Distribution of calretinin-immunoreactivity in the rat entorhinal cortex: Coexistence with GABA

Inhibitory neurons in the entorhinal cortex control information flow between the cortical areas and the hippocampus. We characterized the inhibitory circuits in the rat entorhinal cortex by analyzing the distribution of calretinin-immunoreactivity and its colocalization with glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA). The location of calretinin-immunoreactive (IR) neurons and terminals varies between the different layers and subfields of the entorhinal cortex. The immunopositive neurons can be divided into two major morphological classes: bipolar and multipolar, which have two or more long, aspiny or sparsely spiny dendrites that extend through several layers. In addition, there are unclassified immunopositive neurons that have large lightly stained somata. They are located primarily in layer V. Colocalization analyses with GAD and GABA revealed that approximately 40% (657 out of 1,777) of all calretinin-IR cells within the entorhinal cortex contain GAD or GABA. In layers I–III, over 90% of the calretinin-IR neurons contain GAD or GABA. In layers V–VI, however, most of the calretinin-IR neurons do not colocalize with either GAD or GABA. The distribution patterns of calretinin-immunoreactivity in the entorhinal cortex is consistent with the partitioning of the rat entorhinal cortex into six subfields. Furthermore, calretinin is expressed in a morphologically heterogeneous population of cells in the rat entorhinal cortex which includes both GABAergic and non-GABAergic neurons. J. Comp. Neurol. 378:363–378, 1997. © 1997 Wiley-Liss, Inc.     

GABA neurons in the superficial layers of the rat dorsal cochlear nucleus: light and electron microscopic immunocytochemistry

This article is an application of light and electron microscopic immunocytochemistry to the study of the neuronal circuit of the superficial layers in the rat dorsal cochlear nucleus (DCN). An antiserum against the intrinsic marker glutamate decarboxylase (GAD) is used to identify and map axon terminals and neurons that use gamma aminobutyric acid (GABA) as a neurotransmitter. It is demonstrated that layers 1 and 2 of the DCN contain a very high density of GABAergic boutons, matched only by the granule cell domains of the ventral cochlear nucleus, especially the superficial granule cell domain. These two layers also contain much higher concentrations of GABAergic cell bodies than all other magnocellular regions of the cochlear nuclear complex. Cartwheel and stellate neurons, and probably also Golgi cells, previously characterized in Golgi and electron microscopic investigations, appear immunostained and, therefore, are presumably inhibitory. The synaptic relations between parallel fibers, the axons of granule cells, and cartwheel and stellate neurons are confirmed. The present study also supports the conclusion that stellate cells are coupled to one another by gap junctions. Also scattered in layer 1 are large, GABAergic neurons that occur with irregular frequency and presumably represent displaced Purkinje cells, previously identified with a Purkinje-cell-specific marker. Granule neurons and pyramidal neurons remain unstained, even after topical injection of colchicine, which enhances immunostaining of the other glutamate-decarboxylase-positive cells, and therefore must use transmitters different from GABA. The possible analogies between the spiny cartwheel and the aspiny stellate cells of the DCN and the cerebellar Purkinje and stellate/basket cells are discussed in the light of data from Golgi, electron microscopy, and transmitter imunocytochemistry.     

Synaptic responses of cortical pyramidal neurons to light stimulation in the isolated turtle visual system

The resistance of the turtle brain to hypoxic injury permits a unique in vitro preparation in which the organization and function of visual cortex can be explored. Intracellular recordings from cortical pyramidal neurons revealed biphasic responses to flashes of light, consisting of an early phase (50-100 msec) of concurrent inhibitory and excitatory activation, followed by a longer, inhibitory phase (250-600 msec) composed of summated Cl- -dependent postsynaptic potentials mediated by GABA. This response sequence results from the coactivation of pyramidal and GABAergic non-pyramidal cells, followed by feed-forward and possibly feed-back pyramidal cell inhibition, and is partly dependent on differences in the membrane properties of pyramidal and non-pyramidal neurons.     

