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The monosynaptic targets of different functional types of geniculocortical axons were compared in the primary visual cortex of monkeys. Single thalamocortical axons were recorded extracellularly in the white matter by using horseradish-peroxidase-filled pipettes. Their receptive fields were mapped and classified as corresponding to those of parvi- or magnocellular neurons in the lateral geniculate nucleus. The axons were then impaled and injected intraaxonally with horseradish peroxidase. Two magnocellular (MA) and two parvicellular (PA) axons were successfully recovered and reconstructed in three dimensions. The two MA axons arborised mainly in layer 4C alpha, as did the two PA axons in layer 4C beta. Few collaterals formed varicosities in layer 6. Both MA axons had two large, elongated clumps of bouton (approx. 300-500 x 600-1,200 microns each) and a small clump. One PA axon had two clumps (each with a core appr. 200 microns in diameter); the other had only one (appr. 150-200 microns in axon had 1,380; one MA axon had 3,200 boutons; and those of the more extensive MA axon were not counted. The distribution of postsynaptic targets as well as the number of synapses per bouton has been established for a sample of 150 PA boutons and 173 MA boutons from serial ultrathin sections. The MA axons made on average 2.1 synapses per bouton compared to 1.79 for one PA axon and 2.6 for the other. The sample of boutons taken from the two physiological types of axons contacted similar proportions of dendritic spines (52-68%), shafts (33-47%), and somata (0-3%). The postsynaptic elements were further characterized by immunostaining for GABA. All postsynaptic perikarya and some of the dendrites (4.5-9.5% of all targets) were positive for the amino acid. Near the thalamic synapse GABA-negative dendritic shafts frequently contained lamellar bodies, an organelle identical in structure to spine apparatus. Dendritic shafts and spines postsynaptic to the thalamocortical boutons frequently received an adjacent synapse from GABA-immunoreactive boutons. The similarity between the magno-and parvicellular axons in their targeting of postsynaptic elements, including the GABAergic neurons, suggests that the structural basis of the physiological differences between 4C alpha and 4C beta neurons should be sought in other aspects of the circuitry of layer 4C, such as local cortical circuits, or in the far greater horizontal extent of the thalamocortical and GABAergic axons in layer 4C alpha compared to those in the beta subdivision.

In adult rats with a unilateral 6-hydroxy-dopamine-induced lesion of the nigrostriatal dopamine pathway, grafts of embryonic ventral mesencephalon can establish extensive efferent connections with the previously denervated host neostriatum and can compensate for motor and sensorimotor asymmetries induced by the lesion. The object of this study was to examine the afferent synaptic inputs to grafted dopaminergic neurons, implanted into a cortical cavity overlying the previously denervated caudate-putamen, using electron microscopic immunocytochemistry. The dopaminergic neurons of the grafts in the same animals had previously been shown to re-innervate the host neostriatum, to form synaptic connections therein and to attenuate the lesion-induced motor asymmetry that occurred in response to amphetamine (Freund et al. 1985). In the light microscope, the grafts were found to contain numerous tyrosine hydroxylase-immunoreactive perikarya, dendrites, axons and axonal swellings which had distinct distributions. In addition axons and axonal swellings that were immunoreactive for either substance P or glutamate decarboxylase were present. Electron microscopic analysis of the boutons contacting tyrosine hydroxylase-immunoreactive neurons in the grafts revealed the presence of at least five distinct types of afferent synaptic boutons based on their immunochemistry, morphology, or types of membrane specialization. One type was itself immunoreactive for tyrosine hydroxylase; such synapses are extremely rare in the intact substantia nigra, none were found in the contralateral substantia nigrae or the substantia nigra of a control rat. Three of the remaining types had ultrastructural features that were similar to synaptic terminals that were immunoreactive for substance P or glutamate decarboxylase. These synapses were similar to the types of synapses found contacting dopaminergic neurons in the substantia nigra contralateral to the graft or the substantia nigra of a control rat. The results demonstrate that, in the absence of the normal extrinsic afferent inputs, the intracortical mesencephalic grafts have a well-developed local synaptic circuitry. It is suggested that local circuit regulation of dopaminergic neurons within the graft may, at least in part, be responsible for the maintenance of a normal or close to normal functional activity.

