GABAergic elements in the neuronal circuits of the monkey neostriatum: a light and electron microscopic immunocytochemical study

An antibody raised in rabbits against a GABA-BSA conjugate was used together with the PAP technique to label elements in the neostriatum of three Old World monkeys. Light microscopy revealed numerous immunoreactive medium-size neurons of various staining intensities, some of which had indented nuclei, as well as an occasional large cell. The neuropil showed a plexus of fine processes with frequent puncta. Ultrastructurally, the medium-size GABA-positive neurons were of two types: one with smooth nuclei and scanty cytoplasm, similar to spiny I cells, the other with invaginated nuclear envelopes and more abundant perikaryon, resembling the aspiny type. Correspondingly, labeled dendrites were either spiny or varicose. Some stained axons were myelinated, and the boutons had either large and ovoid, or small and pleomorphic vesicles. All of these boutons formed symmetric synapses, the former type with GABA-positive dendritic shafts but also with unlabeled dendrites; the latter type usually with GABA-negative elements, either dendrites, dendritic spines, or somata. Synapses were also observed between unreactive boutons and immunostained dendrites. Terminals with densely packed, small round vesicles that established asymmetric synapses with spines were always GABA-negative. Glial elements were consistently unlabeled, save for some astroglial endfeet. These findings provide positive evidence for the existence of two classes of GABAergic striatal neurons corresponding to a long-axoned spiny I type and an aspiny interneuron. Furthermore, the simultaneous labeling of GABA-immunoreactive presynaptic and postsynaptic profiles offers possible morphologic bases for the various kinds of intrastriatal inhibitory processes, including the feedforward, feedback, and "autaptic" types.     

GABAergic circuitry in the dorsal division of the cat medial geniculate nucleus

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the thalamus. We used postembedding immunocytochemistry to examine the synaptic organization of GABA-positive profiles in the dorsal superficial subdivision of the cat medial geniculate nucleus (MGN). Three groups of GABA-positive profiles participate in synapses: axon terminals, dendrites, and presynaptic dendrites. The presynaptic GABA-positive terminals target mainly GABA-negative dendrites. The GABA-positive postsynaptic profiles receive input primarily from GABA-negative axons. The results indicate that the synaptic organization of GABA-positive profiles in the dorsal superficial subdivision of the MGN nucleus is very similar to that in other thalamic nuclei.     

The ultrastructural localization of enkephalin and substance P immunoreactivities in the nucleus tractus solitarii of the cat

In the medial and commissural subdivisions of the nucleus tractus solitarii enkephalin and substance P immunoreactivities were localized within synaptic terminals, unmyelinated axons, and neuronal cell bodies. Both enkephalin and substance P immunoreactivities were contained within synaptic terminals which had a mixture of small clear vesicles and dense core vesicles. The presence of dense core vesicles within both the enkephalin- and substance P-immunoreactive terminals was a consistent feature, although they were not associated with the actual synaptic junction. While enkephalin- and substance P-immunoreactive terminals shared a similar morphology, their respective distributions along the dendritic tree were quite distinct. Enkephalin-immunoreactive terminals contacted mainly the cell body and proximal portions of the dendritic tree. In contrast, substance P-immunoreactive terminals synapsed predominantly with spines and shafts of small to medium-sized dendrites. Few substance P-immunoreactive terminals contacted proximal dendrites and they were never presynaptic to the neuronal cell body. This apparent segregation of synaptic terminals on neurons suggests that enkephalin synapses have a more pronounced effect than substance P terminals.     

Targets and Quantitative Distribution of GABAergic Synapses in the Visual Cortex of the Cat

The morphology and postsynaptic targets of GABA-containing boutons were determined in the striate cortex of cat, using a postembedding immunocytochemical technique at the electron microscopic level. Two types of terminals, both making symmetrical synaptic contacts, were GABA-positive. The first type (95% of all GABA-positive boutons) contained small pleomorphic vesicles, the second type (5%) contained larger ovoid vesicles. Furthermore, 99% of all cortical boutons containing pleomorphic vesicles were GABA positive, and all boutons with pleomorphic vesicles made symmetrical synaptic contacts. These results together with previously published stereological data (Beaulieu and Colonnier, 1985, 1987) were used to estimate the density of GABA-containing synapses, which is about 48 million/mm3 in the striate cortex. The postsynaptic targets of GABA positive boutons were also identified and the distribution was calculated to be as follows: 58% dendritic shafts, 26.4% dendritic spines, 13.1% somata and 2.5% axon initial segments. A total of 11% of the postsynaptic targets were GABA immunoreactive and therefore originated from GABAergic neurons. The results demonstrate that the majority of GABAergic synapses exert their action on the membrane of dendrites and spines rather than on the somata and axons of neurons.     

