Synaptic relationships between axon terminals from the mediodorsal thalamic nucleus and gamma-aminobutyric acidergic cortical cells in the prelimbic cortex of the rat

Although the reciprocal interconnections between the prefrontal cortex and the mediodorsal nucleus of the thalamus (MD) are well known, the involvement of inhibitory cortical interneurons in the neural circuit has not been fully defined. To address this issue, we conducted three combined neuroanatomical studies on the rat brain. First, the frequency and the spatial distribution of synapses made by reconstructed dendrites of nonpyramidal neurons were identified by impregnation of cortical cells with the Golgi method and identification of thalamocortical terminals by degeneration following thalamic lesions. Terminals from MD were found to make synaptic contacts with small dendritic shafts or spines of Golgi-impregnated nonpyramidal cells with very sparse dendritic spines. Second, a combined study that used anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and postembedding gamma-aminobutyric acid (GABA) immunocytochemistry indicated that PHA-L-labeled terminals from MD made synaptic junctions with GABA-immunoreactive dendritic shafts and spines. Nonlabeled dendritic spines were found to receive both axonal inputs from MD with PHA-L labelings and from GABAergic cells. In addition, synapses were found between dendritic shafts and axon terminals that were both immunoreactive for GABA. Third, synaptic connections between corticothalamic neurons that project to MD and GABAergic terminals were investigated by using wheat germ agglutinin conjugated to horseradish peroxidase and postembedding GABA immunocytochemistry. GABAergic terminals in the prelimbic cortex made symmetrical synaptic contacts with retrogradely labeled corticothalamic neurons to MD. All of the synapses were found on cell somata and thick dendritic trunks. These results provide the first demonstration of synaptic contacts in the prelimbic cortex not only between thalamocortical terminals from MD and GABAergic interneurons but also between GABAergic terminals and corticothalamic neurons that project to MD. The anatomical findings indicate that GABAergic interneurons have a modulatory influence on excitatory reverberation between MD and the prefrontal cortex.     

Early sequential formation of functional GABA(A) and glutamatergic synapses on CA1 interneurons of the rat foetal hippocampus

During postnatal development of CA1 pyramidal neurons, GABAergic synapses are excitatory and established prior to glutamatergic synapses. As interneurons are generated before pyramidal cells, we have tested the hypothesis that the GABAergic interneuronal network is operative before glutamate pyramidal neurons and provides the initial patterns of activity. We patch-clamp recorded interneurons in foetal (69 neurons) and neonatal P0 (162 neurons) hippocampal slices and performed a morphofunctional analysis of biocytin-filled neurons. At P0, three types of interneurons were found: (i) non-innervated "silent" interneurons (5%) with no spontaneous or evoked synaptic currents; (ii) G interneurons (17%) with GABA(A) synapses only; and (iii) GG interneurons with GABA and glutamatergic synapses (78%). Relying on the neuronal capacitance, cell body size and arborization of dendrites and axons, the three types of interneurons correspond to three stages of development with non-innervated neurons and interneurons with GABA(A) and glutamatergic synapses being, respectively, the least and the most developed. Recordings from both pyramidal neurons and interneurons in foetuses (E18-20) revealed that the majority of interneurons (65%) had functional synapses whereas nearly 90% of pyramidal neurons were quiescent. Therefore, interneurons follow the same GABA-glutamate sequence of synapse formation but earlier than the principal cells. Interneurons are the source and the target of the first synapses formed in the hippocampus and are thus in a position to modulate the development of the hippocampus in the foetal stage.     

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.     