Using a monoclonal antibody to choline acetyltransferase, immunoreactive synaptic boutons were identified in the neostriatum, cingulate cortex, basolateral nucleus of the amygdala, hippocampus and interpeduncular nucleus of the rat. The synapses were generally symmetrical although some asymmetrical membrane specializations were observed. Postsynaptic targets included perikarya, dendritic shafts and dendritic spines.

Glutamate decarboxylase (GAD)-immunoreactive varicosities were found around cell bodies of nonimmunoreactive and immunoreactive neurons in the cat's visual cortex; they also occurred along apical dendrites and axon initial segments of pyramidal neurons. By examination in the electron microscope of structures first identified in the light microscope, it was established that the GAD-immunoreactive varicosities were boutons in symmetrical synaptic contact with pyramidal cells in layers II-IV. More than 90% of 142 boutons surrounding the cell bodies of 20 pyramidal neurons were immunoreactive for GAD. Since such a high proportion of the axosomatic boutons are GAD-immunoreactive, it is likely that the terminals of basket cells are included in this population and so the basket cell probably uses gamma-aminobutyrate as a transmitter, as suggested by previous authors. Almost all the 68 boutons in symmetrical contact with the axon initial segments of six pyramidal neurons could be shown to be GAD-immunoreactive, which makes it very likely that the boutons of axoaxonic cells contain GAD-immunoreactivity. This was established unequivocally for an individual Golgi-impregnated axoaxonic cell by combining Golgi impregnation and immunocytochemistry in the same sections: A Golgi-impregnated axoaxonic cell whose cell body was in layer II gave rise to numerous terminal segments, some of which were examined in the electron microscope after gold-toning. These boutons were in synaptic contact with axon initial segments and not only contained the Golgi precipitate but were also immunoreactive for GAD. It is concluded that the axoaxonic cell in the visual cortex uses gamma-aminobutyrate as a transmitter. An individual axoaxonic cell in layer II/III was filled with horseradish peroxidase by intracellular iontophoresis. The very extensive local axonal field was composed of 330 terminal bouton rows in layer II/III and a sparse descending collateral projection to infragranular layers. A computer-assisted reconstruction of the axonal field in three dimensions revealed the following: The main output of the cell is to pyramidal neurons that lie deeper than the soma; the axonal arborization occupies an area of 400 micron in the anteroposterior axis and extends 200 micron along the mediolateral axis; the terminal bouton rows in layer II/III form clusters about 50 micron wide running approximately at right angles to the border between areas 17 and 18, with an intercluster interval of about 100 micron. These findings suggest that the terminals of an individual axoaxonic cell could be contained within one ocular dominance column but that there may be inhomogeneities in the weighting of the axoaxonic input to pyramidal cells in the supragranular layers.

A miniature multiple thin-film recording sensor was used to measure simultaneously the electrical activity, oxygen content and temperature of brain tissue. The chamber-type potential sensor was an Ag/AgCl electrode covered by an Si3N4 (silicon nitride) chamber. The chamber-type oxygen sensor consisted of an Au-Ag/AgCl two-electrode electrochemical cell embedded in an electrolyte-filled Si3N4 chamber. The temperature sensor was a thin-film germanium resistor. The different sensors were spaced 300 microns apart. Anaesthetics (pentobarbital, chloral hydrate, chlornembutal, halothane) were shown to depress electrical activity and to increase local oxygen tension in the hippocampus. Halothane, but not the other anaesthetics, also increased the current output of the oxygen sensor when tested in saline bath, indicating that the apparent increase in measured oxygen levels during halothane anaesthesia was partly due to an electrochemical effect of halothane on the oxygen sensors. The decrease of tissue oxygen consumption produced by the other anaesthetics is likely to be the result of metabolic depression. Cerebral ischemia, evoked by cauterization of the vertebral arteries and occlusion of the carotid arteries for 30 min, resulted in the disappearance of both spontaneous and evoked electrical activity in the hippocampus and a decrease of both local temperature and oxygen tension. There was a marked overshoot of the oxygen tension to above preocclusion level following the release of the carotid arteries. As soon as electrical activity returned, the oxygen tension fell again, often below the lowest level seen during the ischemic period. This secondary decrease of oxygen level could be reversed by administration of supplementary small doses of anaesthetic. The anaesthetic-induced increase in oxygen tension was accompanied by a marked decrease in electroencephalogram amplitude and frequency. During electrically induced seizures a decrease in hippocampal oxygen content occurred and was accompanied by an increase of local temperature. Since the rectal temperature was kept constant, the changes in temperature are likely to reflect changes in blood perfusion of the recorded area. These findings are in agreement with previous observations made with conventional electrodes. In addition, the miniature size of the chamber-type microelectrode assembly allows a correlated monitoring of parallel physiological changes with high spatial and temporal resolution during anaesthesia, ischemia and epilepsy.

