Pyramidal cell dendrites are the primary targets of calbindin D28k-immunoreactive interneurons in the hippocampus

The axonal arborization and postsynaptic targets of calbindin D28k (CB)-immunoreactive nonprincipal neurons have been studied in the rat dorsal hippocampus. Two types of neurons were distinguished on the basis of soma location, the characteristics of the dendritic tree, and the axon arborisation pattern. Type I cells were located in stratum radiatum of the CA1 and CA3 regions and occasionally in strata pyramidale and oriens. These cells had multipolar or bitufted dendritic trees primarily located in stratum radiatum. Their axons could be followed for a considerable distance, arborised within stratum radiatum, and were covered with regularly spaced small boutons. As demonstrated with postembedding immunogold staining, their axon terminals were γ-aminobutyric acid (GABA) immunoreactive, and formed symmetrical synapses pre-dominantly on proximal and distal dendrites of pyramidal cells (28% and 58%, respectively), and occasionally on spines (9%) or on GABA-positive dendrites (5%). Type II cells were found exclusively in stratum oriens of the CA1 and CA3 regions and possessed large, fusiform cell bodies and long, horizontally oriented dendrites. Their axon initial segments turned towards the alveus and disappeared in a myelin sheet, which was often possible to follow into the white matter. We conclude that type I CB-immunoreactive cells are likely to represent a major source of inhibitory synapses in the dendritic region of pyramidal cells, which are responsible for the control of dendritic electrogenesis. The distribution of local collaterals of type II cells—if they have any—remains unknown, but their main axon is likely to project to the medial septum. © 1996 Wiley-Liss, Inc.     

Local circuit interactions between oriens/alveus interneurons and CA1 pyramidal cells in hippocampal slices: electrophysiology and morphology

Electrophysiological and anatomical techniques were used to determine the role, in the hippocampal circuitry, of local circuit neurons located at the oriens/alveus border (O/A interneurons). Intracellular recording from these cells showed that their response characteristics were clearly nonpyramidal: high input resistance, short membrane time constant, short-duration action potential, pronounced, brief afterhyperpolarizations (AHP), and nondecremental firing during intrasomatic depolarizing current pulses. Intracellular Lucifer yellow (LY) injection and subsequent fluorescence microscopy confirmed their nonpyramidal nature. O/A interneuron somata were bipolar or multipolar; their dendrites projected mostly parallel to the alveus, except for 1 or 2 processes that turned perpendicularly, and ascended through stratum oriens and pyramidale and into radiatum. Their axons were seen to branch profusely in stratum oriens and pyramidale. Simultaneous intracellular recordings from O/A interneurons and CA 1 pyramidal cells showed that pyramidal cells directly excite these interneurons. Major hippocampal afferents also directly excited the O/A interneurons. In a small number of interneuron-pyramidal pairs, stimulation of the O/A interneuron directly inhibited pyramidal cells. In one case, reciprocal connections were observed: The pyramidal cell excited the interneuron, and the interneuron inhibited the pyramidal cell. In 1 interneuron-to-interneuron pair, an inhibitory connection from O/A interneuron to stratum pyramidale interneuron was also observed. With intracellular HRP injections into O/A interneurons and subsequent electron microscopy, we observed that O/A interneuron axons made contacts with pyramidal and nonpyramidal cells. HRP-filled symmetric synaptic contacts were found on pyramidal cell dendrites and somata. HRP-filled axons also made contacts with pyramidal cell initial segments. HRP-filled O/A interneuron axon contacts were also found on nonpyramidal cell dendrites in stratum oriens. These electrophysiological and anatomical results suggest that O/A interneurons make synaptic contact with pyramidal cells and may mediate feedforward and feedback inhibition onto CA 1 pyramidal cells.     

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.     