Kalirin‐7, an important component of excitatory synapses,is regulated by estradiol in hippocampal neurons

Estradiol enhances the formation of dendritic spines and excitatory synapses in hippocampal neurons in vitro and in vivo, but the underlying mechanisms are not fully understood. Kalirin‐7 (Kal7), the major isoform of Kalirin in the adult hippocampus, is a Rho GDP/GTP exchange factor localized to postsynaptic densities. In the hippocampus, both Kal7 and estrogen receptor α (ERα) are highly expressed in a subset of interneurons. Over‐expression of Kal7 caused an increase in spine density and size in hippocampal neurons. To determine whether Kalirin might play a role in the effects of estradiol on spine formation, Kal7 expression was examined in the hippocampus of ovariectomized rats. Estradiol replacement increased Kal7 staining in both CA1 pyramidal neurons and interneurons in ovariectomized rats. Estradiol treatment of cultured hippocampal neurons increased Kal7 levels at the postsynaptic side of excitatory synapses and increased the number of excitatory synapses along the dendrites of pyramidal neurons. These increases were mediated via ERα because a selective ERα agonist, but not a selective ERβ agonist, caused a similar increase in both Kal7 levels and excitatory synapse number in cultured hippocampal neurons. When Kal7 expression was reduced using a Kal7‐specific shRNA, the density of excitatory synapses was reduced and estradiol was no longer able to increase synapse formation. Expression of exogenous Kal7 in hippocampal interneurons resulted in decreased levels of GAD65 staining. Inhibition of GABAergic transmission with bicuculline produced a robust increase in Kal7 expression. These studies suggest Kal7 plays a key role in the mechanisms of estradiol‐mediated synaptic plasticity. © 2010 Wiley‐Liss, Inc.     

Dendroaxonic synapses in the substantia gelatinosa glomeruli of the spinal trigeminal nucleus of the cat

The glomeruli in the substantia gelatinosa layer of the spinal trigeminal nucleus of the cat contain three kinds of dendritic processes. One of these, the type 2 dendrite, contains large synaptic vesicles in its spine heads and in its shaft. The type 2 dendrite receives axodendritic synapses from primary trigeminal afferent (C) axons and an occasional axodendritic synapse from small axonal (P) endings with small synaptic vesicles. The type 2 dendrites in turn form dendroaxonic synapses on the C endings. The dendroaxonic synapse and the axodendritic synapse of the C ending typically occur in reciprocal pairs. The axodendritic synapse usually lies in the depths of scalloped depressions in the surface of the C ending while the dendroaxonic synapse is found on the rim of the depression. Type 1 spines, i.e., dendritic spines receiving axodendritic synapses from the primary ending and lacking synaptic vesicles, also receive dendrodendritic synapses from type 2 dendrites. The types 2 dendrite with its large, rounded synaptic vesicles is considered to be excitatory at its dendroaxonic and dendrodendritic synapses. The type 2 dendrites course from glomerulus to glomerulus receiving their excitatory input through the axodendritic synapses of C axons. A type 2 dendrite, in response to C axon excitation would activate type 1 spines directly through their dendrodendritic synapses (C→2→1) and indirectly by increasing transmitter release at the axodendritic synapses of the C axonal endings through their dendroaxonic synapses (2→C→1). The type 2 dendrites could serve two functions. First, they may prolong transmitter release from the axodendritic synapses of C axonal endings beyond the time of arrival of incoming action potentials because of the reciprocal pairing of dendroaxonic and axodendritic synapses (C?2). Second, they may extend the spatial range of the excitatory output of active primary afferent axons to type 1 spines of glomeruli whose primary afferent axons may be inactive (C→2→1).     

Electron microscopic immunocytochemical study of the distribution of parvalbumin-containing neurons and axon terminals in the primate dentate Gyrus and Ammon's horn