Labelled neurons were identified by autoradiography following tangential intracortical injection of [3H]-gamma-aminobutyrate (GABA). The addition of cis-1,3-aminocyclohexane carboxylic acid to the GABA solution prevented perikaryal labelling. Labelled neurons were found in each injected layer and in addition they were always present directly above the injection track. The labelling of neurons in layer II, and upper III, following injections in layers V. and VI. can be explained by retrograde axonal transport and indicates that some GABA-ergic neurons project vertically. Ninety neurons of different types were Golgi impregnated and examined for selective [3H]-GABA uptake. Sixteen of these were labelled. On the basis of dendritic characteristics they were classified as aspiny multipolar neurons with small, medium or large dendritic fields, sparsely spiny multipolar neurons and one neuron was a bipolar cell. Thus Golgi impregnation of their processes reveals that cortical GABA-ergic neurons are a heterogeneous population. A [3H]-GABA accumulating, aspiny neuron with profoundly branching, "bushy" dendrites and locally arborizing axon in layer VI, was studied in the electron microscope. Its fine structural characteristics were similar to those of other identified non-pyramidal neurons. The existence of several types of cortical GABA-ergic neurons differing in their synaptic connections is discussed.

Solid pieces of embryonic hippocampal tissue were implanted in a cavity formed by aspiration of the fimbria-fornix and the overlying cingulate cortex in adult rats. Six to 8 months after the transplantation, chronic recording electrodes were implanted into the graft and the host hippocampi for the recording of electroencephalogram and unit activity in the freely moving animal. Irregularly occurring sharp waves or electroencephalogram spikes and concurrent synchronous discharge of large groups of neurons dominated the electrical activity of the grafts, in contrast to the situation in normal animals. Light microscopy and GABA immunocytochemistry in the grafts revealed that the three major cell types of the hippocampal formation, i.e. pyramidal neurons, dentate granule cells and GABA-immunoreactive interneurons were present in the hippocampal grafts. At the ultrastructural level, however, significant alterations in connectivity were observed. The most striking finding was the absence or sparse occurrence of synapses on the axon initial segments of pyramidal neurons. The axon initial segments are normally densely covered by GABAergic synapses derived from a specialized type of interneuron, the chandelier or axo-axonic cell. On the other hand, numerous GABA-immunoreactive terminals were found in synaptic contact with somata of pyramidal neurons, suggesting that other types of GABAergic interneurons and their efferent connections may have developed in a normal manner. The cell bodies of pyramidal neurons received, in addition, several asymmetric synapses from GABA-negative terminals. These presumably excitatory synapses are not present on the somata of pyramidal cells in the normally developing hippocampus. We hypothesize that the somatic excitatory synapses originate, at least in part, from the axon collaterals of the neighbouring pyramidal cells in the graft. We suggest that the hyperexcitability of the neuronal circuitry within the graft is due to reduced inhibition (lack of axo-axonic synapses) coupled with increased collateral excitation of the pyramidal neurons.

A method is described for impregnating neurons by the Golgi procedure in sections of brain tissue that are of a thickness 80-100 microns, so allowing a variety of histochemical procedures to be carried out prior to Golgi-impregnation. The method has been applied to the retina of the adult primate and to brain sections incubated to reveal horseradish peroxidase activity with substrates that give electron-dense reaction products. Sections containing neurons that have been filled with horseradish peroxidase by intracellular iontophoresis following electrophysiological characterization, or containing neurons retrogradely labelled by horseradish peroxidase, can be impregnated by this Golgi procedure. Subsequent gold-toning of the Golgi-impregnated neurons makes it possible to study, in the electron-microscope, both Golgi-impregnated and peroxidase-containing neurons and their afferent and efferent synaptic contacts within the same section. The procedure also makes it possible to repeat Golgi impregnation on sections already Golgi-impregnated and gold-toned, or to repeat the Golgi impregnation if the first impregnation is not satisfactory. The procedure is illustrated by examples from the monkey retina, cat visual cortex and rat neostriatum.

