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Pyramidal neurons are covered with dendritic spines, the main postsynaptic targets of excitatory (asymmetrical) synapses. However, the proximal portion of both the apical and basal dendrites is devoid of spines, suggesting a lack of excitatory inputs to this region. In the present study we used electron microscopy to analyse the proximal region of the basal dendrites of supra- and infragranular pyramidal cells to determine if this is the case. The proximal region of 80 basal dendrites sampled from the rat hindlimb representation in the primary somatosensory cortex was studied by electron microscopy. A total of 317 synapses were found within this region of the dendrites, all of which were of the symmetrical type. These results suggest that glutamate receptors, although present in the cytoplasm, are not involved in synaptic junctions in the proximal portion of the dendrites. These data further support the idea that inhibitory terminals exclusively innervate the proximal region of basal dendrites.

Alterations in the numerical density and structure of asymmetrical synapses were examined in thin sections through barrel D4 in six CD/1 mice, including three controls and three sensory deprived animals. Sensory deprivation was effected by once daily trimming of all large mystacial vibrissae on the contralateral side of the snout from P0. The mice were perfuse-fixed at P20, several days following the termination of rapid synaptic growth during barrel development (White et al., Somatosens Mot Res 14: 34-55, 1997). Cerebral hemispheres contralateral to the deprived side were osmicated, sectioned at 40 microm and embedded in plastic for thin sectioning. Sterio's (J Microsc 134: 127-136, 1984) procedure combined with serial thin section analysis (Braendgaard and Gundersen, J Neurosci Meth 18: 39-78, 1986), was applied blindly to systematic random samples of neuropil in barrel hollows and septa. No significant difference in the numerical density, estimated total number, or in the proportion of perforated postsynaptic densities was observed. However, a significant decrease in the diameters of asymmetrical postsynaptic densities was observed in hollow (P < 0.05) and septal (P < 0.05) neuropil of deprived animals. These results demonstrate a significant morphological alteration in asymmetrical synapses of a type consistent with a reduction in synaptic activity consequent to sensory deprivation.

The long term goal of this work is to understand synaptogenesis in homologous regions of the cerebral cortex, i.e. a whisker barrel. Hemispheres of aldehyde perfused mice, at various ages from P6 to P65 (DOB = P0; three each), were osmicated and sectioned at 40 microns parallel to the pia. Barrels were identified, mapped and measured in sections through mid-level layer IV, and then embedded for electron microscopy. The main findings were: (1) Cell bodies and large diameter dendrites thin out in barrel hollows from P6 to P8. (2) Degeneration occurs primarily from P6 to P11, peaking on P8. (3) Single synapses from narrow, tubular axons predominate before P14; afterwards, multiple synapses from bag-like terminals increase in number. (4) The number of spines increases dramatically between P9 and P12. (5) Asymmetrical and symmetrical synapses occur at all ages studied; their junction lengths are not significantly different at any age. (6) Asymmetrical synapse density increases rapidly from P6 to P8, slowly from P9 to P.12, sharply between P13 and P14 along with patterned whisking, slowly to P20 and drops in adults. (7) Synapses onto spiny and non-spiny stellate cell bodies increase markedly from P10 to P20. (8) Changes in density of asymmetrical synapses in neuropil and of symmetrical synapses on spiny stellate cell bodies follow similar sequences but the sequence in neuropil is 72 h earlier. (9) When barrel size is taken into account, synaptogenesis is monotonic, increasing sharply in the second postnatal week followed by a slower increase into adulthood.

Regular spiking (RS) and intrinsically bursting (IB) neurons show distinct differences in their inhibitory responses. Under various conditions, the synaptic responses of RS cells display marked inhibitory postsynaptic potentials (IPSPs), whereas the responses of most IB cells do not (Silva et al: Soc Neurosci Abstr 14:883, 1988; Chagnac-Amitai and Connors: J Neurophysiol 61:747, 62:1149, 1989; Connors and Gutnick: TINS 13:99, 1990). This investigation is designed to determine if differences in the inhibitory responses of RS versus IB cells are reflected in differences in the concentration of inhibitory synapses onto their somata. RS and IB neurons in rat somatosensory cortex were identified by using intracellular recording and labeling, examined with the light microscope, and then serial thin-sectioned prior to examination with the electron microscope. Axonal terminals presynaptic to their somata and proximal dendrites were identified and classified according to criteria described by Peters and coworkers (Peters et al: J Neurocytol 19:584, 1990; Peters and Harriman: J Neurocytol 19:154, 1990; 21:679, 1992). The locations of these boutons were displayed on the surfaces of 3-D reconstructions of the somata and proximal dendrites. The reconstructions were produced directly from the serial thin sections by using a novel, electron microscopic, image-processing computer resource. Our analysis showed no significant difference in the types and concentration of boutons presynaptic to the cell bodies and proximal dendrites of intrinsically bursting versus regular spiking neurons. We conclude that the differences observed in the inhibitory responses of intrinsically bursting versus regular spiking neurons cannot be explained by differences in the concentrations of synapses onto their somata.

