures with highly different connectivity and function despite anatomical proximity.     

Comparative Ultrastructural Analysis of Mitochondria in the CA1 and CA3 Hippocampal Pyramidal Cells Following Global Ischemia in Mongolian Gerbils

Post‐ischemic injury of the hippocampus unrolls at different levels and has both functional and structural implications. The deficiency in neuron energy metabolism is an initiating factor. We performed transmission electron microscopic (TEM) comparative analysis of mitochondria in excitatory spine synapses in CA1 stratum radiatum and CA3 hippocampal areas after 5 min of global cerebral ischemia in Mongolian gerbils, 4 and 7 days after reperfusion. Electron microscopy and unbiased morphometric methods were used to evaluate synaptic plasticity, and the number and size of mitochondria in synaptic terminals. We compared the morphological organization of mitochondria in presynaptic terminals between CA1 and CA3 areas in control and post‐ischemic condition according to the following morphometric parameters: mitochondrial volume fraction, mitochondrial frequency in CA1 and CA3 terminals, mean number of mitochondria per presynaptic terminal, frequency of damaged mitochondria in terminals, and density of presynaptic terminals. Our ultrastructural study revealed statistically significant differences in morphometric parameters between CA1 and CA3 areas in control conditions, as well as in post‐ischemic conditions. Also, we found temporal differences in measured parameters obtained 4 and 7 days after reperfusion. This study showed significant morphological differences in the organization of mitochondria in excitatory spine synapses between CA1 and CA3 areas, which corresponded with already known differences in functionality and sensitivity to the ischemic insult. Our conclusion is that revealed post‐ischemic changes in mitochondrial distribution in presynaptic CA1 and CA3 terminals could be an indicator of hippocampal metabolic dysfunction and synaptic plasticity. Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.     

Ultrastructural double localization of B-50/GAP43 and synaptophysin (p38) in the neonatal and adult rat hippocampus

Summary B-50/GAP43, a neuron-specific phosphoprotein, is highly expressed in developing nervous tissue. Monospecific polyclonal affinity-purified B-50 antibodies were used to document the ultrastructural distribution of B-50 in the hippocampus of 90-day-old (P90) and 1-day-old (P1) rats. Double-labelling immunoprocedures were performed to compare the localization of B-50 and synaptophysin (p38), a protein specific for synaptic vesicles. By immunofluorescence light microscopy B-50 and p38 were similarly distributed in the CA1 neuropil of P90 rats. In contrast, in P1 rats B-50 was more widely distributed than p38. By electron microscopy of P90 rat hippocampus, B-50 was located at the plasma membranes of axon shafts and of p38-immunoreactive axon terminals. Some B-50 was found in the cytosol of axon terminals. B-50 was absent at the plasma membranes of apical dendrites and of pyramidal cells. In the P1 rat hippocampus, B-50 was detected at the plasma membrane of growth cones, axon terminals and axon shafts, but not in their cytosol. The plasma membranes of pyramidal cell bodies and their processes extending into the stratum radiatum were without B-50. B-50-immunoreactive organelles of the lysosomal family were found in the cytosol of pyramidal cells of the hippocampus of P1 and P90 rats. This ultrastructural study shows that during development of the stratum radiatum in the hippocampal field CA1, the localization of B-50 persists at the plasma membrane of axons and axon terminals in P1 and P90 rats. This localization of B-50 is consistent with the suggestion that B-50 acts as a regulator of neurotransmitter release and intracellular messengers.     

Tetanus neurotoxin-insensitive vesicle-associated membrane protein localizes to a presynaptic membrane compartment in selected terminal subsets of the rat brain

Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicular soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) that has been implicated in neurite outgrowth. It has previously been reported that TI-VAMP is localised in the somatodendritic compartment of neurons indicating a role in membrane fusion events within dendrites. Using a newly produced monoclonal antibody to TI-VAMP that improves signal/noise immunodetection, we report that TI-VAMP is also present in subsets of axon terminals of the adult rat brain. Four distinctive populations of labelled axon terminals were identified: 1) the hippocampal mossy fibres of the dentate gyrus and of CA3, 2) the striatal peridendritic terminal plexuses in the globus pallidus (GP), substantia nigra pars reticulata (SNr), 3) peridendritic plexuses in the central nucleus of the amygdala, and 4) the primary sensory afferents in the dorsal horn of the spinal cord. The presynaptic localisation of TI-VAMP in these locations was demonstrated by co-localisation with synaptophysin. Ultrastructural studies showed TI-VAMP labelling over synaptic vesicles in the mossy fibres, whereas it was localised in tubulo-vesicular structures and multivesicular bodies in the pyramidal cell dendrites. The presynaptic localisation of TI-VAMP occurred by P15, so relatively late during development. In contrast, dendritic labelling was most prominent during the early post-natal period. Co-localisation with markers of neurotransmitters showed that TI-VAMP-positive terminals are GABAergic in the GP and SNr and glutamatergic in the mossy fibre system and in the dorsal root afferents. Most of these terminals are known to co-localise with neuropeptides. We found met-enkephalin-immunoreactivity in a sizeable fraction of the TI-VAMP positive terminals in the GP, amygdala, and dorsal horn, as well as in a few mossy fibre terminals. The function of TI-VAMP in subsets of mature axon terminals remains to be elucidated; it could participate in the exocytotic molecular machinery and/or be implicated in particular growth properties of the mature axon terminals. Thus, the presence of TI-VAMP in the mossy fibres may correspond to the high degree of plasticity that characterises this pathway throughout adult life.     