Ultrastructural characterization and GAD co-localization of somatostatin-like immunoreactive neurons in CA1 of rabbit hippocampus

Immunocytochemical techniques have been used to identify a striking interneuronal population which is immunoreactive for the peptide, somatostatin. The cell population, which is seen most densely in stratum oriens and at the oriens/alveus border of the CA1 region of rabbit hippocampus, was characterized in light and electron microscopic observations. The cells have dendrites which extend parallel to and into the alveus, with occasional processes ascending through stratum pyramidale toward the hippocampal fissure. The dendrites receive numerous synaptic contacts directly onto aspinous dendritic shafts. Axon collaterals ramify profusely within the pyramidale region, and among the proximal apical and basal pyramidal cell dendrites in areas of stratum radiatum and stratum oriens. Somatostatin-like immunoreactive terminals make synaptic contact, primarily of the symmetric type, with the somata and proximal dendrites of pyramidal neurons. Somatostatin-like neurons are found at approximately equal density in the hippocampus of immature (8 days postnatal) and mature (30 days postnatal) rabbit. Double-labelling techniques, to identify both somatostatin-like and glutamic acid decarboxylase (GAD) immunoreactive neurons, demonstrated that a large proportion of the somatostatin neurons were also GABAergic.     

Precision and Variability in Postsynaptic Target Selection of Inhibitory Cells in the Hippocampal CA3 Region

Non-pyramidal cells were filled intracellularly with biocytin in the CA3 region of the guinea-pig hippocampus in vitro, within or close to stratum pyramidale. On the basis of camera lucida reconstructions and electron microscopy, six different cell types with distinct laminar distribution of axon terminals could be distinguished. The axon of three axo-axonic cells, three typical basket cells, and atypical basket cells of two types arborized in the perisomatic and proximal dendritlc region of CA3 pyramidal cells. Two cells with axons innervating the distal dendritlc segments of pyramidal cells were also found; one terminated in stratum radiatum and the other in stratum lacunosum-moleculare. Electron microscopy demonstrated that symmetrical synapses were formed by the labelled boutons on axon initial segments, somata, and proximal or distal dendrites of mostly pyramidal neurons. Axo-axonic cells showed absolute target selectivity for axon initial segments, whereas for the other cells the distribution of contacted elements was determined by the laminar distribution of axon terminals. In two cases, where additional cells were labelled with biocytin, multiple (up to nine) light microscopically identified contacts (presumed synaptic contacts) were established by the interneurons on several pyramidal cells and on an axo-axonic cell. Our results show that a restricted set of inhibitory cells, with somata within or close to CA3 stratum pyramldale, possess variable patterns of axonal arborization. Various types of postsynaptic elements are contacted, but precision in selecting certain targets and ignoring others is maintained within a particular cell type and layer. In contrast to the diversity of axonal arbors the structure of the dendritic trees shows no consistent differences, suggesting that the cells may be activated by a similar set of afferents. It seems probable that the innervation of precise regions of postsynaptic pyramidal cells by different types of interneurons–often in conjunction with particular excitatory afferents (Han et at., Eur. J. Neurosci., 5, 395–410, 1993)–underlies functional differences in inhibitory synaptic actions.     

Complete Axon Arborization of a Single CA3 Pyramidal Cell in the Rat Hippocampus, and its Relationship With Postsynaptic Parvalbumin-containing Interneurons

The complete axon arborization of a single CA3 pyramidal cell has been reconstructed from 32 (60 /μm thick) sections from the rat hippocampus following in vivo intracellular injection of neurobiotin. The same sections were double-immunostained for parvalbumin-a calcium-binding protein selectively present in two types of GABAergic interneurons, the basket and chandelier cells-in order to map boutons of the pyramidal cell in contact with dendrites and somata of these specific subsets of interneurons visualized in a Golgi-like manner. The axon of the pyramidal cell formed 15 295 boutons, 63.8% of which were in stratum oriens, 15.4% in stratum pyramidale and 20.8% in stratum radiatum. Only 2.1% of the axon terminals contacted parvalbumin-positive neurons. Most of these were single contacts (84.7%), but double or triple contacts (15.3%) were also found. The majority of the boutons terminated on dendrites (84.1%) of parvalbumin-positive cells, less frequently on cell bodies (15.9%). In order to estimate the proportion of contacts representing synapses, 16 light microscopically identified contacts between boutons of the filled pyramidal cell axon and the parvalbumin-positive targets were examined by correlated electron microscopy. Thirteen of them were found to be asymmetrical synapses, and in the remaining three cases synapses between the labelled profiles could not be confirmed. We conclude that the physiologically effective excitatory connections between single pyramidal cells and postsynaptic inhibitory neurons are mediated by a small number of contacts, mostly by a single synapse. This results in a high degree of convergence and divergence in hippocampal networks.     