Subcellular localization of GABA(B) receptor subunits in rat visual cortex

Although studies in the visual cortex have found gamma-aminobutyric acid B (GABA(B)) receptor-mediated pre- and postsynaptic inhibitory effects on neurons, the subcellular localization of GABA(B) receptors in different types of cortical neurons and synapses has not been shown directly. To provide this information, we have used antibodies against the GABA(B) receptor (R)1a/b and GABA(B)R2 subunits and have studied the localization of immunoreactivities in rat visual cortex. Light microscopic analyses have shown that both subunits are expressed in cell bodies and dendrites of 65-92% of corticocortically projecting pyramidal neurons and in 92-100% of parvalbumin (PV)-, calretinin (CR)-, and somatostatin (SOM)-containing GABAergic neurons. Electron microscopic analyses of immunoperoxidase- and immunogold-labeled tissue revealed staining in the nucleus, cytoplasm and cell surface membranes with both antibodies. Colocalization of both subunits was observed in all of these structures. GABA(B)R1a/b and GABA(B)R2 were concentrated in excitatory and inhibitory synapses and in extrasynaptic membranes. In GABAergic synapses, GABA(B)R1a/b and GABA(B)R2 were more strongly expressed postsynaptically on pyramidal and nonpyramidal cells than presynaptically. In type 1 synapses GABA(B)R1a/b and GABA(B)R2 was found in pre- and postsynaptic membranes. The nuclear localization of GABA(B)R1 and GABA(B)R2 subunits suggests a novel role for neurotransmitter receptors in controlling gene expression. The synaptic colocalization of GABA(B)R1 and GABA(B)R2 indicates that subunits form heteromeric assemblies of the functional receptor in inhibitory and excitatory synapses. Subunit coexpression in GABAergic synapses that include PV-containing and PV-deficient terminals suggests that pre- and postsynaptic GABA(B) receptor activation is provided by several different types of interneurons. The coexpression of both subunits in excitatory synapses suggests a role for GABA(B) receptors in the regulation of glutamate release and raises the question how these receptors are activated in the absence of pre-or postsynaptic GABAergic synaptic inputs to excitatory synapses.     

NOS-positive local circuit neurons are exclusively axo-dendritic cells both in the neo- and archi-cortex of the rat brain

Neuronal nitric oxide synthase (nNOS)-containing neurons and axon terminals were examined in the rat somatosensory and temporal neocortex, in the CA3/a-c areas of Ammon's horn and in the hippocampal dentate gyrus. In these areas, only nonpyramidal neurons were labeled with the antibody against nNOS. Previous observations suggested that all nNOS-positive nonpyramidal cells are GABAergic local circuit neurons, which form exclusively symmetric synapses. In agreement with this, nNOS-positive axon terminals in the hippocampal formation formed symmetric synapses exclusively with dendritic shafts. In the neocortex, in contrast, in addition to the nNOS-positive axon terminals that formed synapses with unlabeled spiny and aspiny dendrites and with nNOS-positive aspiny dendrites, a small proportion of the nNOS-positive axon terminals formed symmetric synapses with dendritic spines. These results suggest that nNOS-positive local circuit neurons form a distinct group of axo-dendritic cells displaying slightly different domain specificity in the archi- and neocortex. However, nNOS-positive cells show no target selectivity, because they innervate principal cells and local circuit neurons. Afferents to the NOS-positive cells display neither domain nor target selectivity, because small unlabeled terminals formed synapses with both the soma or dendrites of nNOS-positive neurons and an adjacent unlabeled dendrite or spine in both the hippocampal formation and in neocortex.     

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.     