Glutamatergic components of the retrosplenial granular cortex in the rat

The ultrastructural characteristics, distribution and synaptic relationships of identified, glutamate-enriched thalamocortical axon terminals and cell bodies in the retrosplenial granular cortex of adult rats is described and compared with GABA-containing terminals and cell bodies, using postembedding immunogold immunohistochemistry and transmission electron microscopy in animals with injections of cholera toxin- horseradish peroxidase (CT-HRP) into the anterior thalamic nuclei. Anterogradely labelled terminals, identified by semi-crystalline deposits of HRP reaction product, were approximately 1 microm in diameter, contained round, clear synaptic vesicles, and established asymmetric (Gray type I) synaptic contacts with dendritic spines and small dendrites, some containing HRP reaction product, identifying them as dendrites of corticothalamic projection neurons. The highest densities of immunogold particles following glutamate immunostaining were found over such axon terminals and over similar axon terminals devoid of HRP reaction product. In serial sections immunoreacted for GABA, these axon terminals were unlabelled, whereas other axon terminals, establishing symmetric (Gray type II) synapses were heavily labelled. Cell bodies of putative pyramidal neurons, containing retrograde HRP label, were numerous in layers V-VI; some were also present in layers I-III. Most were overlain by high densities of gold particles in glutamate but not in GABA immunoreacted sections. These findings provide evidence that the terminals of projection neurons make synaptic contact with dendrites and dendritic spines in the ipsilateral retrosplenial granular cortex and that their targets include the dendrites of presumptive glutamatergic corticothalamic projection neurons.     

The GABA-immunoreactive neurons in the interlaminar regions of the cat lateral geniculate nucleus: light and electron microscopic observations

Summary GABA-immunoreactive cells located in the interlaminar zone between the A and A1 laminae of cat LGN were studied at the LM and EM levels. The mean perikaryal size of these neurons was larger than that of GABA-immunoreactive cells in the A-laminae of LGN. Interlaminar GABA+ cells examined in plastic semithin sections of LGN after massive injections of HRP in the striate and extrastriate visual cortex were not retrogradely labeled with reaction products (as previously reported for the GABA+ cells in the laminar regions of LGN) suggesting that these cells do not project to the visual cortex. Serial EM analysis of two partially reconstructed interlaminar GABA+ cells showed that they receive synaptic inputs from RLD terminals of axon collaterals of geniculo-cortical relay cells, from cortical (RSD) terminals, from inhibitory (F) axon terminals, and from other undetermined terminals, but not from retinal (RLP) axon terminals. These data suggest that the GABAergic cells in the interlaminar zones of LGN participate as interneurons in recurrent inhibitory circuits in LGN. The synaptic inputs to these cells and ultrastructural features, notably somatic spines and dendrites oriented predominantly orthogonal to the projection lines in LGN, are similar to those of neurons of the perigeniculate nucleus.     

包埋前免疫电镜双标技术在神经解剖学研究中的应用

目的　在超微结构水平观察两种神经递质在纤维终末内的共存或一种神经递质与其相应受体之间的关系。　方法　包埋前免疫电镜双重标记技术———酶标法和免疫金 银标记法相结合的方法。　结果　在免疫反应双重标记的纹状体切片上 ,电镜下观察到大量的SP样 (过氧化物酶免疫反应产物 )阳性终末和SP受体 (SPR ,免疫金 银标记颗粒 )样阳性神经元的胞体和树突 ,同时可见部分SP样阳性轴突终末分别与SPR样阳性神经元的胞体或树突形成对称性轴 体或轴 树突触联系。而在三叉神经脊束核尾侧亚核切片上 ,电镜下可观察到大量的两种囊泡膜谷氨酸转运体 ,即DNPI样 (过氧化物酶免疫反应产物 )和VGluT1样 (免疫金 银标记颗粒 )阳性轴突终末 ,同时还观察到DNPI样和VGluT1样双标的轴突终末与阴性树突形成非对称性突触。　结论　包埋前免疫电镜双重标记技术敏感性较高 ,组织的抗原性保存好 ,特别是在神经解剖学研究中 ,用于研究两种神经递质在同一个细胞或终末内的共存或分析神经递质与其相应受体之间的联系中有独到之处。     