Subdivisions in the Multiple GABAergic Innervation of Granule Cells in the Dentate Gyrus of the Rat Hippocampus

The sources of GABAergic innervation to granule cells were studied to establish how the basic cortical circuit is implemented in the dentate gyrus. Five types of neuron having extensive local axons were recorded electrophysiologically in vitro and filled intracellularly with biocytin (Han et al., 1993). They were processed for electron microscopy in order to reveal their synaptic organization and postsynaptic targets, and to test whether their terminals contained GABA. (1) The hilar cell, with axon terminals in the commissural and association pathway termination field (HICAP cell), formed Gray's type 2 (symmetrical) synapses with large proximal dendritic shafts (n= 18), two-thirds of which could be shown to emit spines, and with small dendritic branches (n= 6). Other boutons of the HICAP neuron were found to make either Gray's type 1 (asymmetrical) synapses (n= 4) or type 2 synapses (n= 6) with dendritic spines. Using a highly sensitive silver-intensified immunogold method for the postembedding visualization of GABA immunoreactivity, both the terminals and the dendrites of the HICAP cell were found to be immunopositive, whereas its postsynaptic targets were GABA-immunonegative. The dendritic shafts of the HICAP cell received synapses from both GABA-negative and GABA-positive boutons; the dendritic spines which densely covered the main apical dendrite in the medial one-third of the molecular layer received synapses from GABA-negative boutons. (2) The hilar cell, with axon terminals distributed in conjunction with the perforant path termination field (HIPP cell), established type 2 synapses with distal dendritic shafts (n= 17), most of which could be shown to emit spines, small-calibre dendritic profiles (n= 2) and dendritic spines (n= 6), all showing characteristics of granule cell dendrites. The sparsely spiny dendrites of the HIPP cell were covered with many synaptic boutons on both their shafts and their spines. (3) The cell with soma in the molecular layer had an axon associated with the perforant path termination field (MOPP cell). This GABA-immunoreactive cell made type 2 synapses exclusively on dendritic shafts (n= 20), 60% of which could be shown to emit spines. The smooth dendrites of the MOPP cell were also restricted to the outer two-thirds of the molecular layer, where they received both GABA-negative and GABA-positive synaptic inputs. (4) The extensive axonal arborization of the dentate basket cell terminated mainly on somata (n= 26) and proximal dendrites (n= 9) in the granule cell layer, and some boutons made synapses on somatic spines (n= 6); all boutons established type 2 synapses. (5) The dentate axo-axonic cell established type 2 synapses (n= 14) exclusively on axon initial segments of granule cells in the granule cell layer, and on initial segments of presumed mossy cells in the hilus. The results demonstrate that granule cells receive inputs from the local circuit axons of at least five distinct types of dentate neuron terminating in mutually exclusive domains of the cell's surface in four out of five cases. Four of the cell types (HICAP cell, MOPP cell, basket cell, axo-axonic cell) contain GABA, and the HIPP cell may also be inhibitory. The specific local inhibitory neurons terminating in conjunction with particular excitatory amino acid inputs to the granule cells (types 1 – 3) are in a position to interact selectively with the specific inputs on the same dendritic segment. This arrangement provides a possibility for the independent regulation of the gain and long-term potentiation of separate excitatory inputs, through different sets of GABAergic local circuit neurons. The pairing of excitatory and inhibitory inputs may also provide a mechanism for the downward reseating of excitatory postsynaptic potentials, thereby extending their dynamic range.     