Sections of the cat's visual cortex were stained by an antiserum to glutamate decarboxylase using the peroxidase-antiperoxidase method; they were then impregnated by the section Golgi procedure and finally the Golgi deposit was replaced by gold. Neurons containing glutamate decarboxylase immunoreactivity were found in all layers of the visual cortex, without any obvious pattern of distribution. Fifteen immunoreactive neurons were also Golgi-impregnated and gold-toned, which enabled us to study the morphology and synaptic input of identified GABAergic neurons. These neurons were found to be heterogeneous both with respect to the sizes and shapes of their perikarya and the branching patterns of their dendrites. All the immunoreactive, Golgi-impregnated neurons had smooth dendrites, with only occasional protrusions. The synaptic input of glutamate decarboxylase-immunoreactive neurons was studied in the electron microscope. Immunoreactive neurons received immunoreactive boutons forming symmetrical synapses on their cell bodies. The Golgi-impregnation made it possible to study the input along the dendrites of immunoreactive neurons. One of the large neurons in layer III whose soma was immunoreactive was also Golgi-impregnated: it received numerous non-immunoreactive asymmetrical synaptic contacts along its dendrites and occasional ones on its soma. The same neuron also received a few boutons forming symmetrical synaptic contacts along its Golgi-impregnated dendrites; most of these boutons were immunoreactive for glutamate decarboxylase. Glutamate decarboxylase-immunoreactive boutons were also found in symmetrical synaptic contact with non-immunoreactive neurons that were Golgi-impregnated. A small pyramidal cell in layer III was shown to receive several such boutons along its somatic membrane. It is concluded that the combination of immunoperoxidase staining and Golgi impregnation is technically feasible and that it can provide new information. The present study has shown that there are many morphologically distinct kinds of aspiny GABAergic neurons in the visual cortex; that the predominant type of synaptic input to the dendrites of such neurons is from boutons forming asymmetrical synapses, but that some of the GABAergic neurons also receive a dense symmetrical synaptic input on their cell bodies, and occasional synapses along their dendrites, from the boutons of other GABAergic neurons. These findings provide a morphological basis, firstly, for a presumed powerful excitatory input to GABAergic interneurons and, secondly, for the disinhibition which has been postulated from electrophysiological studies to occur in the cat's visual cortex.

Chandelier or axo-axonic cells (AACs) are specialized interneurons terminating on the axon initial segments of pyramidal neurons. Two AACs have been localized by Golgi impregnation, one in the CA1 region of the hippocampus and one in the visual cortex of cat, for structural analysis and for the identification of their transmitter. They had 323 and 268 terminal bouton rows, respectively, probably making synapses with an equal number of initial segments. The distribution of the dendrites of the hippocampal cell was strikingly similar to that of pyramidal cells suggesting a similar input. Using an antiserum to GABA and postembedding GABA-immunocytochemistry, developed for Golgi-impregnated neurons, both cells were found to be GABA-immunoreactive. The strategic location of their synapses and the presence of GABA in AACs suggest that in normal cortical tissue they play a major role in GABA-mediated inhibition.

3H-GABA was injected into restricted regions of visual areas 1 and 2 (cortical areas 17 and 18) on the lateral surface of the occipital lobe in monkeys. The injected tissue was processed for Golgi impregnation and gold toning. Sections containing Golgi-impregnated neurons were re-embedded, sectioned at 1 micron, and prepared for autoradiography to reveal neurones that had selectively accumulated 3H-GABA. Golgi-impregnated pyramidal, spiny stellate and aspiny nonpyramidal neurons were examined for 3H-GABA accumulation. Out of 47 aspiny non-pyramidal neurons 16 were labelled by 3H-GABA. The other cell types did not accumulate the amino acid. Twelve of the labelled neurons were drawn. Eight were bitufted neurons with their dendrites oriented predominantly radially, three were small multipolar neurons, and one could be reconstructed only partially. One neuron had a locally arborizing axon in layer III. Two bitufted, Golgi-impregnated neurons in layer II and upper III of area 18 were labelled from GABA injection radially beneath in layer VI, providing evidence for earlier suggestions that in the monkey's visual cortex the cells in the upper layers which project radially and accumulate 3H-GABA are aspiny non-pyramidal cells. The results indicate the existence of different types of putative GABA-ergic interneurons.