Gamma-aminobutyric acid-immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies

The distribution of GABA-immunoreactive neurons and axonal varicosities was investigated in the hippocampal region of the rat brain by means of an indirect peroxidase immunocytochemical method with recently developed anti-GABA antibodies. The immunolabeling was found to be restricted to nervous structures: neuronal cell bodies, dendrites and axon terminals. Myelinated axons showing GABA-immunoreactivity were also observed. GABA-immunoreactive neurons were found in great number in the stratum pyramidale, the superficial part of the stratum oriens and the deep part of the stratum radiatum in the Ammon's horn. Less were found in the other regions; rare labeled cells were observed in the superficial part of the stratum radiatum and the middle part of the stratum oriens. The dentate gyrus exhibited numerous labeled cells in the granular layer, few in the hilus, rare in the molecular later. A high density of GABA-immunoreactive terminals was found at the limit of the stratum oriens with the alveus, in the stratum pyramidale and in the stratum lacunosum. A lower density of labeled fibers was observed in the other areas. The somata and proximal dendrites of pyramidal and granular cells were encompassed by characteristics pericellular arrangements of GABA-immunoreactive varicosities. Ultrastructural observations revealed a diffuse immunoreaction product spread over the cytoplasm and the nucleus without specific relationship with the organelles, and immunoreactive aggregates in the cytoplasm. Labeled dendrites often showed enlargements displaying the immunoreaction whereas thinner segments were devoid of it. They received numerous asymmetrical synapses from unlabeled axon terminals. GABA-immunoreactive terminals were filled with small clear vesicles with immunopositive membranes and were observed in symmetrical contact with somata and dendrites.     

Combined golgi and electron microscopic study on the synapses formed by double bouquet cells in the visual cortex of the cat and monkey

The morphology of certain Golgi-stained cells was examined in the striate and peristriate cortex of the cat and in the striate cortex of the rhesus monkey. Neurons in layer III were selected on the basis of their characteristic vertical axon bundles, which are 20–150 μ in diameter and traverse layers II–V Selected neurons were examined under the electron microscope to characterize their synapses and to establish their postsynaptic targets. It was found that double bouquet cells form symmetrical or type II synapses. In the cat the postsynaptic membrane specialization was more extensive than in the monkey. After removing the Golgi precipitate from boutons of two cells in the cat, small pleomorphic and flattened vesicles were found in the boutons Earlier suggestions that double bouquet cells make synapses preferentially with spines of apical dendrites could not be confirmed. Out of 66 boutons in area 17 of the cat, 86.4% formed synapses with dendritic shafts, many of them belonging to nonpyramidal cells, 9% with perikarya of nonpyramidal cells, and only 4.6% with spines. Out of 19 synapses examined in area 18, 74% were contacting dendritic shafts and the rest contacted spines. In the monkey 60% of a total of 35 double bouquet cell synapses made synapses with dendritic shafts. A different type of double bouquet cell with densely spiny dendrites is also described in layer IV of the monkey striate cortex. This neuron formed asymmetrical synapses It is suggested that layer III double bouquet cells with vertical axon bundles are probably inhibitory and act on other nonpyramidal cells and certain parts of pyramidal cells.     