Fine structure of the optic fiber termination layer in the tectum of the teleost Rutilus: a stereological and morphometric study

The stratum fibrosum et griseum superficiale (SFGS) of the Rutilus optic tectum, which receives a massive fiber projection from the contralateral retina, was studied by electron microscopy. The qualitative and quantitative analysis of the laterodorsal (LD) portion of the stratum involved both a stereological examination of the different elements and a morphometric study of the various profiles containing synaptic vesicles (PCSVs). The relative volume of each element in the LD SFGS was as follows: myelinated and unmyelinated axons, 6.6%; PCSVs, 38%; dendrites without vesicles, spines, and cell bodies, 41.7%; glia, 10.5%. With the fixation employed, 35% of PCSVs showed spheroidal synaptic vesicles. These profiles could be subdivided into three types: (1) S1 (23.5%) represented optic terminals, since they degenerated after retinal ablation or were labeled after intraocular injection of HRP or [3H] proline. Three subgroups of S1 were identified: S1m--profiles containing clear mitochondria;S1c--profiles that were contiguous with S1m and lacked mitochondria;S1i--isolated profiles without mitochondria. (2) S2 (9.3%) were characterized mainly by their dark mitochondria. (3) S3 (2.2%) corresponded to small nonvisual terminals that were isolated and lacked mitochondria. The PCSVs with pleiomorphic synaptic vesicles (65%) were subdivided into three groups: P1 (38%), P2 (19%), and P3 (8%). P1 and P2 were axonal in nature; P2 could be distinguished from P1 by a greater density of synaptic vesicles. P3 was of dendritic origin. Analysis of synaptic patterns revealed a small number of serial synapses. The presynaptic elements were optic boutons, whereas the intermediate profiles were dendrites with synaptic vesicles (P3). Results are compared with ultrastructural data obtained in the superficial tectal layers of other teleosts and other vertebrate groups.     

Cartwheel neurons of the dorsal cochlear nucleus: a Golgi-electron microscopic study in rat

Cartwheel neurons in rat dorsal cochlear nucleus (DCN) were studied by Golgi impregnation-electron microscopy. Usually situated in layers 1-2, cartwheel neurons (10-14 micrometers in mean cell body diameter) have dendritic trees predominantly in layer 1. The dendrites branch at wide angles. Most primary dendrites are short, nontapering, and bear only a few sessile spines. Secondary and tertiary dendrites are short, curved, and spine-laden. The perikaryon forms symmetric synapses with at least two kinds of boutons containing pleomorphic vesicles. The euchromatic nucleus is indented and has an eccentric nucleolus. The cytoplasm shows several small Nissl bodies, a conspicuous Golgi apparatus, and numerous subsurface and cytoplasmic cisterns of endoplasmic reticulum with a narrow lumen, joined by mitochondria in single or multiple assemblies. In primary dendrites mitochondria are situated peripherally, while in distal branches they become ubiquitous and relatively more numerous. Dendritic shafts usually form symmetric synapses with boutons that contain pleomorphic vesicles. The majority of the dendritic spines are provided with a vesiculo-saccular spine apparatus. All dendritic spines have asymmetric synapses. Most of these are formed with varicosities of thin, unmyelinated fibers (presumably axons of granule cells) running parallel to the long axis of the DCN or radially. These varicosities contain round, clear synaptic vesicles. On the initial axon segment few symmetric synapses are present. The axon acquires a thin myelin sheath after a short trajectory. Cartwheel neurons outnumber all other neurons in layers 1-2 (with the exception of granule cells), and presumably correspond to type C cells with thinly myelinated axons described by Lorente de Nó. The axons of these neurons provide a dense plexus in the superficial layers without leaving the DCN. The possible functional role of cartwheel neurons is discussed.     

Synaptic maturation of the Xenopus retinotectal system: Effects of brain‐derived neurotrophic factor on synapse ultrastructure

Synaptogenesis is a dynamic process that involves structural changes in developing axons and dendrites as synapses form and mature. The visual system of Xenopus laevis has been used as a model to study dynamic changes in axons and dendrites as synapses form in the living brain and the molecular mechanisms that control these processes. Brain‐derived neurotrophic factor (BDNF) contributes to the establishment and refinement of visual connectivity by modulating retinal ganglion cell (RGC) axon arborization and presynaptic differentiation. Here, we have analyzed the ultrastructural organization of the Xenopus retinotectal system to understand better the maturation of this synaptic circuit and the relation between synapse ultrastructure and the structural changes in connectivity that take place in response to BDNF. Expression of yellow fluorescent protein (YFP) followed by preembedding immunoelectron microscopy was used to identify RGC axons specifically in living tadpoles. Injection of recombinant BDNF was used to alter endogenous BDNF levels acutely in the optic tectum. Our studies reveal a rapid transition from a relatively immature synaptic circuit in which retinotectal synapses are formed on developing filopodial‐like processes to a circuit in which RGC axon terminals establish synapses with dendritic shafts and spines. Moreover, our studies reveal that BDNF treatment increases the number of spine synapses and docked vesicle number at YFP‐identified synaptic sites within 24 hours of treatment. These fine structural changes at retinotectal synapses are consistent with the role that BDNF plays in the functional maturation of synaptic circuits and with dynamic, rapid changes in synaptic connectivity during development. J. Comp. Neurol. 518:972–989, 2010. © 2009 Wiley‐Liss, Inc.     