Laminar and cellular localization of cytochrome oxidase in the cat striate cortex

Cytochrome oxidase (C.O.) was histochemically localized in the cat striate cortex at the light and electron microscopic levels. The results indicate that the oxidative metabolic activity within the cat striate cortex may vary between (1) different laminae, (2) neurons and glia, (3) different neuron types, (4) dendrite and soma of the same cell, (5) different types of dendrites, (6) different segments of the same dendrite, and (7) different classes of symmetric and asymmetric axon terminals. Maximal laminar C.O. staining was localized within geniculoreceptive layer IV. Darkly reactive neurons include the large (presumed corticotectal) pyramids of layer V, and various classes of large and medium-sized presumed GABAergic nonpyramidal cells sparsely distributed throughout layers II-VI. The small and medium-sized pyramids of layers II, III, V, and VI, as well as many of the smaller presumed GABAergic neurons, were only lightly or moderately reactive. The darkly reactive neurons tended to be those that received convergent or proximally localized asymmetric axosomatic synapses, implying that they are strongly driven by excitatory synaptic input. The darkly reactive nonpyramids resembled those that form GAD+, symmetric axosomatic synapses with pyramidal cells. The dark reactivity of the symmetric synaptic terminals indicates that they mediate strong inhibition of neuronal discharge. The dark reactivity of a class of large asymmetric terminals in layer IV is likely to represent highly active geniculocortical terminals. The predominant distribution of elevated C.O. reactivity in dendrites is correlated with reported sites of (1) convergent excitatory synaptic input, (2) maximal field potentials, (3) highly active ion transport, and (4) Na+, K+-ATPase.     

Appearance of putative amino acid neurotransmitters during differentiation of neurons in embryonic turtle cerebral cortex.

Pyramidal and nonpyramidal neurons can be recognized early in the development of the cerebral cortex in both reptiles and mammals, and the neurotransmitters likely utilized by these cells, glutamate and gamma-aminobutyric acid, or GABA, have been suggested to play critical developmental roles. Information concerning the timing and topography of neurotransmitter synthesis by specific classes of cortical neurons is important for understanding developmental roles of neurotransmitters and for identifying potential zones of neurotransmitter action in the developing brain. We therefore analyzed the appearance of GABA and glutamate in the cerebral cortex of embryonic turtles using polyclonal antisera raised against GABA and glutamate. Neuronal subtypes become immunoreactive for the putative amino acid neurotransmitters GABA and glutamate early in the embryonic development of turtle cerebral cortex, with nonpyramidal cells immunoreactive for GABA and pyramidal cells immunoreactive for glutamate. The results of controls strongly suggest that the immunocytochemical staining in tissue sections by the GABA and glutamate antisera corresponds to fixed endogenous GABA and glutamate. Horizontally oriented cells in the early marginal zone (stages 15-16) that are GABA-immunoreactive (GABA-IR) resemble nonpyramidal cells in morphology and distribution. GABA-IR neurons exhibit increasingly diverse morphologies and become distributed in all cortical layers as the cortex matures. Glutamate-immunoreactive (Glu-IR) cells dominate the cellular layer throughout development and are also common in the subcellular layer at early stages, a distribution like that of pyramidal neurons and distinct from that of GABA-IR nonpyramidal cells. The early organization of embryonic turtle cortex in reptiles resembles that of embryonic mammalian cortex, and the immunocytochemical results underline several shared as well as distinguishing features. Early GABA-IR nonpyramidal cells flank the developing cortical plate, composed primarily of pyramidal cells, shown here to be Glu-IR. The earliest GABA-IR cells in turtles likely correspond to Cajal-Retzius cells, a ubiquitous and precocious cell type in vertebrate cortex. Glutamate-IR projection neurons in vertebrates may also be related. The distinctly different topographies of GABA and glutamate containing cells in reptiles and mammals indicate that even if the basic amino acid transmitter-containing cell types are conserved in higher vertebrates, the local interactions mediated by these transmitters may differ. The potential role of GABA and glutamate in nonsynaptic interactions early in cortical development is reinforced by the precocious expression of these neurotransmitters in turtles, well before they are required for synaptic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)     

GABAergic innervation of alpha type II calcium/calmodulin-dependent protein kinase immunoreactive pyramidal neurons in the rat basolateral amygdala