The precise location of physiologically identified specific afferent input on the different types of cell in the visual cortex and the identification of the neurotransmitters of these cells are essential to a better understanding of the first stage of cortical processing. A combination of anatomical, neurochemical, and physiological methods was used to identify the cortical neurones that receive synaptic input from X- and Y-type afferents, which are thought to originate from cells of the lateral geniculate nucleus. One method relied on chance contacts made between single physiologically characterised axons, which had been injected with horseradish peroxidase (HRP), and the processes of cells impregnated by the Golgi method. These experiments revealed that both X and Y axons formed synapses on the dendrites of spiny stellate cells in layer 4. Y axons in both areas 17 and 18 established multiple synaptic contacts on basal dendrites of layer 3 pyramidal cells. One X axon contacted the apical dendrite of a layer 5 pyramidal cell and one Y axon contacted the dendrite of a large cell with smooth dendrites in layer 3. The maximum number of synapses made between one axon and a single postsynaptic cell was eight, although in most cases it was only one. It was concluded that one axon only provides a small fraction of the geniculate afferent input to an individual cell. A second method revealed that the somata in layer 4 in synaptic contact with the HRP-filled axon terminals were GABA-immunoreactive, and therefore might be involved in inhibitory processes. From light microscopic data it was found that somata receiving contacts from X axons in area 17 were significantly smaller (average diameter 15 microns) than those contacted by the Y axons in areas 17 and 18 (average diameter 24 microns). Somatic contacts were extremely rare in layer 6. These data show that the X and Y afferents may activate separate subsets of inhibitory neurones.

A subpopulation of gamma-aminobutyric acid (GABA) containing neurons was reported to contain cholecystokinin-immunoreactive material in the visual cortex of cat [Somogyi et al., J. Neurosci. (1984) 4, 2590-2603]. In the present study pre-embedding immunocytochemistry was used to identify which of the several types of presumed GABAergic nonpyramidal cells in areas 17 and 18 contain cholecystokinin immunoreactivity. Most of the cholecystokinin-immunoreactive somata were found in layers II-III, they were less frequent in layers I and VI, and relatively rare in layers IV and V. The distribution and density of the axon terminals resembled that of the cell bodies. Two well defined types of cholecystokinin-immunoreactive neuron were distinguished: (1) double bouquet cells in layers II-III with vertically projecting axons, and (2) small basket cells with local axons either restricted to layers II-III, or descending to layer V. Additional cholecystokinin-positive cells showed features of bitufted or multipolar neurons in layers II-VI and horizontal cells in layer I, but these cells could be defined less well due to partial staining. Cholecystokinin-immunoreactive dendrites were found to run horizontally in layer I for several hundred micrometers. Some of the cholecystokinin-immunoreactive cells in layer VI had very long dendrites ascending radially up to layer III, as did their axons. A few cholecystokinin-immunoreactive cells appeared to have two axons and this was confirmed by electron microscopy. All cholecystokinin-immunoreactive neurons and terminals were separated from the basal lamina of blood vessels by glial endfeet. Random samples of boutons from each layer as well as identified terminals traced to their origin from local neurons were examined in the electron microscope. All of the boutons established symmetrical (type II) synaptic contacts with dendritic shafts, spines or somata. Quantitative electron microscopy of the postsynaptic targets of double bouquet cells and small basket cells demonstrated clear differences between these two types of neuron; basket cells having a higher proportion of their terminals in synaptic contact with somata. The findings that several distinct types of cortical neurons, as defined by their synaptic connections, contain cholecystokinin-immunoreactive material and that identified axons of all examined neurons form type II synaptic contacts suggests that the majority, if not all cholecystokinin-positive boutons forming type II contacts originate from local cortical cells. The distribution of targets postsynaptic to cholecystokinin-positive neurons is compared to those of cells labelled by other methods.