Synaptic input to dentate granule cell basal dendrites in a rat model of temporal lobe epilepsy

In patients with temporal lobe epilepsy some dentate granule cells develop basal dendrites. The extent of excitatory synaptic input to basal dendrites is unclear, nor is it known whether basal dendrites receive inhibitory synapses. We used biocytin to intracellularly label individual granule cells with basal dendrites in epileptic pilocarpine-treated rats. An average basal dendrite had 3.9 branches, was 612 microm long, and accounted for 16% of a cell's total dendritic length. In vivo intracellular labeling and postembedding GABA-immunocytochemistry were used to evaluate synapses with basal dendrites reconstructed from serial electron micrographs. An average of 7% of 1,802 putative synapses were formed by GABA-positive axon terminals, indicating synaptogenesis by interneurons. Ninety-three percent of the identified synapses were GABA-negative. Most GABA-negative synapses were with spines, but at least 10% were with dendritic shafts. Multiplying basal dendrite length/cell and synapse density yielded an estimate of 180 inhibitory and 2,140 excitatory synapses per granule cell basal dendrite. Based on previous estimates of synaptic input to granule cells in control rats, these findings suggest an average basal dendrite receives approximately 14% of the total inhibitory and 19% of excitatory synapses of a cell. These findings reveal that basal dendrites are a novel source of inhibitory input, but they primarily receive excitatory synapses.     

Ultrastructural analysis of synaptic relationships of intracellularly stained pyramidal cell axons in piriform cortex

Axons of pyramidal cells in piriform cortex stained by intracellular injection of horseradish peroxidase (HRP) have been analyzed by light and electron microscopy. Myelinated primary axons give rise to extensive, very fine caliber (0.2 micron) unmyelinated collaterals with stereotyped radiating branching patterns. Serial section electron microscopic analysis of the stained portions of the collateral systems (initial 1-2 mm) revealed that they give rise to synaptic contacts on dendritic spines and shafts. These synapses typically contain compact clusters of large, predominantly spherical synaptic vesicles subjacent to asymmetrical contacts with heavy postsynaptic densities. On the basis of comparisons with Golgi material and intracellularly stained dendrites, it was concluded that dendritic spines receiving synapses from the proximal portions of pyramidal cell axon collaterals originate primarily from pyramidal cell basal dendrites. Postsynaptic dendritic shafts contacted closely resemble dendrites of probable GABAergic neurons identified in antibody and [3H]-GABA uptake studies. Electron microscopic examination of pyramidal cell axon initial segments revealed a high density of symmetrical synaptic contacts on their surfaces. Synaptic vesicles in the presynaptic boutons were small and flattened. It is concluded that pyramidal cells synaptically interact over short distances with other pyramidal cells via basal dendrites and with deep nonpyramidal cells that probably include GABAergic cells mediating a feedback inhibition. This contrasts with long associational projections of pyramidal cells that terminate predominantly on apical dendrites of other pyramidal cells.     

Presynaptic neurotrophin-3 increases the number of tectal synapses,vesicle density,and number of docked vesicles in chick embryos