Postsynaptic-GABAergic inhibition of non-pyramidal neurons in the guinea-pig hippocampus

Intracellular recording and staining was applied to study non-pyramidal neurons in the guinea-pig hippocampus. To avoid accidental impalement of pyramidal or granule cells, two hippocampal regions known to be devoid of pyramidal or granule cells were chosen. In transverse and longitudinal slices, neurons of the deep hilar region (zone 4 of Amaral3), and in transverse slices, neurons of the stratum lacunosum-moleculare (CA3) were impaled. The intracellular staining with Lucifer Yellow revealed that of 20 neurons stained in these zones all were non-pyramidal neurons. Hilar neurons, situated just below the granular layer, differed from granule cells and CA3 neurons with respect to their action potential waveform and their current/voltage relationship. In contrast to granule cells, hilar neurons exhibited spontaneous bursts in the presence of bicuculline (25 microM). In all neurons impaled in the hilar region and the stratum lacunosum-moleculare (n = 42), inhibitory postsynaptic potentials could be elicited. These inhibitory postsynaptic potentials were blocked by bicuculline. In transverse slices, perforant path stimulation elicited inhibition preceding excitation in hilar neurons and excitation preceding inhibition in granule cells. Since non-pyramidal neurons are likely to be inhibitory neurons, our data suggest that GABAergic neurons in the hilus or in the stratum lacunosum-moleculare are controlled by inhibitory GABAergic synapses. This was verified by immunocytochemistry using antibodies against glutamate decarboxylase, the gamma-aminobutyric acid synthetizing enzyme. In both hippocampal regions studied, glutamate decarboxylase-positive synaptic terminals on glutamate decarboxylase-positive cells were observed. It is concluded that disinhibition is an important feature of information processing in the hippocampus, and that disinhibition is mediated by GABAergic synapses on GABAergic neurons.     

A study of the rat septohippocampal pathway using anterograde transport of horseradish peroxidase

The projection of the septohippocampal pathway in the rat was studied using anterograde transport of horseradish peroxidase. This technique provides a number of advantages over other methods including the ability to differentiate between terminal and preterminal axon labeling, a very high ‘signal to noise’ ratio, and a short delay in obtaining results. As applied to the septohippocampal projection, anterograde transport of horseradish peroxidase reveals a dense septal input to the dentate hilus and stratum oriens of CA3, a modest input to the dentate molecular layer and stratum radiatum of CA3, and a very sparse input to stratum oriens and stratum lacunosum-moleculare of CA1 with most labeling in this field confined to axons passing through it. In addition, our results suggest that a septal projection to the supragranular region of the dentate is present only within the rostral pole of the hippocampal formation. Potential artifacts such as labeling of fibers-of-passage and ‘collateral’ filling do not appear to interfere with the results but transneuronal transport of horseradish peroxidase may occur when large amounts of the protein are injected.     

Ultrastructural distribution of glycinergic and GABAergic neurons and axon terminals in the rat dorsal cochlear nucleus,with emphasis on granule cell areas

A knowledge of neurotransmitters in the neurons of the rat cochlear nuclear complex is of importance in understanding the function of auditory circuits. Using post-embedding ultrastructural immunogold labelling, the distribution of glycinergic and GABAergic neurons and axonal terminals has been studied in the molecular, fusiform and polymorphic layers of the rat dorsal cochlear nucleus (DCN). This technique is not limited by the penetration of antibodies into the nervous tissue as in pre-embedding methods, and allows a fine neurochemical mapping of the nervous tissue. Numerous glycinergic and GABAergic axon terminals contain pleomorphic and flat synaptic vesicles, and are present in all layers (1, 2, 3) of the dorsal cochlear nucleus. Glycine and GABA-negative large terminals (mossy fibres) are mainly seen in granule cell areas of layer 2 (fusiform layer). Mossy fibres contact the dendrites of GABA- and glycine-negative granule cells and of the few unipolar brush cells (excitatory neurons). The least common cells in the granule cell areas are GABAergic and glycinergic Golgi-stellate neurons. In unipolar brush cells, aggregations of vesicles seem to be the origin of their characteristic ringlet-bodies. Golgi-stellate cells send their inhibitory terminals to the dendrites of granule and unipolar brush cells, occasionally directly to mossy fibres. Small or (less frequently) large GABAergic terminals contact the soma or the main dendrite of unipolar brush cells. The circuit of a hypothetical functional unit of neurons in the DCN is proposed. The inputs from auditory tonotopic or non-auditory non-tonotopic mossy fibres eventually reach pyramidal cells through axons from the granule cells or unipolar brush cells. Pyramidal cells convey an excitatory signal from the DCN to higher mesencephalic nuclei for further elaboration of the acoustic signal.     