Neurons in areas 17/18a and 17/18b of mouse cerebral cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) transported from severed callosal axons in the contralateral hemisphere. Terminals of the local axon collaterals of labeled neurons (intrinsic terminals) were identified in the border regions of area 17 with areas 18a and 18b, and their distribution and synaptic connectivity were determined. Also examined were the synaptic connections of extrinsic callosal axon terminals labeled by lesion-induced degeneration consequent to the severing of callosal fibers. A postlesion survival time of 3 days was chosen because by this time the extrinsic terminals were all degenerating, whereas the intrinsic terminals were labeled by horseradish peroxidase. Both intrinsic and extrinsic callosal axon terminals occurred in all layers of the cortex where, with rare exception, they formed asymmetrical synapses. Layers II and III contained the highest concentrations of intrinsic and extrinsic callosal axon terminals. Analyses of serial thin sections through layers II and III in both areas 17/18a and 17/18b yielded similar results: 97% of the intrinsic (1,412 total sample) and of the extrinsic (414 total sample) callosal axon terminals synapsed onto dendritic spines, likely those of pyramidal neurons; the remainder synapsed onto dendritic shafts of both spiny and nonspiny neurons. Thus, the synaptic output patterns of intrinsic vs. extrinsic callosal axon terminals are strikingly similar. Moreover, the high proportion of axospinous synapses formed by both types of terminal (97%) contrasts with the proportion of asymmetrical axospinous synapses that occurs in the surrounding neuropil where about 64% of the asymmetrical synapses are onto spines. This result is in accord with previous quantitative studies of the synaptic connectivities of callosal projection neurons in mouse somatosensory cortex, and lends additional weight to the hypothesis that axonal pathways are highly selective for the types of elements with which they synapse.

This is one of a series of papers aimed at identifying the synaptic output patterns of the local and distant projections of subgroups of pyramidal neurons. The subgroups are defined by the target site to which their main axon projects. Pyramidal neurons in areas 1 and 40 of mouse cerebral cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) transported from severed callosal axons in the contralateral hemisphere. Terminals of the local axon collaterals of these neurons ("intrinsic" terminals) were identified in somatosensory areas 1 and 40, and their distribution and synaptic connectivity were examined. Also examined were the synaptic connections of "extrinsic" callosal axon terminals labeled by lesion induced degeneration consequent to the severing of callosal fibers. A post-lesion survival time of 3 days was chosen because by this time the extrinsic terminals were all degenerating, whereas the intrinsic terminals were labeled by HRP. Both intrinsic and extrinsic callosal axon terminals occurred in all layers of the cortex where they formed only asymmetrical synapses. Layers II and III contained the highest concentrations of both types of callosal axon terminal. Analyses of serial thin sections through layers II and III in both areas 1 and 40 yielded similar results: 97% of the extrinsic (277 total sample) and of the intrinsic (1215 total sample) callosal axon terminals synapsed onto dendritic spines, likely those of pyramidal neurons; the remainder synapsed onto dendritic shafts of both spiny and nonspiny neurons. Thus the synaptic output patterns of intrinsic vs. extrinsic callosal axon terminals are strikingly similar. Moreover, the high proportion of axospinous synapses formed by both types of terminal contrasts with the proportion of asymmetrical, axospinous synapses that occur in the surrounding neuropil where only about 80% of the asymmetrical synapses are onto spines. This result is in accord with previous quantitative studies of the synaptic connectivities of both extrinsic and intrinsic axonal pathways in the cortex (White and Keller, 1989: Cortical Circuits; Boston: Birkhauser): in all instances, axonal pathways are highly selective for the types of elements with which they synapse.