Tyrosine hydroxylase-immunoreactive fibres in the rat neostriatum were studied in the electron microscope in order to determine the nature of the contacts they make with other neural elements. The larger varicose parts of such fibres contained relatively few vesicles and rarely displayed synaptic membrane specializations; however, thinner parts of axons (0.1-0.4 micron) contained many vesicles and had symmetrical membrane specializations, indicative of en passant type synapses. By far the most common postsynaptic targets of tyrosine hydroxylase-immunoreactive boutons were dendritic spines and shafts, although neuronal cell bodies and axon initial segments also received such input. Six striatonigral neurons in the ventral striatum were identified by retrograde labelling with horseradish peroxidase and their dendritic processes were revealed by Golgi impregnation using the section-Golgi procedure. The same sections were also developed to reveal tyrosine hydroxylase immunoreactivity and so we were able to study immunoreactive boutons in contact with the Golgi-impregnated striatonigral neurons. Each of the 280 immunoreactive boutons examined in the electron microscope displayed symmetrical synaptic membrane specializations: 59% of the boutons were in synaptic contact with the dendritic spines, 35% with the dendritic shafts and 6% with the cell bodies of striatonigral neurons. The dendritic spines of striatonigral neurons that received input from immunoreactive boutons invariably also received input, usually more distally, from unstained boutons that formed asymmetrical synaptic specializations. A study of 87 spines along the dendrites of an identified striatonigral neuron showed that the most common type of synaptic input was from an individual unstained bouton making asymmetrical synaptic contact (53%), while 39% of the spines received one asymmetrical synapse and one symmetrical immunoreactive synapse. It is proposed that the spatial distribution of presumed dopaminergic terminals in synaptic contact with different parts of striatonigral neurons has important functional implications. Those synapses on the cell body and proximal dendritic shafts might mediate a relatively non-selective inhibition. In contrast, the major dopaminergic input that occurs on the necks of dendritic spines is likely to be highly selective since it could prevent the excitatory input to the same spines from reaching the dendritic shaft. One of the main functions of dopamine released from nigrostriatal fibres might thus be to alter the pattern of firing of striatal output neurons by regulating their input.

Consequences of transient (15-20 min) ischemia on the neuronal activity of the dentate gyrus and hippocampal CA 1 region were investigated in chronically implanted Sprague-Dawley rats. Forebrain ischemia was produced by occlusion of the carotids for 15 or 20 min, following cauterization of the vertebral arteries. Following the release of the carotids, both spontaneous and evoked activity showed a steady but partial recovery, reaching a maximum 12 to 24 h after the ischemic insult. From this plateau, both the power of rhythmic slow activity recorded during walking and the power of slow delta activity obtained during alert immobility decreased monotonically, with large changes occurring between postischemic days 2 and 4. The changes in spontaneous activity were accompanied by a decrease and eventual disappearance of the Schaffer collateral evoked responses in CA 1. Perforant path volleys were less efficient in activating the granule cells following ischemia compared to baseline levels. This decreased responsiveness was paralleled by a relative impairment of paired pulse depression. Neurophysiological signs of spontaneous or evoked neuronal hyperexcitability were not observed at any time point during the 8 postischemic days. Neuronal damage in the CA 1 region varied from moderate to complete loss of pyramidal cells. In addition, degenerating neurons were also observed in the hilus of the dentate gyrus. These findings do not support the "overwork" version of the excitoxic hypothesis of delayed neuronal damage and indicate that the cause of ischemic cell death should be sought in factors other than neuronal hyperactivity.

There are numerous hypotheses for the role of the axon collaterals of pyramidal cells. Most hypotheses predict that pyramidal cells activate specific classes of postsynaptic cells. We have studied the postsynaptic targets of two layer III pyramidal cells, that were of special interest because of their clumped axon arborization near, and also 0.4-1.0 mm from the cell body, in register in both layers III and V. 191 terminations from four sites (layers III and V, both in the column of the cell and in distant clumps) were analysed by electron microscopy. Only one bouton contacted a cell body and that was immunoreactive for GABA. The major targets were dendritic spines (84 and 87%), and the remainder were dendritic shafts. Of these 13 were classed as pyramidal-like (P), 8 smooth cell-like (S) and three could not be classified. Four of five S types, but none of the seven P types tested were immunoreactive for GABA, supporting the fine structural classification. The putative inhibitory cells therefore formed not more than 5% of the postsynaptic targets, and their activation could only take place through the convergence of pyramidal cells onto a select population of GABA cells. The results show that the type of pyramidal cells with clumped axons studied here make contacts predominantly with other pyramidal cells. Thus the primary role of both the intra and intercolumnar collateral systems is the activation of other excitatory cells.