Electron microscopic study of GABA-immunoreactive neuronal processes in the superficial gray layer of the rat superior colliculus: their relationships with degenerating retinal nerve endings

Summary GABA-immunoreactive neuronal elements were detected in the stratum griseum superficiale or superficial gray layer of the rat superior colliculus in an electron microscopic study, using postembedding immunocytochemistry with protein A-gold as a marker. In addition to neuronal somata, two types of GABA-immunoreactive neuronal processes were observed. Numerous profiles of axon terminals (1 m in diameter) with clear round or pleomorphic synaptic vesicles and mitochondria were found to establish mostly symmetrical synaptic contacts with GABA-immunonegative dendrites of various diameters. Some axosomatic synapses could also be observed. The gold particle density in this axon terminal compartment was between seven and 13 times the background level. The stratum griseum superficiale also included GABA-immunoreactive dendrites, some of which contained clear synaptic vesicles. These dendritic profiles always formed the presynaptic component of dendrodendritic synaptic contacts. The density of the gold particles in the dendritic compartment, taken as a whole, was between three and 13 times the background level. Furthermore, the relationship between the GABA-immunoreactive neuronal elements and degenerating retinal nerve endings identified in the left stratum griseum superficiale following enucleation of the right eye was investigated after a 7-day survival period. The profiles of degenerating retinal nerve endings (0.7 m in diameter) were found to be devoid of any specific labelling. Most of the retinal boutons established axodendritic synapses of the asymmetrical type with an immunonegative dendrite, which was also contacted in some cases by a GABA-immunopositive axon terminal. Other retinal endings were presynaptic to GABA-immunopositive dendritic profiles with synaptic vesicles, some of which were found to contact in turn an unlabelled dendrite, thereby completing serial synaptic relationships. More rarely, retinal endings formed the presynaptic component of possible axoaxonic synapses with GABA-positive terminals presumed to be axonic in nature. It can be concluded that the retinal input to the superficial gray layer often converges with a GABAergic axonal input on a dendritic target, the neurotransmitter specificity of which is unknown. In other cases, retinal terminals synaptically contact GABA-immunolabelled conventional and presynaptic dendrites and probably also some axon terminals; this might provide an anatomical substrate for the control of GABA release from these GABAergic processes. These results indicate that transmitter GABA plays an important role in retinocollicular transmission.     

Fine structures of nerve fibers with corticotropin-releasing factor-like immunoreactivity in the rat lateral septum

The fine structures of nerve fibers with corticotropin-releasing factor (CRF)-like immunoreactivity in the rat lateral septum were investigated by means preembedding immunoelectron microscopy. A number of CRF axon terminals formed synapses with cell bodies of non-immunoreactive septal neurons. They occasionally had broad terminal bulges whose subregions showed little or no immunoreactivity for CRF. CRF axon terminals were also in synaptic contact with non-immunoreactive dendrites or dendritic spines. Some dendrites with CRF were postsynaptic to non-immunoreactive axon terminals.     

Mu-opioid receptors facilitate the propagation of excitatory activity in rat hippocampal area CA1 by disinhibition of all anatomical layers

Hippocampal mu-opioid receptors (MORs) have been implicated in memory formation associated with opiate drug abuse. MORs modulate hippocampal synaptic plasticity acutely, when chronically activated, and during drug withdrawal. At the network level, MORs increase excitability in area CA1 by disinhibiting pyramidal cells. The precise inhibitory interneuron subtypes affected by MOR activation are unknown; however, not all subtypes are inhibited, and specific interneuron subtypes have been shown to preferentially express MORs. Here we investigate, using voltage-sensitive dye imaging in brain slices, the effect of MOR activation on the patterns of inhibition and on the propagation of excitatory activity in rat hippocampal CA1. MOR activation augments excitatory activity evoked by stimulating inputs in stratum oriens [i.e., Schaffer collateral and commissural pathway (SCC) and antidromic], stratum radiatum (i.e., SCC), and stratum lacunosum-moleculare (SLM; i.e., perforant path and thalamus). The augmented excitatory activity is further facilitated as it propagates through the CA1 network. This was observed as a proportionately larger increase in amplitudes of excitatory activity at sites distal from where the activity was evoked. This facilitation was observed for excitatory activity propagating from all three stimulation sites. The augmentation and facilitation were prevented by GABAA receptor antagonists (bicuculline, 30 microM), but not by GABAB receptor antagonists (CGP 55845, 10 microM). Furthermore, MOR activation inhibited IPSPs in all layers of area CA1. These findings suggest that MOR-induced suppression of GABA release onto GABAA receptors augments all inputs to CA1 pyramidal cells and facilitates the propagation of excitatory activity through the network of area CA1.