Five green monkeys were examined with light and electron microscopic preparations to explore the regional differences in the distribution of parvalbumin (PV)-positive neurons and axon terminals in the primate hippocampus. PV-positive neurons were mainly found in the hilus of the dentate gyrus and the strata oriens and pyramidale of Ammon's horn. In electron microscopic preparations, the PV-positive cells displayed nuclear infoldings, intranuclear rods, a large rim of perikaryal cytoplasm with numerous organelles and both asymmetric and symmetric axosomatic synapses. One prominent PV-positive cell type in CA1 was a large multipolar neuron that resembled the large basket cells of the neocortex. Although most PV-positive dendrites were aspiny and postsynaptic to numerous axon terminals, some PV-positive dendrites in the molecular layer of the dentate gyrus displayed filipodia-like appendages with no synapses or spines that were postsynaptic to multiple axon terminals. The PV-positive dendrites in the hilus and stratum oriens were apposed at specialized junctions that resembled gap junctions. PV-positive axons were concentrated in the principal cell layers, and formed axosomatic, axodendritic, and axon initial segment synapses. In cases where these axons were observed to appose the surface of granule cells for a long length, only one axosomatic symmetric synapse per cell was found. In the hilus, PV-positive axon terminals formed synapses onto thorny excrescences of spiny cells. Both semithin sections and electron microscopic preparations indicated that more PV-positive axon terminals formed symmetric axosomatic synapses with pyramidal cells in CA2 than in CA1 and CA3. Also, CA2 displayed a unique plexus of PV-positive axon terminals in stratum lacunosum moleculare. These results indicate that the PV-positive hippocampal cells form a subset of GABAergic local circuit neurons, including the basket and chandelier cells. The ubiquitous finding of PV-positive dendrites linked by gap junctions throughout the dentate gyrus and Ammon's horn adds further data to indicate that this subset of GABAergic neurons is linked electrotonically. The synaptic organization of PV-positive neurons in the hippocampus suggests their participation in both feedback and feedforward inhibition. The PV-positive neurons in the hippocampus are only a proportion of the basket and chandelier cells, whereas virtually all of these cells in neocortex are PV-positive. © 1993 Wiley-Liss, Inc.     

High level of mGluR7 in the presynaptic active zones of select populations of GABAergic terminals innervating interneurons in the rat hippocampus

The release of neurotransmitters is modulated by presynaptic metabotropic glutamate receptors (mGluRs), which show a highly selective expression and subcellular location in glutamatergic terminals in the hippocampus. Using immunocytochemistry, we investigated whether one of the receptors, mGluR7, whose level of expression is governed by the postsynaptic target, was present in GABAergic terminals and whether such terminals targeted particular cells. A total of 165 interneuron dendritic profiles receiving 466 synapses (82% mGluR7a-positive) were analysed. The presynaptic active zones of most GAD-(77%) or GABA-positive (94%) synaptic boutons on interneurons innervated by mGluR7a-enriched glutamatergic terminals (mGluR7a-decorated) were immunopositive for mGluR7a. GABAergic terminals on pyramidal cells and most other interneurons in str. oriens were mGluR7a-immunonegative. The mGluR7a-decorated cells were mostly somatostatin- and mGluR1alpha-immunopositive neurons in str. oriens and the alveus. Their GABAergic input mainly originated from VIP-positive terminals, 90% of which expressed high levels of mGluR7a in the presynaptic active zone. Parvalbumin-positive synaptic terminals were rare on mGluR7a-decorated cells, but on these neurons 73% of them were mGluR7a-immunopositive. Some type II synapses innervating interneurons were immunopositive for mGluR7b, as were some type I synapses. Because not all target cells of VIP-positive neurons are known it has not been possible to determine whether mGluR7 is expressed in a target-cell-specific manner in the terminals of single GABAergic cells. The activation of mGluR7 may decrease GABA release to mGluR7-decorated cells at times of high pyramidal cell activity, which elevates extracellular glutamate levels. Alternatively, the presynaptic receptor may be activated by as yet unidentified endogenous ligands released by the GABAergic terminals or the postsynaptic dendrites.     

Axon arbors and synaptic connections of a vulnerable population of interneurons in the dentate gyrus in vivo