To determine whether presynaptically derived neurotrophins may contribute to synaptic plasticity, we examined whether neurotrophin-3 (NT-3) changed the number, size, vesicle content, or vesicle distribution of synapses within the retinorecipient layers of the chick optic tectum. In this system, endogenous NT-3 derives presynaptically from retinal ganglion cell axons. Retinotectal synapses comprise the majority of synapses in superficial tectal layers, as demonstrated by destruction of retinotectal input by intraocular application of the drug monensin. To examine the effect of increased or decreased levels of NT-3, either exogenous NT-3 or monoclonal NT-3 blocking antibodies were injected into the optic tectum of 19-day-old chick embryos, spiked with radiolabeled protein to verify the success of injections and estimate effective concentrations. After 48 hours, the ultrastructure of superficial tectal layers was analyzed and compared with samples from control tecta injected with cytochrome C. NT-3 increased the number of synapses, synaptic vesicles/profile, synaptic vesicle densities, the number of docked vesicles, and the length of the synaptic profile. Deprivation of anterogradely transported endogenous NT-3 with NT-3 antibodies resulted in the opposite effect: decreased numbers of synapses, decreased vesicle densities, and decreased numbers of docked vesicles. Brain-derived neurotrophic factor (BDNF) had a largely different effect than NT-3. BDNF increased the density of vesicles and deprivation of endogenous TrkB ligands with TrkB fusion protein reduced the density of vesicles in the synapses, without effects on synapse number or docked vesicles. We conclude that anterogradely transported NT-3 affects synapse strength in a way that differs from that of presumably postsynaptic-derived BDNF.     

Isthmotectal axons make ectopic synapses in monocular regions of the tectum in developing Xenopus laevis frogs.

During the development of binocular maps in the tectum of Xenopus laevis, axons that relay input from the ipsilateral eye via the nucleus isthmi undergo a prolonged period of shifting connections. This shifting accompanies the dramatic change in eye position that takes place as the laterally placed eyes of the tadpole move dorsofrontally. There is a concomitant expansion of the proportion of tectum that receives contralateral retinotectal input corresponding to the binocular portion of the visual field. Electrophysiological recording demonstrates that ipsilateral units are present in those rostral tectal zones, and anatomical methods show that the isthmotectal axons arborize densely in the rostral region but also extend sparser branches into the caudal zone, which is occupied by contralateral inputs with receptive fields in the monocular zone of the visual field. A mechanism that aligns the ipsilateral and contralateral maps is activity-dependent stabilization of isthmotectal axons that exhibit firing patterns correlated with those of nearby retinotectal axons. In order for activity patterns to function in stabilizing correct connections and promoting the withdrawal of incorrect connections, synaptic communication of some sort is hypothesized to be essential. We have investigated whether isthmotectal axons make morphologically identifiable synapses during development and where such synapses are located. We find evidence for morphologically identifiable synapses in all regions of the tectum, along with many growth cones and structures that are probably immature synapses. As in the adult, the synapses contain round, clear vesicles, have asymmetric specializations, and terminate on structures that appear to be dendrites. In both adult and tadpole, the rarity of serial synapses involving isthmotectal terminals suggests that the interactions between retinotectal and isthmotectal inputs are mediated by postsynaptic dendrites.     

The lateral reticular nucleus of the opossum (Didelphis virginiana). I. Conformation,cytology and synaptology

When viewed in Nissl preparations, the lateral reticular nucleus (LRN) of the opossum can be divided into three subgroups: a medial internal portion, a lateral external portion and a rostral trigeminal division. Neurons within the internal division measure 13-45 μ in their greatest dimension whereas those within the external and trigeminal portions measure 11-32 μ and 14-27 μ respectively. Golgi impregnations reveal that many neurons in all three subdivisions display a radial dendritic pattern although some of the nerve cells within the external division have dendrites which orient mainly in a ventromedial to dorsolateral direction. The cell bodies of LRN neurons are relatively spine-free. However, a small percentage of neurons exhibit clusters of sessile spines on proximal and more distal dendritic segments. No locally ramifying axons or axon collaterals were found within the LRN. Synaptic terminals within the LRN were divided into four categories: (1) small terminals measuring 2.5 μ or less containing agranular spherical vesicles; (2) small terminals (2.5 μ or less) with agranular pleomorphic synaptic vesicles, i.e., a mixture of spherical and elliptical synaptic vesicles; (3) small terminals (2.5 μ or less) containing agranular spherical or pleomorphic vesicles with a variable number (4-27) of dense core vesicles; and (4) large terminals (greater than 2.5 μ) which contain agranular spherical synaptic vesicles and a variable number of dense core vesicles (1-17). Dendritic diameters were measured from Golgi impregnations and correlated with cross-sectioned profiles in electron micrographs to help determine the post-synaptic distribution of synaptic endings. Small terminals containing agranular spherical or pleomorphic synaptic vesicles contact the soma and entire dendritic tree in each portion of the nucleus, whereas the small terminals containing dense core vesicles are usually located on distal dendrites or spines. Some large terminals make multiple synaptic contacts with a cluster of spines, others contact groups of small (distal) dendrites. In order to identify two of the major afferent systems to the LRN, 15 adult opossums were subjected to either a cervical spinal cord hemisection or a stereotaxic lesion of the red nucleus. Two days subsequent to spinal hemisection, large terminals in the caudal part of the ipsilateral LRN exhibit either an electron dense or filamentous reaction. Their postsynaptic loci are spines and shafts of proximal dendrites or a number of distal dendrites and spines. In addition, small terminals containing spherical agranular synaptic vesicles undergo an electron dense reaction in the same areas. Their postsynaptic loci are proximal or distal dendrites. Two days subsequent to rubral lesions, small terminals containing agranular spherical synaptic vesicles undergo a dark reaction in rostral portions of the contralateral nucleus. They contact intermediate or distal dendrites and occasionally spines.     