Pyramidal neurons in the mouse SmI cortex were labeled by the retrograde transport of horseradish peroxidase (HRP) injected into the ipsilateral MsI cortex. Terminals of the local axon collaterals of these neurons (CC terminals) were identified in SmI, and their distribution and synaptic connectivity were examined. To avoid confusion, terminals in SmI cortex labeled by the anterograde transport of HRP injected into MsI were eliminated by lesion-induced degeneration. Lesions of MsI were made 24 hours after the injection of HRP; postlesion survival time was 4 days. Most CC axon terminals occurred in layers III and V where they formed asymmetrical synapses. Of 139 CC synapses in layer III and 104 in layer V, approximately 13% were formed with dendritic shafts. Reconstruction of 19 of these dendrites from serial thin sections showed them to originate from both spiny and nonspiny neurons. Most synapses of CC terminals (about 87%) were onto dendritic spines. In contrast, White and Keller (1987) demonstrated that terminals belonging to the local axon collaterals of corticothalamic (CT) projection cells synapse mainly with dendritic shafts of nonspiny neurons: 92% onto shafts, the remainder onto spines. The distribution of asymmetical synapses onto spines and dendritic shafts was analyzed for neuropil in layers III, IV, and V. Depending on the layer, from 34 to 46% of the asymmetrical synapses in the neuropil were onto dendritic shafts. Results showing that CC and CT terminals form proportions of axodendritic vs. axospinous synapses that differ from each other, and from the neuropil, indicate that local axon collaterals are highly selective with regard to their postsynaptic elements.

The thalamocortical and other synapses of the apical dendrites of corticostriatal projection neurons in mouse primary somatosensory cortex (SmI) were examined by combining anterograde degeneration with the retrograde transport of horseradish peroxidase (HRP). Electrolytic lesions were made in the ventrobasal thalamus, followed 3 days later by injections of 40% HRP into the ipsilateral caudate-putamen nucleus. The next day, the mice were perfused and the SmI cortex ipsilateral to the lesion and injection sites was chopped at 125-micrometer and reacted for HRP using a CoCl2-DAB method. HRP-labeled corticostriatal cells in SmI cortex were medium-sized pyramidal cells, having somata located in the superficial portion of layer V and apical dendrites extending into layer I. Seven corticostriatal cells were serially thin sectioned and the layer IV portions of their apical dendrites were reconstructed. Each apical dendrite formed only one or two thalamocortical synapses (0.3 to 0.9% of their synapses in layer IV) indicating that corticostriatal neurons may be minimally responsive to direct synaptic input from the specific thalamic nuclei. Each apical dendrite formed about 12.6 asymmetrical synapses for every symmetrical synapse, suggesting that the relative numbers of excitatory and inhibitory synapses impinging on apical dendrites belonging to an individual class of neurons may be specified.

The distribution of thalamocortical (TC) and other synapses involving spiny stellate neurons in layer IV of the barrel region of mouse primary somatosensory cortex (SmI) was examined in seven male CD/1 mice. TC axon terminals were labeled by lesion-induced degeneration, which has been shown to label reliably all TC synapses in mouse barrel cortex. Spiny stellate neurons, labeled by Golgi impregnation and gold toning, were identified with the light microscope prior to thin sectioning and electron microscopy. Analysis of eight dendritic segments from seven spiny stellate neurons showed that most of their synapses are with their dendritic spines, rather than with their shafts. Axospinous synapses are primarily of the asymmetrical type, whereas axodendritic synapses are mainly of the symmetrical type. Dendrites of spiny stellate neurons consistently form thalamocortical synapses, most of which involve spine heads rather than spine stalks or dendritic shafts. From 10.4% to 22.9% of all asymmetrical synapses with dendrites of spiny stellate neurons involve TC axon terminals. In general, this is a higher range than the ranges that characterize the TC synaptic connectivity of dendrites belonging to other types of neurons, implying that spiny stellate neurons are perhaps more strongly influenced by TC synaptic input than other types of cortical neurons examined previously. Spines involved in TC synapses were distributed irregularly along each of the stellate cell dendrites; about half of the interspinous intervals between these spines were about 5 microns or less. Modulations of the efficacy of TC synaptic input to dendrites of layer IV spiny stellate neurons are discussed in the light of recently reported computer simulated analyses of axospinous synaptic connections.