Although calcium/calmodulin-dependent protein kinase II (CaMK) has been shown to play a critical role in long-term potentiation (LTP) and emotional learning mediated by the basolateral amygdala, little is known about its cellular localization in this region. We have utilized immunohistochemical methods to study the neuronal localization of CaMK, and its relationship to gamma-aminobutyric acid (GABA)-ergic structures, in the rat basolateral amygdala (ABL). Light microscopic observations revealed dense CaMK staining in the ABL. Although the cell bodies and proximal dendrites of virtually every pyramidal cell appeared to be CaMK(+), the cell bodies of small nonpyramidal neurons were always unstained. Dual localization of CaMK and GABA immunoreactivity with confocal immunofluorescence microscopy revealed that CaMK and GABA were found in different neuronal populations in the ABL. CaMK was contained only in pyramidal neurons; GABA was contained only in nonpyramidal cells. At the ultrastructural level, it was found that CaMK was localized to pyramidal cell bodies, thick proximal dendrites, thin distal dendrites, most dendritic spines, axon initial segments, and axon terminals forming asymmetrical synapses. These findings suggest that all portions of labeled pyramidal cells, with the exception of some dendritic spines, can exhibit CaMK immunoreactivity. By using a dual immunoperoxidase/immunogold-silver procedure at the ultrastructural level, GABA(+) axon terminals were seen to innervate all CaMK(+) postsynaptic domains, including cell bodies (22%), thick (>1 microm) dendrites (34%), thin (<1 microm) dendrites (22%), dendritic spines (17%), and axon initial segments (5%). These findings indicate that CaMK is a useful marker for pyramidal neurons in ultrastructural studies of ABL synaptology and that the activity of pyramidal neurons in the ABL is tightly controlled by a high density of GABAergic terminals that target all postsynaptic domains of pyramidal neurons.     

Calretinin immunoreactivity in the cerebral cortex of the lizard Psammodromus algirus: A light and electron microscopic study

The present study describes the distribution and structural features of calretinin-immunoreactive neurons and fiber plexuses in the cerebral cortex of a lacertid lizard, at the light and electron microscopic levels, and also examines the colocalization of calretinin with parvalbumin and gamma-aminobutyric acid (GABA) in certain cortical regions. Calretinin-immunoreactive neurons are present throughout the cerebral cortex of Psammodromus and can be classified according to morphological and neurochemical criteria. Neurons in the medial cortex are small, spine-free and lack parvalbumin, whereas in the lateral cortex, calretinin-immunoreactive neurons display sparsely spiny dendrites and also lack parvalbumin. The dorsomedial and dorsal cortices contain most of the calretinin cortical neurons, which were located almost exclusively in the deep plexiform layer. These neurons are large, with an extensive spine-free dendritic tree. Most of the calretinin-immunoreactive neurons of dorsomedial and dorsal cortices are GABAergic and contain parvalbumin. Calretinin-immunoreactive fibers form two main afferent systems in the cortical areas. One probably intrinsic inhibitory system, arising from the calretinin and parvalbumin GABAergic neurons in the dorsomedial and dorsal cortices, makes symmetrical synapses on the soma and proximal dendrites of neurons located in the cell layers of the same cortical areas. The other system is formed by extremely thin axons running within the superficial plexiform layers of the medial, dorsomedial and dorsal cortices. These axons make asymmetrical synapses on dendrites or dendritic spines. We suggest that this system, probably extrinsic excitatory, arises from neurons located in the basal forebrain. J. Comp. Neurol. 382:382-393, 1997. © 1997 Wiley-Liss Inc.     

Distribution of GABAergic neurons and axon terminals in the macaque striate cortex