Immunocytochemical localization of choline acetyltransferase within the rat neostriatum: a correlated light and electron microscopic study of cholinergic neurons and synapses

Monoclonal antibodies to choline acetyltransferase (ChAT) were used in an immunocytochemical study to characterize putative cholinergic neurons and synaptic junctions in rat caudate-putamen. Light microscopy (LM) revealed that ChAT-positive neurons are distributed throughout the striatum. These cells have large oval or multipolar somata, and exhibit three to four primary dendrites that branch and extend long distances. Quantitative analysis of counterstained preparations indicated that ChAT-positive neurons constitute 1.7% of the total neuronal population. Electron microscopy (EM) of immunoreactive neurons initially studied by LM revealed somata characterized by deeply invaginated nuclei and by abundant amounts of organelle-rich cytoplasm. Surfaces of ChAT-positive neurons are frequently smooth, but occasional somatic protrusions and dendritic spines occur. Although infrequently observed, axons of ChAT-positive neurons branch, receive synapses, and become myelinated. Unlabeled boutons make both symmetrical and asymmetrical synapses with ChAT-positive somata and proximal dendrites, but are more numerous on distal dendrites. In addition, some unlabeled terminals form asymmetrical synapses with ChAT-positive somata and dendrites that are distinguished by prominent subsynaptic dense bodies. Light microscopy demonstrated a dense distribution of ChAT-positive fibers and punctate structures in the striatum, and these structures appear to correlate, respectively, with labeled preterminal axons and presynaptic boutons identified by EM. ChAT-positive boutons contain pleomorphic vesicles, and make symmetrical synapses primarily with unlabeled dendritic shafts. Furthermore, they establish synaptic contacts with somata, dendrites and axon initial segments of unlabeled neurons that ultrastructurally resemble medium spiny neurons. These observations, together with the results of other investigations, suggest that medium spiny GABAergic projection neurons receive a cholinergic innervation that is probably derived from ChAT-positive striatal cells. The results of this study also indicate that cholinergic neurons within caudate-putamen belong to a single population of cells that have large somata and extensive sparsely spined dendrites. Such neurons, in combination with dense concentrations of ChAT-positive fibers and terminals, are the likely basis for the large amounts of ChAT and acetylcholine detected biochemically within the neostriatum.     

Synaptic organization of cortico-cortical connections from the primary visual cortex to the posteromedial lateral suprasylvian visual area in the cat

The synaptic organization of the projection from the cat striate visual cortex to the posteromedial lateral suprasylvian cortical area (PMLS) was examined. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was iontophorectically delivered into area 17, and anterogradely labeled fibers were revealed in PMLS by means of an immunocytochemical detection method. Most axons and presumptive terminal swellings were found in layers III and IV. The neuronal elements (n = 190) that were postsynaptic to anterogradely labeled boutons were quantitatively analyzed. All anterogradely labeled cortico-cortical boutons (n = 182) established type 1 synapses. The results show that 83% of the postsynaptic targets were dendritic spines, probably belonging to pyramidal cells. Dendritic shafts constituted 17% of the targets. The dendritic shafts postsynaptic to cortico-cortical boutons were studied for the presence of gamma-aminobutyric acid (GABA) with a postembedding immunogold method. Most dendritic shafts (85%) that were tested were found to be GABA-positive, demonstrating that they originate from local inhibitory neurons. Taking into account that most postsynaptic targets were spines and extending the results of the immunocytochemical testing to the total population of postsynaptic dendrites, it was calculated that at least 14% of targets originated from GABA-positive cells. Thus cortico-cortical axons establish direct monosynpatic connections mainly with pyramidal and to a lesser extent with GABAergic nonpyramidal neurons in area PMLS, providing both feedforward excitation and feedforward inhibition to a visual associational area known to be involved in the processing of motion information. The results are consistent with previously demonstrated deficits in physiological properties of neurons in PMLS following removal of cortico-cortical afferents.     