The predominant gamma-aminobutyric acid (GABA)ergic neuron class in the hilus of the dentate gyrus consists of spiny somatostatinergic interneurons. We examined the axon projections and synaptic connections made by spiny hilar interneurons labeled with biocytin in gerbils in vivo. Axon length was 152-497 mm/neuron. Sixty to 85% of the axon concentrated in the outer two thirds of the molecular layer of the dentate gyrus. The septotemporal span of the axon arbor extended over 48-82% of the total hippocampal length, which far exceeds the septotemporal span of axons of granule cells whose complete axon arbors extended over 15-29%. A three-dimensionally reconstructed 216-microm-long spiny hilar interneuron axon segment in the outer third of the molecular layer formed an average of 1 synapse every 5.1 microm. Of the 42 symmetric (inhibitory) synapses formed by the reconstructed segment, 88% were with spiny dendrites of presumed granule cells, and 67% were with dendritic spines that also receive an asymmetric (excitatory) contact from an unlabeled axon terminal. Postembedding GABA-immunocytochemistry revealed that 55% of the GABAergic synapses in the outer third of the molecular layer were with spines. Therefore, in the outer molecular layer, spiny hilar interneurons form synaptic contacts that appear to be positioned to exert inhibitory control near sites of excitatory synaptic input from the entorhinal cortex to granule cell dendritic spines. These findings demonstrate far-reaching, yet highly specific, connectivity of individual interneurons and suggest that the loss of spiny hilar interneurons, as occurs in temporal lobe epilepsy, may contribute to hyperexcitability in the hippocampus.     

Ultrastructure of stratum lacunosum-moleculare interneurons of hippocampal CA1 region

Intracellular recordings were obtained from nonpyramidal neurons (interneurons) in stratum lacunosum-moleculare (L-M) of the CA1 region of guinea pig hippocampal slices. These interneurons had response characteristics that distinguish them from pyramidal cells and other interneuron types: the L-M neurons had relatively broad action potentials with large spike afterhyperpolarizations, high input resistance and little spike-firing adaptation, and low spontaneous activity. Lucifer Yellow (LY) and horseradish peroxidase (HRP) were injected intracellularly into physiologically identified L-M interneurons, and the cells were characterized morphologically using light and electron microscopy. L-M somata were fusiform-shaped (15 x 25 micron), had multiple processes, and were located at the border between stratum (str.) lacunosum-moleculare and str. radiatum. L-M dendrites coursed through str. lacunosum-moleculare and projected into str. radiatum. L-M axons made axodendritic synaptic contacts primarily in str. lacunosum-moleculare and str. radiatum, but also in str. moleculare of the dentate gyrus. These axodendritic synaptic contacts were made onto spiny dendritic processes (presumably pyramidal cell and granule cell dendrites) and onto aspinous dendrites (presumably interneuron dendrites), and appeared to be of the symmetric type (type 2), characteristic of inhibitory synapses. In separate groups of animals, selective lesions were made of afferents to the CA1 and dentate regions of hippocampus, and subsequent degeneration of contacts and L-M interneuron somata and dendrites was examined at the ultrastructural level. Fibers originating from contralateral and ipsilateral CA3 region, and from ipsilateral entorhinal cortex, were found to make synaptic contact onto presumed L-M interneurons. Degenerating terminals appeared to be of the asymmetric type (type 1), characteristic of excitatory synapses. These morphological data are consistent with electrophysiological results showing that L-M interneurons can mediate feedforward inhibition of CA1 pyramidal cells.     

Somatostatin interneurons delineate the inner part of the external plexiform layer in the mouse main olfactory bulb

Neuropeptides play a major role in the modulation of information processing in neural networks. Somatostatin, one of the most concentrated neuropeptides in the brain, is found in many sensory systems including the olfactory pathway. However, its cellular distribution in the mouse main olfactory bulb (MOB) is yet to be characterized. Here we show that ≈95% of mouse bulbar somatostatin‐immunoreactive (SRIF‐ir) cells describe a homogeneous population of interneurons. These are restricted to the inner lamina of the external plexiform layer (iEPL) with dendritic field strictly confined to the region. iEPL SRIF‐ir neurons share some morphological features of Van Gehuchten short‐axon cells, and always express glutamic acid decarboxylase, calretinin, and vasoactive intestinal peptide. One‐half of SRIF‐ir neurons are parvalbumin‐ir, revealing an atypical neurochemical profile when compared to SRIF‐ir interneurons of other forebrain regions such as cortex or hippocampus. Somatostatin is also present in fibers and in a few sparse presumptive deep short‐axon cells in the granule cell layer (GCL), which were previously reported in other mammalian species. The spatial distribution of somatostatin interneurons in the MOB iEPL clearly outlines the region where lateral dendrites of mitral cells interact with GCL inhibitory interneurons through dendrodendritic reciprocal synapses. Symmetrical and asymmetrical synaptic contacts occur between SRIF‐ir dendrites and mitral cell dendrites. Such restricted localization of somatostatin interneurons and connectivity in the bulbar synaptic network strongly suggest that the peptide plays a functional role in the modulation of olfactory processing. J. Comp. Neurol. 518:1976–1994, 2010. © 2010 Wiley‐Liss, Inc.     