Distribution patterns of individual medial lemniscal axons in the ventrobasal complex of the monkey thalamus

Somatostatin-immunoreactive neurons in the rat neostriatum were studied by correlated light and electron microscopy using the peroxidase-antiperoxidase immunocytochemical technique. Immunoreactivity was localized in neuronal perikarya and processes. The perikarya were of spindle or fusiform shape (average length 16.9 microns) and were found in all parts of the neostriatum. From each neuron there arose two to four straight immunoreactive dendritelike processes, which could frequently be traced as far as about 130 microns from their perikaryon. Immunoreactive varicose axonlike processes were occasionally found, some of which were proximal axons of identified immunoreactive cells. Nine of the light microscopically identified neurons showing somatostatin-immunoreactivity were studied in the electron microscope; two of them had proximal axons with varicosities. Each neuron had an oval or elongated nucleus, which was always indented. These morphological features correspond well to those of certain "medium-size aspiny" neurons classified by Golgi studies. Although the immunoreactive endproduct was diffusely located throughout the neuron, it was characteristically located in the saccules and large granules (diameter 133 nm) of the Golgi apparatus, and large immunoreactive vesicles of similar size to those in the Golgi apparatus frequently occurred in all parts of axon. Very little synaptic input was found on the perikarya and dendrites of somatostatin-immunoreactive neurons. The perikarya and proximal dendrites received both symmetrical and asymmetrical synaptic input, while the distal dendrites usually received boutons that formed asymmetrical contacts. The somatostatin-immunoreactive boutons contained pleomorphic electron-lucent vesicles (diameter 39.3 nm) and a few large immunoreactive granular vesicles; these boutons always formed symmetrical synapses. Their postsynaptic targets were dendritic shafts, spines, and unclassified dendritic profiles. On the other hand, the varicosities of identified proximal axons of somatostatin-positive neurons did not form typical synapses, since they lacked clusters of small vesicles, but some of them were in direct apposition (via membrane specializations) to unlabelled perikarya or dendrites. It is concluded that somatostatin is a useful marker for a particular type of neuron in the neostriatum. The presence of somatostatin immunoreactivity in synaptic boutons is consistent with the view that somatostatin could be a neurotransmitter in the neostriatum.     

Monosynaptic cortical input and local axon collaterals of identified striatonigral neurons. A light and electron microscopic study using the golgi-peroxidase transport-degeneration procedure

Following the injection of horseradish peroxidase into the ipsilaeral substantia nigra, 36 retrogradely labelled neurons in the striatum were characterized (in three rats) by Golgi staining and gold toning: each neuron was of the medium-size, densely spinous type. Prior to the injection of horseradish peroxidase, two of the rats had had lesions placed in the ipsilateral motor cortex, the third rat had had a lesion placed in the ipsilateral frontal and prefrontal cortex. In the electron microscope, degenerating boutons of cortical neurons were found in asymmetrical synaptic contact with the spines of proximal and distal dendrites of all six of the identified striatonigral neurons that were studied. Some of the degenerating boutons were small (diameter 0.1–0.3 μ), while others were larger (1–2 μ). An individual dendrite of a striatonigral neuron was in synaptic contact with very few degenerating boutons Local axon collaterals im the striatum could be traced from two of the identified striatonigral neurons that received degenerating cortical boutons. These were studied in the electron microscope; their boutons formed symmetrical synapses with spines or dendritic shafts of other striatal neurons. The synaptic boutons contained large, clear, round and pleomorphic vesicles. The postsynaptic targets of these boutons morphologically resemble the dendrites of medium-size spiny neurons It is concluded that afferents from the cortex make monosynaptic contact with the dendritic spines of medium-size spiny striatonigral neurons and that such neurons have local axon collaterals in the striatum that form synapses with other spiny neurons.     