Specific thalamic afferents to visual areas 17 and 18 were physiologically classified as X or Y type and injected with horseradish peroxidase (HRP). The axons were examined under the light microscope and were then processed for correlated electron microscopy. X axons arborized in area 17 and in the border between area 17 and 18. The X axons all formed terminals throughout layer 6, but were heterogeneous in their distribution in layer 4. They either occupied the entire width of sublayers 4A and 4B or were strongly biased toward layer 4A. Y axons also arborized in layers 4 and 6, but in area 17 they did not form boutons in sublamina 4B. Some Y axons projected only to area 18; others branched and arborized in both areas 17 and 18. Only the collaterals of one X axons were found to enter area 18; all the others were restricted to area 17. Y axons formed three to four separate patches of boutons about 300-400 microns in diameter, while all but one X axon formed a single elongated patch. Y axons had thicker main branches (3-4 microns) than X axons (1.5-2.5 microns) at their point of entry to the cortex. The main axon trunks and their medium-calibre collaterals were myelinated, but the preterminal segments were unmyelinated and studded with boutons. Each X or Y axon contacted about seven to ten somata, but Y axons made more contacts per soma (three to six) than did X axons (two to three). In addition to somatic synapses, both X and Y axons formed asymmetric (type 1) synapses on dendritic spines and shafts, with spines forming the most frequent targets (80%). Each Y bouton made, on average, 1.64 synapses in area 17 and 1.79 synapses in area 18, whereas each X bouton made only 1.27 synapses on average. Although there are proportionally fewer Y axons than X axons entering area 17, the Y axons provide as many synapses as the X axons because of their larger arbors and multisynaptic boutons.

Several lines of evidence suggest that gamma-aminobutyric acid is an inhibitory neurotransmitter in the cerebral cortex. To study the intracortical projection of neurons that selectively accumulate this amino acid, we injected radioactive gamma-aminobutyric acid into the upper layers of the striate cortex of monkeys along tracks at an oblique angle to the pia. Sections from the injected area were then processed by a combination of autoradiography and Golgi impregnation to reveal the distribution of labeled neurons and their morphological characteristics. Labeled neurons always occurred around the injection site in each layer. In addition, a consistent radial pattern of perikaryal labeling was observed in layers IVc-VI below the injection track in layers I-IVa. The closer the injection track was to the pia the deeper the peak density of labeled cells appeared. After injection in layers IVa and the lower part of III, the highest number of labeled neurons was in layer IVc; after injection in the upper part of layer III, most labeled neurons were in layer V; and, after injection in layers I and II, the proportion of labeled neurons increased in the lower part of layer V and in layer VI. All these neurons in the infragranular layers are presumably labeled by retrograde axonal transport via the labeled fiber bundles that extended from upper to lower layers. Thirty-four Golgi-stained neurons of various types were also examined for retrograde labeling. Two were labeled, and both were aspiny stellate cells in layer V. The arrangement of these putative GABAergic neurones, with axons that ascend from lower to upper layers in a regular pattern and arborize locally, would enable them to mediate inhibition within cortical columns and between neighboring columns.

[3H]GABA was injected into the visual cortex of rats in vivo. The labeled amino acid was demonstrated by autoradiography using semithin sections of Golgi material. Selective accumulation was seen in the perikarya of Golgi-stained, gold-toned, aspinous stellate neurons. Spine-laden pyramidal-like cells did not show labeling. This method gives direct information about the dendritic arborization of a neuron, and its putative transmitter, and allows the identification of its synaptic connections.