Differential synaptic distribution of the AMPA-GluR2 subunit on GABAergic and non-GABAergic neurons in the basolateral amygdala

The cellular and ultrastructural distribution patterns of the AMPA glutamate receptor subunit, GluR2, were determined in the rat basolateral amygdala. GluR2 immunoreactivity was widely and uniformly distributed in the basolateral nucleus, with both pyramidal and non-pyramidal neurons labelled. In fact, double label immunohistochemical analyses demonstrated that over 90% of the GABAergic interneurons were labelled for GluR2. Electron microscopic analyses further confirmed the presence of GluR2 in the soma and dendrites of GABAergic interneurons as well as in the soma, spines and dendritic shafts of pyramidal cells. As in our parallel study in the rat hippocampus, immunogold analyses revealed that GluR2 immunoreactivity was frequently preferentially located at asymmetric synapses on both pyramidal cell spines and shafts, as well as the dendritic processes and soma of GABAergic interneurons. However, the number of immunogold particles per labelled synapse on GABAergic neurons was significantly lower than at similar labelled asymmetric synapses on spines of presumed pyramidal cells. Given that the presence of GluR2 within the AMPA receptor complex decreases calcium flux, these data indicate that GABAergic local circuit neurons might possess AMPA receptors with higher calcium permeability on average than pyramidal cells, as has been suggested for hippocampus. Such cell class-specific differences in the subunit representation and resultant channel properties of AMPA receptors have implications for response properties as well as selective vulnerability of neurons within the basolateral nucleus of the amygdala.     

Dendrodendritic synapses in the cat reticularis thalami nucleus: a structural basis for thalamic spindle synchronization

Dendrodentritic synapses have been found between the dendrites of reticularis thalami neurons. These synapses were symmetric and the presynaptic element contained pleomorphic vesicles. A few cases of reciprocal dendrodendritic synapses were also observed. Given the morphological features of the synapses and the well-established GABAergic nature of reticularis neurons it is concluded that reticularis cell dendrites form a local inhibitory network. The functional implications of this type of organization for the synchronization of spindle oscillations are discussed and a new hypothesis is proposed.     

Analysis of gamma-aminobutyric acidergic synaptic contacts in the thalamic reticular nucleus of the monkey

Gamma-aminobutyric acidergic (GABAergic) neurons in the thalamic reticular nucleus (TRN) spontaneously generate a synchronous bursting rhythm during slow-wave sleep in most mammals. A previous study at the electron microscopic level in cat anterior TRN has suggested that synchronous bursting activity could result from the large number of presumably GABAergic dendrodendritic synaptic contacts. However, little is known about the synaptology of the monkey thalamic reticular nucleus and whether it contains dendrodendritic contacts. To address this issue, we examined tissue obtained from Macaca fascicularis that was prepared for electron microscopy using postembedding techniques to demonstrate GABA immunoreactivity. Examination of the anterior (motor) and posterior (somatosensory) portions of the TRN disclosed the following: The majority of synaptic contacts (87.5% of 958) were formed by axon terminals showing no GABA immunoreactivity and making asymmetric synaptic contacts on dendrites or cell bodies. A further 6.4% of synaptic contacts was composed of GABA-immunoreactive presynaptic terminals making symmetric contacts with the dendrites of TRN neurons. The majority resembled the pleomorphic vesicle containing F-terminals seen in the dorsal thalamus and known to originate from axons of TRN. A subset or possible second class did not resemble any previously described class of GABA-immunoreactive terminals in the TRN. Both classes of these terminals making symmetric contacts may originate wholly or partially within the nucleus. There was one dendrodendritic synaptic contact and only a small number (3.2%) of axodendritic contacts with synaptic vesicles visible both pre- and postsynaptically. We conclude that dendrodendritic contacts are probably not responsible for the synchronized bursting neuronal activity seen in the slow-wave sleep of monkeys, and that, if TRN neurons are coupled synaptically, the most likely mechanism is through the synapses formed by recurrent axon collaterals of TRN neurons onto TRN dendrites. © 1994 Wiley-Liss, Inc.     