Antisera to glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) have been used to characterize the morphology and distribution of presumed GABAergic neurons and axon terminals within the macaque striate cortex. Despite some differences in the relative sensitivity of these antisera for detecting cell bodies and terminals, the overall patterns of labeling appear quite similar. GABAergic axon terminals are particularly prominent in zones known to receive the bulk of the projections from the lateral geniculate nucleus; laminae 4C, 4A, and the cytochrome-rich patches of lamina 3. In lamina 4A, GABAergic terminals are distributed in a honeycomb pattern which appears to match closely the spatial pattern of geniculate terminations in this region. Quantitative analysis of axon terminals that contain flat vesicles and form symmetric synaptic contacts (FS terminals) in lamina 4C beta and in lamina 5 suggest that the prominence of GAD and GABA axon terminal labeling in the geniculate recipient zones is due, at least in part, to the presence of larger GABAergic axon terminals in these regions. GABAergic cell bodies and their initial dendritic segments display morphological features characteristic of nonpyramidal neurons and are found in all layers of striate cortex. The density of GAD and GABA immunoreactive neurons is greatest in laminae 2-3A, 4A, and 4C beta. The distribution of GABAergic neurons within lamina 3 does not appear to be correlated with the patchy distribution of cytochrome oxidase in this region; i.e., there is no significant difference in the density of GAD and GABA immunoreactive neurons in cytochrome-rich and cytochrome-poor regions of lamina 3. Counts of labeled and unlabeled neurons indicate that GABA immunoreactive neurons make up at least 15% of the neurons in striate cortex. Layer 1 is distinct from the other cortical layers by virtue of its high percentage (77-81%) of GABAergic neurons. Among the other layers, the proportion of GABAergic neurons varies from roughly 20% in laminae 2-3A to 12% in laminae 5 and 6. Finally, there are conspicuous laminar differences in the size and dendritic arrangement of GAD and GABA immunoreactive neurons. Lamina 4C alpha and lamina 6 are distinguished from the other layers by the presence of populations of large GABAergic neurons, some of which have horizontally spreading dendritic processes. GABAergic neurons within the superficial layers are significantly smaller and the majority appear to have vertically oriented dendritic processes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Striatal NPY-Containing Neurons Receive GABAergic Afferents and may also Contain GABA: An Electron Microscopic Study in the Rat

Dual labelling methods were applied to localize simultaneously neuropeptide Y (NPY) and glutamate decarboxylase (GAD) immunoreactivities on ultrathin sections of the rat caudate-putamen (CP). By means of a double peroxidase-anti-peroxidase technique, using 3,3'-diaminobenzidine and benzidine dihydrochloride as chromogens in animals with no colchicine pretreatment, GAD immunoreactivity was found to be present in terminals only whereas NPY immunoreactivity was detected in neurons displaying the features of aspiny type cells and processes. With this approach, we observed numerous synaptic associations of the symmetrical type between GAD-immunoreactive (-Ir) axonal boutons and NPY-Ir cell bodies and dendrites. By combining immunoperoxidase and radioimmunocytochemical labelling in animals pretreated with colchicine, NPY was again detected in a single population of aspiny type neurons whereas GAD immunoreactivity was observed in neurons which could be classified as aspiny and spiny on the basis of their ultrastructural characteristics. All the cells of the aspiny type displaying clear-cut NPY immunoreactivity were also found to be GAD-positive. Some other neurons of both the aspiny and the spiny type were found to be immunoreactive to GAD alone. GAD/NPY dually labelled terminals were also observed and some axo-axonic appositions between GAD- and NPY-Ir terminals were also detected. All in all, these data show that NPY aspiny type neurons of the rat CP receive GABAergic afferents and provide morphological support for two hypotheses: that NPY is co-localized with GABA in some cell bodies, dendrites and axons, and that presynaptic interactions may occur between NPY and GABAergic neuronal systems.     

Evidence for GABAergic interneurons in the red nucleus of the painted turtle.