Synaptic connections of intracellularly filled clutch cells: a type of small basket cell in the visual cortex of the cat

Light and electron microscopic quantitative analysis was carried out on a type of neuron intracellularly filled with horseradish peroxidase. Two cells were studied in area 17, one of which was injected intra-axonally, and its soma was not recovered. One cell was studied in area 18. The two somata were on the border of layers IVa/b; they were radially elongated and received synapses from numerous large boutons with round synaptic vesicles. The dendrites were smooth and remained largely in layer IV. The cells can be recognised on the basis of their axonal arbor, which was restricted to layer IV (90-95% of boutons) with minor projections to layers III, V, and VI. Many of the large, bulbous boutons contacted neuronal somata, short collaterals often forming "claw"-like configurations around cells. The name "clutch cell" is suggested to delineate this type of neuron from other aspiny multipolar cells. Computer-assisted reconstruction of the axon showed that in layer IV the axons occupied a rectangular area about 300 X 500 microns, elongated anteroposteriorly in area 17 and mediolaterally in area 18. The distributions of synaptic boutons and postsynaptic cells were patchy within this area. A total of 321 boutons were serially sectioned in area 17. The boutons formed type II synaptic contacts. The postsynaptic targets were somata (20-30%), dendritic shafts (35-50%), spines (30%), and rarely axon initial segments. Most of the postsynaptic somata tested were not immunoreactive for GABA and their fine structural features suggest that they are spiny stellate, star pyramidal, and pyramidal neurons. The characteristics of most of the postsynaptic dendrites and spines also suggest that they belong to these spiny neurons. A few of the postsynaptic dendrites and somata exhibited characteristics of cells with smooth dendrites and these somata were immunoreactive for GABA. It is suggested that clutch cells are inhibitory interneurons exerting their effect mainly on layer IV spiny neurons in an area localised perhaps to a single ocular dominance column. The specific laminar location of the axons of clutch cell also suggests that they may be associated with the afferent terminals of lateral geniculate nucleus cells, and could thus be responsible for generating some of the selective properties of neurons of the first stage of cortical processing.     

Stratum radiatum giant cells: a type of principal cell in the rat hippocampus

Neurons of a distinct type in CA1 area stratum radiatum of the rat hippocampus have been found to express a direct cellular form of long-term potentiation (LTP, Maccaferri & McBain, 1996, J. Neurosci. 16, 5334), but their functional identity, i.e. whether interneuron or principal cell, remained unknown. Whole cell recording from hippocampal slices in vitro was combined with light and electron microscopy to answer this question. LTP was robustly induced by a pairing protocol and physiological properties were measured in radiatum giant cells (RGCs) using biocytin containing pipettes. Reconstruction of the cells' dendritic and axonal arbor revealed morphological properties similar to CA1 pyramidal cells with some characteristic differences. They typically had two large diameter apical dendrites, or when only one dendrite arose, it soon bifurcated. Apical dendrites formed a dendritic tuft in stratum lacunosum-moleculare and the dendrites, but not the somata, were densely covered with conventional spines. The axon arose from the basal pole of the soma, descended to stratum oriens and emitted several axon terminals bearing collaterals that travelled horizontally, remaining in stratum oriens. The main, myelinated axon trunks turned towards the fimbria. In the electron microscope axon terminals were found to form asymmetrical synapses on postsynaptic dendritic shafts and dendritic spines in stratum oriens. The dendrites received asymmetrical synapses, mostly on their spines. The axon initial segments also received several synapses, a feature never observed on interneurons. All the above characteristics support the conclusion that RGCs are excitatory principal neurons.     

Commissural afferents to the rat hippocampus terminate on vasoactive intestinal polypeptide-like immunoreactive non-pyramidal neurons. An EM immunocytochemical degeneration study

In the rat hippocampus, bipolar non-pyramidal neurons in stratum radiatum and stratum oriens and multipolar neurons in stratum lacunosum-moleculare react for vasoactive intestinal polypeptide (VIP) immunostaining, but pyramidal cells do not. Such bipolar VIP-like immunoreactive neurons in strata radiatum and oriens of regio superior were studied by electron microscopy for synaptic contacts with commissural afferents. The commissural fibers were identified by their anterograde degeneration induced by contralateral fimbria transections 2 days before sacrifice. Electron-dense degenerated boutons of commissural origin were found in synaptic contact with the cell bodies and dendrites of the VIP-like immunoreactive non-pyramidal cells.     