Axon terminals from the nucleus isthmi pars parvocellularis control the ascending retinotectofugal output through direct synaptic contact with tectal ganglion cell dendrites

The optic tectum in birds and its homologue the superior colliculus in mammals both send major bilateral, nontopographic projections to the nucleus rotundus and caudal pulvinar, respectively. These projections originate from widefield tectal ganglion cells (TGCs) located in layer 13 in the avian tectum and in the lower superficial layers in the mammalian colliculus. The TGCs characteristically have monostratified arrays of brush‐like dendritic terminations and respond mostly to bidimensional motion or looming features. In birds, this TGC‐mediated tectofugal output is controlled by feedback signals from the nucleus isthmi pars parvocellularis (Ipc). The Ipc neurons display topographically organized axons that densely ramify in restricted columnar terminal fields overlapping various neural elements that could mediate this tectofugal control, including the retinal terminals and the TGC dendrites themselves. Whether the Ipc axons make synaptic contact with these or other tectal neural elements remains undetermined. We double labeled Ipc axons and their presumptive postsynaptic targets in the tectum of chickens (Gallus gallus) with neural tracers and performed an ultrastructural analysis. We found that the Ipc terminal boutons form glomerulus‐like structures in the superficial and intermediate tectal layers, establishing asymmetric synapses with several dendritic profiles. In these glomeruli, at least two of the postsynaptic dendrites originated from TGCs. We also found synaptic contacts between retinal terminals and TGC dendrites. These findings suggest that, in birds, Ipc axons control the ascending tectal outflow of retinal signals through direct synaptic contacts with the TGCs. J. Comp. Neurol. 524:362–379, 2016. © 2015 Wiley Periodicals, Inc.     

Ultrastructural localization of neurotransmitter immunoreactivity in mossy cell axons and their synaptic targets in the rat dentate gyrus

Electrophysiologically identified and intracellularly biocytin-labeled mossy cells in the dentate hilus of the rat were studied using electron microscopy and postembedding immunogold techniques. Ultrathin sections containing a labeled mossy cell or its axon collaterals were reacted with antisera against the excitatory neurotransmitter glutamate and against the inhibitory neurotransmitter γ-aminobutyric acid (GABA). From single- and double-immunolabeled preparations, we found that 1) mossy cell axon terminals made asymmetric contacts onto postsynaptic targets in the hilus and stratum moleculare of the dentate gyrus and showed immunoreactivity primarily for glutamate, but never for GABA; 2) in the hilus, glutamate-positive mossy cell axon terminals targeted GABA-positive dendritic shafts of hilar interneurons and GABA-negative dendritic spines; and 3) in the inner molecular layer, the mossy cell axon formed asymmetric synapses with dendritic spines associated with GABA-negative (presumably granule cell) dendrites. The results of this study support the view that excitatory (glutamatergic) mossy cell terminals contact GABAergic interneurons and non-GABAergic neurons in the hilar region and GABA-negative granule cells in the stratum moleculare. This pattern of connectivity is consistent with the hypothesis that mossy cells provide excitatory feedback to granule cells in a dentate gyrus associational network and also activate local hilar inhibitory elements. Hippocampus 1997;7:559–570. © 1997 Wiley-Liss, Inc.     

Synaptic organization of projections from basal forebrain structures to the mediodorsal thalamic nucleus of the rat.