Immunocytochemical and electrophysiological evidence supporting the presence of GABAergic interneurons in the turtle red nucleus is presented. Injections of HRP into the spinal cord produced labeling of large neurons in the contralateral red nucleus. The peroxidase-antiperoxidase (PAP) method revealed smaller cells immunoreactive to an antibody against glutamate decarboxylase (GAD), the synthetic enzyme for the inhibitory neurotransmitter GABA, that were interspersed among larger immunonegative neurons. Similar small neurons were densely immunostained by antibodies to GABA-glutaraldehyde conjugates obtained from different sources and applied according to pre-embedding and postembedding protocols. Rubrospinal neurons retrogradely labeled with HRP measured 16 and 27 microns in mean minor and major cell body diameters, while GABA-like immunopositive neurons situated within the red nucleus measured 7 and 13 microns. There was very little overlap in soma size between the two cell populations. Therefore, we suggest that the GAD- and GABA-positive neurons may be local inhibitory interneurons. This notion is further supported by observations of pre-embedding immunostaining for GAD and postembedding immunostaining for GABA showing that the turtle red nucleus is amply innervated by immunoreactive axon terminals. These puncta are closely apposed to cell bodies and dendrites of both immunonegative large neurons and immunopositive small neurons. Moreover, immunogold staining at the electron microscopic level demonstrated that GABA-like immunoreactive axon terminals with pleomorphic synaptic vesicles formed symmetric synapses with cell bodies and dendrites of the two types of red nucleus cells. These ultrastructural features are commonly assumed to indicate inhibitory synapses. A moderately labeled bouton with round vesicles and asymmetric synapses was also observed. In addition, the two types of red nucleus neurons received asymmetric axosomatic and axodendritic synapses with GABA-negative boutons provided with round vesicles, features usually associated with excitatory functions. To obtain electrophysiological evidence for inhibition, intracellular recordings from red nucleus neurons were conducted using an in vitro brainstem-cerebellum preparation from the turtle. Small, spontaneous IPSPs were recorded from 7 out of 14 red nucleus cells studied. These morphological and physiological results provide strong support for concluding that the turtle red nucleus, like its mammalian counterpart, contains GABAergic inhibitory interneurons. While we have not identified the main source of input to these interneurons, in view of the scarce development of the reptilian cerebral cortex, this input is unlikely to come from the motor cortex as it does in mammals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural Double-Labelling Study of Dopamine Terminals and GABA-Containing Neurons in Rat Anteromedial Cerebral Cortex

The aim of this study was to identify, at the ultrastructural level, the neuronal targets of dopamine afferents to the medial prefrontal and the anterior cingulate cortex of the adult rat. Since, in addition to pyramidal neurons, the cortical neuronal population mainly consists of GABAergic nonpyramidal intrinsic neurons, the simultaneous visualization of both dopamine- and GABA-containing neurons should leave the pyramidal neurons as the only unlabelled dopamine postsynaptic target. In this context, we used a double labelling immunocytochemical procedure: a pre-embedding PAP immunostaining to visualize monoclonal conjugated-dopamine (DA) antibody, followed by postembedding immunogold staining with a polyclonal conjugated-GABA antibody. In a single section sampling of 369 DA-immunoreactive (DA-IR) varicosities observed and the GABA-containing elements, 75% of the DA-IR terminals showed no indication of any contact with a GABA neuron. Twenty-five per cent were found in nonsynaptic contiguity with a GABA-immunoreactive neuronal element: axon, dendrite or cell body. When a DA varicosity was in nonsynaptic contiguity with a neuronal perikaryon (5% of cases), this cell was GABA positive. Ten per cent of the DA varicosities were contiguous to a GABA axon, but axoaxonic synapses in either direction were never observed. A symmetrical synapse between a DA varicosity and a GABA-containing dendrite was observed only once. The other 13 DA-IR terminals exhibiting a clear synaptic junction were apposed to nonGABA-containing dendrites, spines and shafts. Triads were observed in which a DA varicosity, forming or not a symmetrical synapse, was apposed to an unlabelled dendrite already receiving a symmetrical junction from another unlabelled axon. These data confirm and extend previous results designating the pyramidal cell dendritic tree as the main synaptic target of DA cortical afferents in rat and primate cerebral cortex. However, a direct effect of dopamine on a subpopulation of intrinsic GABA neurons cannot be excluded.     