An ultrastructural study of the synaptic contacts ofα1-motoneuron axon collaterals. II. Contacts in lamina VII

Horseradish peroxidase (HRP) was injected intracellularly in triceps surae α-motoneurons. The axons and axon collaterals of these neurons were traced light and electron microscopically. Synaptic boutons of collaterals in the ventral part of Rexed's lamina VII were studied ultrastructurally. The boutons exhibited spherical synaptic vesicles and made synaptic contacts with cell bodies and proximal dendrites of neurons assumed to be Renshaw cells and with dendrites of unknown origin. The observations are discussed in relation to earlier qualitative and quantitative studies on the other known synaptic contacts of the α-motor axons, both in the central and peripheral nervous system.     

Synaptic relationships involving local axon collaterals of pyramidal neurons in the cat motor cortex

The intracortical synaptic relationships of pyramidal neurons in the cat motor cortex were studied by intracellular recording and labeling techniques. Neurons that responded with monosynaptic excitatory postsynaptic potentials (EPSPs) to microstimulation in the somatosensory cortex were identified by intracellular recordings. Long-term potentiation (LTP) was evoked in all of these neurons (n = 15), following tetanic stimulation (50 Hz, 5 s) of their afferents from the somatosensory cortex. Three of these cells (cells A-C) were identified as pyramidal neurons, following intracellular injections of Neurobiotin. The intracortical axon collaterals of these labeled cells arborized extensively, forming terminal clusters both in clse proximity to the parent soma and along their long, horizontal branches. Terminal clusters in both the proximal and in the distal termination zones of each of the cells were studied by electron microscopy. In their proximal arborization zones, the axon collaterals of the labeled pyramidal neurons synapsed preferentially with dendritic spines belonging to other pyramidal cells. In contrast, in their distal terminal clusters, the axon collateals of each of the cells formed synapses in different proportions with different postsynaptic targets. The distal axon collaterals of cell A formed 86% of their synapses with pyramidal neurons; those of cell B formed 64% of their synapses with pyramidal cells, the remaining synapses with the dendritic shafts and somata of nonpyramidal neurons, and those of cell C provided most of their output (68%) to nonpyramidal, presumably inhibitory neurons. These findings suggest a high selectivity of intrinsic axon collaterals to form specific patterns of synapses. The patterns of synaptic interactions formed by these intrinsic axon collaterals may be a substrate for shaping and modulating representation maps in the motor cortex. © 1993 Wiley-Liss, Inc.     

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.     

Spiny calretinin-immunoreactive neurons in the hilus and CA3 region of the rat hippocampus: Local axon circuits,synaptic connections,and glutamic acid decarboxylase 65/67 mRNA expression

We have used the Golgi method and Golgi electron microscopic techniques to analyze the axonal arborization and efferent connections of spiny calretinin-immunoreactive neurons in the CA3 region and hilus of the rat hippocampal formation. In the hilus, the axons of spiny calretinin-immunoreactive neurons sent out numerous collaterals that arborized in the hilar region and the molecular layer. In the CA3 region, these axons extended mainly to the stratum radiatum and pyramidal layer but also to the stratum oriens and stratum lacunosum-moleculare. Axonal varicosities were distributed widely throughout the axonal collaterals. Electron microscopic studies revealed that the axon terminals of spiny calretinin-immunoreactive neurons established synaptic contacts mainly with dendritic shafts. We next analyzed the expression of glutamic acid decarboxylase (GAD65/67) mRNAs in spiny nonpyramidal neurons that were identified by calretinin immunoreactivity. We found that spiny calretinin-positive neurons in the CA3 region and hilus of the rat hippocampal formation expressed the two isoforms of GAD: GAD65 and GAD67 mRNAs. These findings show that the spiny calretinin-immunoreactive neurons of hippocampus give rise to local axonal arborizations, suggesting that they are inhibitory. J. Comp. Neurol. 404:438–448, 1999. © 1999 Wiley-Liss, Inc.     