The innervation of parvalbumin-containing interneurons by VIP-immunopositive interneurons in the primary somatosensory cortex of the adult rat

gamma-Aminobutyric acid (GABA)ergic interneurons of neocortex consist of many subgroups with extremely heterogeneous morphological, physiological and molecular properties. To explore the putative effect of the vasoactive intestinal polypeptide-immunopositive (VIP +) neurons on neocortical circuitry, the number and distribution of VIP + boutons were analysed on somatodendritic domains of 272 parvalbumin immunopositive (PV +) 3D-reconstructed neurons. The synaptic nature of 91% of somatic and 76% of dendritic contacts was verified by electron microscopy. The target PV + neurons were separated in two significantly different groups by means of cluster analysis. The first group (Cluster 1, 26%) received on average five times more VIP + synapses than those of the second group. The second group (Cluster 2, 74%) contained cells that were poorly innervated by VIP + boutons or did not have either somatic or dendritic or any VIP innervation at all. The cells of Cluster 1 had a soma size and total dendritic length significantly smaller than that of Cluster 2, however, they received three times more dendritic synapses, which resulted in a five times higher VIP + synaptic density on dendrites. Our results showed that although most of the PV + cells are innervated by VIP + boutons at a varying degree, some 6% of PV + cells received no input from VIP + interneurons. This suggests a refined morphological basis to influence the majority of the PV + interneurons, which are very effectively controlling pyramidal cell firing. Together with metabolic and neuromodulatory effects of VIP, this would probably result in an enhanced responsiveness of the latter cell type to tactile stimuli.     

Ultrastructural study of nitric oxide synthase-containing striatal neurons and their relationship with parvalbumin-containing neurons in rats

Single- and double-label electron microscopic immunocytochemistry was used to examine the ultrastructure of striatal neurons containing nitric oxide synthase (NOS⁺) and evaluate the synaptic relationship of NOS⁺ striatal neurons with those containing parvalbumin (PV⁺). In both the single-label and double-label studies, NOS⁺ perikarya were observed to possess polylobulated nuclei. In the single-label studies, NOS⁺ terminals were seen forming synaptic contacts with dendritic shafts and dendritic spines that did not contain NOS, but not with NOS⁺ perikarya or dendrites. In the double-label studies (using diaminobenzidine and silver intensified immunogold as markers), nitric oxide synthase and parvalbumin immunoreactions were found in two different populations of medium-sized aspiny striatal neurons. The PV⁺ axon terminals were seen forming symmetric synapses on the dendritic spines of neurons devoid of PV or NOS labeling, on PV⁺ dendrites, and on NOS⁺ soma and dendrites. In contrast, NOS⁺ terminals were not observed to form synaptic contacts with the dendrites or soma of either PV⁺ or NOS⁺ neurons. These findings suggest that NOS⁺ striatal interneurons form synaptic contact with the spines and presumably the dendrites of striatal projection neurons, but not with the dendrites or soma of PV⁺ or NOS⁺ striatal interneurons. NOS⁺ neurons do, however, receive synaptic input from PV⁺ neurons.     