The synaptic organization of the mediodorsal thalamic nucleus (MD) in the rat was studied with the electron microscope, and correlated with the termination of afferent fibers labeled with wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Presynaptic axon terminals were classified into four categories in MD on the basis of the size, synaptic vesicle morphology, and synaptic membrane specializations: 1) small axon terminals with round synaptic vesicles (SR), which made asymmetrical synaptic contacts predominantly with small dendritic shafts; 2) large axon terminals with round vesicles (LR), which established asymmetrical synaptic junctions mainly with large dendritic shafts; 3) small to medium axon terminals with pleomorphic vesicles (SMP), which formed symmetrical synaptic contacts with somata and small-diameter dendrites; 4) large axon terminals with pleomorphic vesicles (LP), which made symmetrical synaptic contacts with large dendritic shafts. Synaptic glomeruli were also identified in MD that contained either LR or LP terminals as the central presynaptic components. No presynaptic dendrites were identified. In order to identify terminals arising from different sources, injections of WGA-HRP were made into cortical and subcortical structures known to project to MD, including the prefrontal cortex, piriform cortex, amygdala, ventral pallidum and thalamic reticular nucleus. Axons from the amygdala formed LR terminals, while those from the prefrontal and insular cortex ended exclusively in SR terminals. Fibers labeled from the piriform cortex formed both LR and SR endings. Based on their morphology, all of these are presumed to be excitatory. In contrast, the axons from the ventral pallidum ended as LP terminals, and those from the thalamic reticular nucleus formed SMP terminals. Both are presumed to be inhibitory. At least some terminals from these sources have also been identified as GABAergic, based on double labeling with anterogradely transported WGA-HRP and glutamic acid decarboxylase (GAD) immunocytochemistry.     

Fine structure and synaptic connections of the common spiny neuron of the rat neostriatum: A study employing intracellular injection of horseradish peroxidase

Medium-sized spiny neurons of the rat neostriatum, identified by intracellular injection of horseradish peroxidase, were examined at both light and electron microscopic levels. These neurons were characterized by their heavy investment of dendritic spines, beginning about 20 μm from the soma and continuing to the tips of the dendrites. Their axons arose from the soma or from a large dendritic trunk very near the soma, and tapered rapidly to form a main axonal branch from which arose several smaller initial collaterals. These arborized extensively throughout an area of about the same size as, and highly overlapping with, the dendritic field of the cell, while the main axon could be followed for distances of up to 1 mm in the direction of the globus pallidus. Three major synaptic types were seen in contact with spiny neurons. Boutons containing small round synaptic vesicles formed synapses exclusively with spiny regions of the dendrites, and most of these were axo-spinous. Small, very pleomorphic synaptic vesicles characterized a second bouton type of unknown origin, which made contacts with somata, initial segments, and dendrites, but not dendritic spines. Boutons containing large pleomorphic synaptic vesicles had the most widespread distribution, contacting all regions including dendritic spines. Spines receiving these contacts also were postsynaptic to boutons containing small round vesicles. Axon collaterals of spiny cells formed synapses with large pleomorphic vesicles and made synapses with somata, initial segments of axons, dendrites, and dendritic spines of striatal neurons, including other spiny cells.     

Direct connections between dendritic terminals of tectal ganglion cells and glutamate-positive terminals of presumed optic fibres in layers 4-5 of the optic tectum of Gallus domesticus. A light- and electron microscopic study

The principal afferent fibres of the avian optic tectum are the optic fibres of retinal origin. They terminate on the contralateral side, in the external layers (2-7) of the optic tectum (called optic layers) turning into these layers from the external surface. The terminal branchings of the optic fibres develop four densely innervated areas in layers 2, 3, 4-5 and 7. Their terminals are large and of various appearance in the different areas. In the middle third of the optic layer (in layers 4-5), thin dendritic terminal sections of tectal ganglion cells (according to Ramòn y Cajal) of layer 13 terminate into bunches. Phaseolus vulgaris lectin immunotracer corroborates these dendritic endings (further: dendritic terminals) of tectal ganglion cells. The direct connections between these dendritic terminals and the supposed optic fibres were studied under electron microscope and it was found that the large terminals of optic fibres containing round synaptic vesicles establish asymmetrical synapses with several dendritic profiles, among them Phaseolus lectin labelled dendritic terminals of ganglion cells. This result morphologically supports the former physiological observation of a direct synaptic transmission between optic fibres and ganglion cells of layer 13. In addition, on the dendritic terminals of ganglion cells, symmetrical synapses established by GABA-positive terminals were found. The optic terminals, the GABA-immunopositive terminals and the dendritic terminals of ganglion cells form complex synaptic units surrounded with glial sheath, and thus they establish glomerulus-like synaptic units. The size of the dendritic tree and the branching pattern of the dendrites of ganglion cells point to divergence and convergence in visual transmission.