Oxytocin receptor agonists enhance inhibitory synaptic transmission in the rat hippocampus by activating interneurons in stratum pyramidale

Oxytocin probably plays a role as a neurotransmitter/neuromodulator in the hippocampus of the rat. Oxytocin binding sites are present in the subiculum and CA1 region and oxytocin can excite a class of CA1 nonpyramidal neurons. In the present work we characterized the effect of oxytocin on hippocampal synaptic transmission. Whole-cell recordings were obtained from pyramidal neurons, in conditions of nearly symmetrical chloride concentrations. The selective oxytocin receptor agonist, [Thr4,Gly7]-oxytocin (TGOT), caused an increase in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in virtually all neurons. These peptide-enhanced IPSCs were blocked by bicuculline, but not by strychnine, and reversed near 0 mV, indicating that they were mediated by gamma-aminobutyric acid (GABA)A receptors. On average, TGOT caused a nearly threefold increase in the frequency and almost a doubling in the amplitude of spontaneous IPSCs. TGOT did not influence the frequency and the amplitude of miniature IPSCs or spontaneous excitatory postsynaptic currents (EPSCs), and had no effect on evoked IPSCs. The peptide did not affect the basic membrane properties of pyramidal neurons or their GABA sensitivity. Thus, TGOT facilitated inhibitory transmission by exerting an excitatory action on the soma and/or dendrites of GABAergic interneurons. Extracellular recordings were performed in interneurons located in various hippocampal strata. Their sensitivity to TGOT was compared to that of substance P (SP). Interneurons in stratum pyramidale were excited both by TGOT and by SP. By contrast, stratum radiatum interneurons responded to SP but not to TGOT. In stratum oriens, half of the interneurons responded to SP, but only a minority to TGOT. Thus, oxytocin-responsive interneurons appear to be preferentially located in close vicinity of pyramidal neurons.     

Commissural fibers terminate on non-pyramidal neurons in the guinea pig hippocampus — a combined Golgi/EM degeneration study

The combined Golgi/EM method was applied to guinea pig hippocampi with acute anterograde degeneration of the commissural afferents in order to identify possible synaptic contacts between commissural terminals and non-pyramidal neurons. Degenerating, electron-dense terminals of commissural origin were found in synaptic contact with both perikarya and dendrites of two identified non-pyramidal neurons in regio superior, namely a basket cell and a bipolar neuron in stratum oriens. The observed connection may form the morphological basis for the physiologically observed feed-forward inhibition of the pyramidal cells.     

Fine structural studies of cholecystokinin-8-like immunoreactive neurons and axon terminals in the nucleus of tractus solitarius of the rat

Cholecystokinin (CCK)-8-like immunoreactive structures in the nucleus of tractus solitarius (NTS) were studied by using the peroxidase-antiperoxidase (PAP) immunohistochemical method. Immunoreactivity was localized in cell bodies and nerve fibers. The perikarya were oval or fusiform (average length 13 micron) and were mostly located in the dorsal half of the medial subnucleus of the NTS at the level of the area postrema (AP). One to three straight immunoreactive dendritelike processes emerged from the perikarya. Neurons that had first been identified under light microscopy were also studied by electron microscopy. Each neuron had a moderate amount of cytoplasm and an oval or elongated nucleus that was eccentrically located in the soma. A few synaptic inputs were found on the CCK immunoreactive perikarya, while a moderate number were seen on both proximal and distal dendrites. These neurons received both asymmetrical and symmetrical synaptic inputs. The immunoreactive dendrites were most frequently in asymmetrical synaptic contact with nonreactive boutons (max. 2.7 micron in diameter) containing fairly densely packed, small round vesicles. CCK immunoreactive boutons located in the NTS at the level of the AP were analyzed using electron microscopy; these boutons formed asymmetrical synaptic contact with other neuronal elements. Their postsynaptic targets were immunoreactive and nonreactive perikarya and dendrites. These data suggest that CCK-containing afferents might affect the neurotransmission of heterogenous types of solitary neurons.