Neuropeptide Y in the cerebral cortex and the caudate-putamen nuclei: ultrastructural basis for interactions with GABAergic and non-GABAergic neurons

In the cerebral cortex and caudate-putamen (CP) nuclei, neuropeptide Y (NPY) immunoreactivity is detectable within 1-2% of all neurons. The NPY-immunoreactive neurons are interneuronal and are believed to be mostly GABAergic in the cerebral cortex but not in the CP nuclei. Thus NPY and GABA may play different roles in the circuitry within these 2 regions. We tested this possibility by comparing the ultrastructure of NPY-containing neurons between (1) cortex (somatosensory and anterior cingulate areas) versus dorsolateral CP; and (2) GABAergic versus non-GABAergic NPY neurons within each area. Single coronal sections through the rat forebrain were dually labeled for GABA and NPY by combining immunoautoradiography with the immunoperoxidase method. NPY-containing neurons with or without GABA occurred throughout the rostrocaudal portions of CP and all laminae of somatosensory and anterior cingulate cortex. Comparisons between the areas confirmed that somata and terminals dually labeled for GABA and NPY were more prevalent in the cortex. NPY terminals lacking detectable GABA immunoreactivity also were found within the cortex, thus suggesting additional heterogeneity in cortical NPY innervation. The ultrastructural features of NPY perikarya in both regions were morphologically similar regardless of whether the cells also contained GABA. Most synaptic inputs to NPY neurons occurred at distal dendrites. In comparison to neighboring neurons, synaptic inputs to proximal dendrites and somata of NPY neurons of cortex and CP were rare, suggesting that fewer and weaker inputs may modulate the excitability of NPY-containing neurons. In both regions, nearly all NPY- and NPY-GABA-labeled terminals formed symmetric junctions suggestive of inhibitory action. The majority of these junctions were with dendrites containing neither NPY nor GABA. NPY terminals formed few contacts on proximal dendrites and somata of GABAergic neurons (8% of 179 contacts in cortex; 12% of 73 contacts in CP) which, unlike most singly-labeled GABAergic neurons, were sparsely innervated. Thus, NPY may play a more prominent role in modulation of certain GABAergic neurons than would be predicted by the observed frequency of NPY-to-GABA contacts in the two regions. One notable regional difference was the greater prevalence in cortex of axoaxonic associations between NPY-immunoreactive terminals and other terminals, some of which also contained NPY. These nonsynaptic associations may be involved in the modulation of (1) the release of NPY by another transmitter or (2) NPY's modulation of release of other transmitters in cortex.     

GABAergic microcircuits in the neostriatum

The vast majority of neostriatal neurons and intrinsic intrastriatal synapses are GABAergic, the latter arising from axon collaterals of spiny projection neurons and from GABAergic interneurons. An important feature of the functional organization of the neostriatum has long been assumed to be the existence of a widespread lateral inhibitory network mediated by the axon collaterals of spiny projection neurons. However, these collateral connections have recently been demonstrated electrophysiologically to be relatively weak--in contrast to feedforward interneuronal inhibition, which exerts strong effects on spike timing in spiny neurons. These new data are incompatible with current "winner-take-all" models of lateral inhibitory function in the neostriatum, and they force a modification of established concepts of the functional roles of feedback inhibition in this nucleus.     

Patterns of synaptic input to layer 4 of cat striate cortex

Although cells in layer 4 of cat striate cortex represent the first stage in the cortical processing of visual information, they have considerably more complicated receptive field properties than the afferents to the layer from the lateral geniculate nucleus. In considering how these properties are generated, we have focused on the intrinsic cortical circuitry, and particularly on the projection to layer 4 from layer 6. Layer 6 pyramidal cells were injected with horseradish peroxidase and examined at the light and electron microscopic level. The labeled axon terminals were found to form asymmetric synapses and to show a strong preference for contacting dendritic shafts. Serial reconstruction of dendrites postsynaptic to labeled layer 6 cell axon terminals showed that a large proportion of the postsynaptic dendrites belonged to smooth and sparsely spiny stellate cells, suggesting a selective innervation of these cell types. In contrast, the geniculate projection to layer 4 made synapses primarily with dendritic spines and, as a result, the large majority of terminals ended on spiny cells. Since smooth and sparsely spiny stellate cells are thought to mediate inhibition within the cortex, we suggest that one effect of the layer 6 to layer 4 projection could be to contribute to inhibitory features of the receptive fields of layer 4 cells.     