The synaptic connections of a specific type of identified cortical interneuron, the axo-axonic cell, were studied using Golgi methods. In the light-microscope axo-axonic cells were demonstrated in certain layers of the primary and secondary visual cortex of rat, cat and monkey, in the motor cortex of cat and in the subiculum and pyriform cortex of rat. The dendrites originating from the oval soma were oriented radially in a lower and upper spray within a cylinder about 100-150 microns wide. Electron-microscopy of Golgi impregnated, gold-toned axo-axonic cells showed predominantly but not exclusively asymmetrical synaptic contacts on their dendrites and spines, few synaptic contacts on the perikarya some of which were asymmetrical, and no synaptic contacts on the axon initial segment. The axon usually arborized within the vicinity of the cell's own dendritic field in an area 100-200 microns in diameter. In the kitten motor cortex the axon of a neuron in layer III descended to layer VI, providing a columnar arborization. The axon formed specialized, 10-50 microns long terminal segments invariably oriented parallel with the axon initial segment of pyramidal cells. All 85 identified symmetrical-type synaptic contacts, deriving from 31 specialized terminal segments, were found exclusively on the axon initial segment of pyramidal neurons. Rare, lone boutons of axo-axonic cells also made synaptic contact only with axon initial segments, confirming the exclusive target specificity of these cells. In identified gold-toned boutons, flattened pleomorphic vesicles were present. Electron-microscopy showed that axons ending in specialized terminal segments may originate from myelinated fibres, indicating that Golgi impregnation has revealed only part of the axon. Counting of axon terminal segments, each of which was in contact with the axon initial segment of a pyramidal neuron, revealed 166 pyramidal neurons receiving input from a partially reconstructed axo-axonic cell in the motor cortex of the kitten, and 67 from another cell in the visual cortex of the cat. The convergence of five axo-axonic cells onto one pyramidal cell was demonstrated in the striate cortex of the cat by counting all synaptic contacts on three initial segments. Cells from a one-month-old kitten were compared with those of the adult. The axon of the developing neurons was more diverse, having many growth cones and filopodia which made no specialized membrane contacts. However, the developing specific terminal segments formed synapses only with axon initial segments. It is concluded that the presence of axo-axonic cells in all the species and cortical areas we have examined suggests their association with the structural design of pyramidal cells, wherever the latter occur, and with their participation in the information processing of pyramidal cells. Axo-axonic cells are uniquely endowed with the means of simultaneously influencing the action potential at the site of origin in groups of pyramidal cells...

The hippocampus, in particular the neocortex-hippocampus-neocortex circuit, is widely believed to be crucial in memory. Information flow in this circuit is strongly influenced by relatively sparse afferents derived from subcortical centres, such as the septum, involved in arousal, emotions and autonomic control. A powerful mechanism, by which numerically small inputs can produce profound effects, is feed-forward inhibition, that is, the activation of local inhibitory interneurons, which, in turn, control the activity of large populations of principal cells in the hippocampus. An example is the cholinergic input to the hippocampus from the septum, which is likely to be involved in feed-forward operations. Here, we demonstrate the existence of a circuit underlying another powerful mechanism of subcortical control of hippocampal information processing. We show that GABA-containing afferents originating in the septum innervate most of the GABA-containing interneurons in the hippocampus, making many synaptic contacts with each of them. Activation of the GABA-containing neurons in the septum is likely to lead to disinhibition of the principal neurons in the hippocampal formation and so this pathway is probably crucial in the induction of hippocampal electrical activity patterns, and may be involved in NMDA (N-methyl-D-aspartate) receptor-mediated functions, such as memory, in a permissive manner.

The ultrastructural features and synaptic relationships of cholecystokinin (CCK)-immunoreactive cells of rat and cat hippocampus were studied using the unlabeled antibody immunoperoxidase technique and correlated light and electron microscopy. CCK-positive perikarya of variable shape and size were distributed in all layers and were particularly concentrated in stratum pyramidale and radiatum: the CCK-immunoreactive neurons were nonpyramidal in shape and the three most common types had the morphological features of tufted, bipolar, and multipolar cells. Electron microscopic examination revealed that all the CCK-positive boutons established symmetrical (Gray's type II) synaptic contacts with perikarya and dendrites of pyramidal and nonpyramidal neurons. The origin of some of the boutons was established by tracing fine collaterals that arose from the main axon of two CCK-immunostained cells and terminated in the stratum pyramidale; these collaterals were then examined in the electron microscope. The axon of one such neuron exhibited a course parallel to the pyramidal layer and formed pericellular nets of synaptic boutons upon the perikarya of pyramidal neurons. This pattern of axonal arborization is very similar to that of some of the basket cells, previously suggested to be the anatomical correlate for pyramidal cell inhibition. Typical dendrites of pyramidal cells also received symmetrical synaptic contacts from CCK-immunoreactive boutons, and some of these boutons could be shown to originate from a local neuron in stratum radiatum. Many CCK-immunoreactive cells received CCK-labeled boutons upon their soma and dendritic shafts. Synaptic relationship, established by multiple "en passant" boutons, was observed between CCK-positive interneurons of the stratum lacunosum-moleculare and radiatum. The soma and dendrites of the CCK-immunostained neurons also received symmetrical and asymmetrical synapses from nonimmunoreactive boutons. These results indicate that the CCK-immunoreactive neurons participate in complex local synaptic interactions in the hippocampus.