Synaptic integration of newly generated neurons in rat dissociated hippocampal cultures

In the dentate gyrus of the hippocampus new neurons are born from precursor cells throughout development and into adulthood. These newborn neurons hold significant potential for self-repair of brain damage caused by neurodegenerative disease. However, the mechanism by which newborn neurons integrate into the brain is not understood due to a lack of knowledge of the molecular and functional characteristics of the synapses formed by newborn neurons. Here we report that dissociated hippocampal cultures continue to produce new granule cells in vitro that fire action potentials and become synaptically integrated into the existing network of mature hippocampal neurons. Quantification of the expression of synaptic proteins at newborn and mature granule cell synapses revealed synapse development onto newborn neurons occurs sequentially with initial synaptic contacts evident from 6 days after cell birth. These data also showed that the dendrites of newborn neurons have a high density of Piccolo and Bassoon puncta on them and therefore have a high potential to be integrated into the neuronal network through new synaptic connections. Electrophysiological recordings from newborn neurons reveal these synapses are functional within 10 days of cell birth. GABAergic input synapses were found to mature faster in newborn neurons than glutamatergic synapses where sequential recruitment of postsynaptic glutamate receptors occurred. Group I metabotropic glutamate receptors (mGluR1/5) were present at higher levels compared with ionotropic glutamate receptors (NMDA and AMPA receptors), suggesting that metabotropic and ionotropic receptors play differential roles at glutamatergic synapses in the integration and the maturation of newborn neurons. These data show that dissociated hippocampal cultures can provide a useful model system in which to study the integration of newborn neurons into existing neuronal circuits to increase our understanding of how the function of newborn neuron synapses could contribute to restoring damaged neuronal networks.     

Ultrastructural study of cholecystokinin-immunoreactive cells and processes in area CA1 of the rat hippocampus

We used light and electron microscopic immunocytochemical methods to examine the structure of neuronal perikarya and processes containing cholecystokinin-like immunoreactivity (CCK-IR) in area CA1 of the rat hippocampus. The morphology of stained perikarya, their positions within all laminae, and the orientation of their dendrites indicate that CCK-IR is located in interneurons. These cells were seen in the electron microscope to have deeply folded nuclei and to receive both symmetric and asymmetric synaptic junctions on their cell somata and dendritic shafts. Their dendrites are essentially spine-free, but form bulges at the site of some asymmetric synaptic junctions. Axonal varicosities containing CCK-IR make symmetric synaptic junctions with cell somata and dendritic shafts of both pyramidal and non-pyramidal neurons. In addition, CCK-IR varicosities form symmetric junctions with unstained non-pyramidal neurons and with CCK-IR cells, suggesting either recurrent innervation of one cell on itself or interaction between interneurons. The presence of CCK-IR varicosities and synaptic junctions on pyramidal cells is in agreement with physiological data which indicate that CCK has a direct postsynaptic action. The observation of CCK-IR varicosities forming synaptic junctions on non-pyramidal cells suggests that CCK might also modify the response of interneurons.     

Parvalbumin immunoreactivity in the thalamus of guinea pig: Light and electron microscopic correlation with gamma-aminobutyric acid immunoreactivity

The relationship of the calcium binding protein parvalbumin (PV) with gamma-aminobutyric acidergic (GABAergic) neurons differs within different thalamic nuclei and animal species. In this study, the distribution of PV and GABA throughout the thalamus of the guinea pig was investigated at the light microscopic level by using immunoperoxidase methods. Intense PV labelling was found in all the GABAergic neurons of the reticular nucleus and in scattered GABAergic neurons in the anteroventral nucleus, whereas GABAergic interneurons in the ventrobasal and lateral geniculate nuclei were not PV labelled. At the electron microscopic level, preembedding immunuperoxidase for PV was combined with postembedding immunogold for GABA. In the ventrobasal nucleus, four types of profiles were recognized: 1) terminals with flattened vesicles and forming symmetric synapses, which were labelled with both PV and GABA and could therefore be identified as afferents from the reticular nucleus; 2) boutons morphologically similar to presynaptic dendrites of interneurons, labelled only with GABA; 3) large terminals with round vesicles and asymmetric synapses, labelled only with PV, which contacted GABAergic presynaptic dendrites in glomerular arrangements and resembled ascending excitatory afferents; and 4) terminals unlabelled by either antiserum. In the ventrobasal nucleus of the guinea pig a double immunocytochemical labelling permits therefore the differentiation of two populations of GABAergic vesicle-containing profiles, i. e., the terminals originating from reticular nucleus (that are double labelled) and the presynaptic dendrites originating from interneurons (that are GABA-labelled only). The possibility to differentiate GABAergic inputs from the reticular nucleus and from interneurons can shed light to the functional interpretation of synaptic circuits in thalamic sensory nuclei. © 1994 Wiley-Liss, Inc.     