GABAergic neurons of the rat dorsal hippocampus express muscarinic acetylcholine receptors

The expression of muscarinic acetylcholine receptors (mAChRs) in glutamic acid decarboxylase (GAD)-positive cells in the different strata of CA1, CA3, and the dentate gyrus (DG) of the dorsal hippocampus is examined by way of quantitative immunofluorescent double labeling employing M35, the monoclonal antibody raised against purified mAChR protein. Of all GAD-positive neurons, 97.5% express mAChRs. Conversely, 92.9% of the muscarinic cholinoceptive nonpyramidal neurons express GAD. These results indicate that the vast majority of the γ-aminobutyric acid (GABA)ergic neurons express mAChRs. In addition to GAD, parvalbumin (PARV) and somatostatin (SOM) are two neurochemical substances notably expressed in GABAergic neurons. In order to examine whether the entire muscarinic cholinoceptive nonpyramidal cell group can be characterized by these three GABAergic markers, a cocktail of GAD, PARV, and SOM was used in a fluorescent double-labeling experiment with M35. These results show that 97.2% of all muscarinic cholinoceptive nonpyramidal neurons can be neurochemically characterized by the content of GAD, PARV, and SOM. In conclusion, nearly all GABAergic cells express mAChRs and, conversely, virtually the entire muscarinic cholinoceptive nonpyramidal cell group belongs to the GABAergic cell population. This study, therefore, provides anatomical evidence for an extensive neuronal connectivity of the hippocampal muscarinic cholinoceptive nonpyramidal system and the inhibitory GABAergic circuitry.     

Laminar distribution and neuronal targets of GABAergic axon terminals in cat primary auditory cortex (AI)

The form, density, and neuronal targets of presumptive axon terminals (puncta) that were immunoreactive for gamma-aminobutyric acid (GABA) or its synthesizing enzyme, glutamic acid decarboxylase (GAD), were studied in cat primary auditory cortex (AI) in the light microscope. High-resolution, plastic-embedded material and frozen sections were used. The chief results were: (1) There was a three-tiered numerical distribution of puncta, with the highest density in layer Ia, an intermediate number in layers Ib–IVb, and the lowest concentration in layers V and VI, respectively. (2) Each layer had a particular arrangement: layer I puncta were fine and granular (less than 1 μm), and layer V and VI puncta were mixed in size and predominantly small. (3) The form and density of puncta in every layer were distinctive. (4) Immunonegative neurons received, in general, many more axosomatic puncta than immunopositive cells, with the exception of the large multipolar (presumptive basket) cells, which invariably had many puncta in layers II–VI. (5) The number of puncta on the perikarya of GABAergic neurons was sometines related to the number of puncta in the layer, and in other instances it was independent of the layer. Thus, while layer V had a proportion of GABAergic neurons similar to layer IV, it had only a fraction of the number of puncta: perhaps the intrinsic projections of supragranular GABAergic cells are directed toward layer IV, as those of infragranular GABAergic neurons may be. Since puncta are believed to be the light microscopic correlate of synaptic terminals, they can suggest how inhibitory circuits are organized. Even within an area, the laminar puncta patterns may reflect different inhibitory arrangements. Thus, in layer I the fine, granular endings could contact preferentially the distal dendrites of pyramidal cells in deeper layers. The remoteness of such terminals from the spike initation zone contrasts with the many puncta on all pyramidal cell perikarya and the large globular endings on basket cell somata. Basket cells might receive feed-forward disinhibition, pyramidal cells feed-forward inhibition, and GABAergic non-basket cells would be the target of only sparse inhibitory axosomatic input. Such arrangements imply that the actions of GABA on AI neurons are neither singular nor simple and that the architectonic locus, laminar position, and morphological identity of a particular neuron must be integrated for a more refined view of it role in cortical circuitry. © 1994 Wiley-Liss, Inc.