Enkephalin‐containing interneurons are specialized to innervate other interneurons in the hippocampal CA1 region of the rat and guinea‐pig

Enkephalins are known to have a profound effect on hippocampal inhibition, but the possible endogenous source of these neuropeptides, and their relationship to inhibitory interneurons is still to be identified. In the present study we analysed the morphological characteristics of met-enkephalin-immunoreactive cells in the CA1 region of the rat and guinea-pig hippocampus, their coexistence with other neuronal markers and their target selectivity at the light and electron microscopic levels. Several interneurons in all subfields of the hippocampus were found to be immunoreactive for met-enkephalin. In the guinea-pig, fibres arising from immunoreactive interneurons were seen to form a plexus in the stratum oriens/alveus border zone, and basket-like arrays of boutons on both enkephalin-immunoreactive and immunonegative cell bodies in all strata. Immunoreactive boutons always established symmetric synaptic contacts on somata and dendritic shafts. Enkephalin-immunoreactive cells co-localized GABA, vasoactive intestinal polypeptide and calretinin. Postembedding immunogold staining for GABA showed that all the analysed enkephalin-immunoreactive boutons contacted GABAergic postsynaptic structures. In double-immunostained sections, enkephalin-positive axons were seen to innervate calbindin D28k-, somatostatin-, calretinin- and vasoactive intestinal polypeptide-immunoreactive cells with multiple contacts. Based on these characteristics, enkephalin-containing cells in the hippocampus are classified as interneurons specialized to innervate other interneurons, and represent a subset of vasoactive intestinal polypeptide- and calretinin-containing cells. The striking match of ligand and receptor distribution in the case of enkephalin-mediated interneuronal communication suggests that this neuropeptide may play an important role in the synchronization and timing of inhibition involved in rhythmic network activities of the hippocampus.     

Differential regulation of spontaneous and evoked inhibitory synaptic transmission in somatosensory cortex by retinoic acid

Retinoic acid (RA), a developmental morphogen, has emerged in recent studies as a novel synaptic signaling molecule that acts in mature hippocampal neurons to modulate excitatory and inhibitory synaptic transmission in the context of homeostatic synaptic plasticity. However, it is unclear whether RA is capable of modulating neural circuits outside of the hippocampus, and if so, whether the mode of RA's action at synapses is similar to that within the hippocampal network. Here we explore for the first time RA's synaptic function outside the hippocampus and uncover a novel function of all‐trans retinoic acid at inhibitory synapses. Acute RA treatment increases spontaneous inhibitory synaptic transmission in L2/3 pyramidal neurons of the somatosensory cortex, and this effect requires expression of RA's receptor RARα both pre‐ and post‐synaptically. Intriguingly, RA does not seem to affect evoked inhibitory transmission assayed with either extracellular stimulation or direct activation of action potentials in presynaptic interneurons at connected pairs of interneurons and pyramidal neurons. Taken together, these results suggest that RA's action at synapses is not monotonous, but is diverse depending on the type of synaptic connection (excitatory versus inhibitory) and circuit (hippocampal versus cortical). Thus, synaptic signaling of RA may mediate multi‐faceted regulation of synaptic plasticity. In addition to its classic roles in brain development, retinoic acid (RA) has recently been shown to regulate excitatory and inhibitory transmission in the adult brain. Here, the authors show that in layer 2/3 (L2/3) of the somatosensory cortex (S1), acute RA induces increases in spontaneous but not action‐potential evoked transmission, and that this requires retinoic acid receptor (RARα) both in presynaptic PV‐positive interneurons and postsynaptic pyramidal (PN) neurons.