Reciprocal axonal projections between homotypic areas of the vibrissal region of mouse primary motor cortex (MsI) (Porter and White: Neurosci. Lett. 47:37-40, '84) suggested the existence of reciprocal synaptic connections between callosal projection neurons and callosal afferents. In the present study, the retrograde transport of horseradish peroxidase (HRP) was combined with lesion-induced degeneration to identify synapses between callosal afferents and callosal neurons in the corresponding region of the contralateral cortex. The procedure was as follows: MsI was injected with HRP and aspirated on the following day. After 4 days, the animals were perfused and motor cortex was processed for HRP according to a variation of the Adams (Brain Res. 176:33-47,'77) technique, and postfixed in OsO4. The methods used consistently filled fine dendritic branches and spines with dense reaction product, thus allowing examination of synaptic contacts with these processes. All callosal projection neurons were identified as pyramidal neurons, having somata in cortical layers II/III and V. Labeled cells from each of the two levels were prepared for electron microscopy, and that part of each cell's apical dendrite that traversed the superficial cortical layers, where most callosal axons terminate, was cut in an unbroken series of thin sections. Micrographs were taken of all labeled profiles in each thin section, and tracings of the profiles were assembled to reconstruct the apical dendrites. Data on the distribution, type, and amount of callosal and other synapses with the shaft and spines of the apical dendrites were obtained by examining the reconstructions. In addition, profiles of basal dendrites of layer II/III cells were examined in thin sections to ascertain the numbers of callosal and other synapses formed with their shafts and spines. The proportion of synapses that each dendrite formed with callosal axon terminals was compared to the concentration of callosal afferents in the neuropil. Dendrites of both layer II/III and layer V pyramidal cells synapsed with callosal axon terminals. The apical and basal dendrites of layer II/III neurons formed a similar proportion of their synapses with callosal afferents, and this was similar to the concentration of callosal synapses in the surrounding neuropil. Segments of apical dendrites belonging to layer V and layer II/III neurons course through neuropil containing nearly the same concentration of degenerating callosal terminals, but the layer V cells form fewer callosal synapses.(ABSTRACT TRUNCATED AT 400 WORDS)

A Golgi-impregnated spiny stellate cell was selected from layer IV of SmI cortex in a mouse whose ipsilateral ventrobasal complex had been lesioned. The neuron was gold-toned, thin sectioned and then reconstructed in three dimensions using wooden sheets of appropriate thickness. These procedures enabled the numbers and distribution of thalamocortical and other synapses onto the reconstructed neuron to be determined. Results show the cell body to be roughly spherical and to receive 49 symmetrical synapses and four synapses which are intermediate between the asymmetrical and symmetrical type. A single, clearly asymmetrical axosomatic synapse is made by a degenerating, thalamocortical axon terminal. Five primary dendrites and their branches were reconstructed and, interestingly, these processes are distinctly elliptical in cross-section. The reconstructed dendrites receive 68 symmetrical synapses onto their shafts and 373 synapses onto spines of which 359 are asymmetrical and 14 symmetrical. Forty-eight, or about 13%, of the asymmetrical axospinous synapses are made by degenerating, thalamocortical axon terminals. An intriguing finding is that in many regions of the dendritic tree, two or more spines involved in thalamocortical synapses are attached to the dendritic shaft at intervals of 5 +/- 0.5 micron.

Lesion-induced degeneration was combined with the retrograde transport of horseradish peroxidase (HRP) to examine the thalamocortical and other synaptical relationships of corticothalamic projection cells in mouse SmI cortex. Injections containing 40% HRP were placed in the ventrobasal thalamus and the next day, electrolytic lesions were made of the injection site. About four days later, the animals were perfused with aldehydes and SmI cortex ipsilateral to the injection and lesion sites was tissue chopped and reacted for HRP. The somata of HRP-filled corticothalamic cells in SmI cortex had diameters of about 10 micrometers and occurred in the upper half of layer VI and in the lower half of layer V; their apical dendrites usually terminated within or just below layer IV, but in some instances, extended nearly to the pial surface. Reconstructions of serial thin sections through the layer IV portions of the apical dendrites of seven well-labelled corticothalamic cells showed them to form 7 to 20% of their synapses with degenerating thalamocortical axon terminals. In contrast, the proportion of thalamocortical synapses formed by the layer IV dendrites of corticostriatal (Hersch & White, in preparation) and corticocortical (White & Hersch, 1981) projection cells ranges from 0.3 to 0.9% and from 1.5 to 6.8%, respectively. We interpret these findings to indicate that pyramidal cell dendrites are specified to form characteristic proportions of thalamocortical synapses.

Immunocytochemical methods were used to examine GABAergic neurons in the barrel region of the mouse primary somatosensory cortex. GABAergic neurons occur in all layers of the barrel cortex but are more concentrated in the upper portion of layers II/III and in layers IV and VI. Nine cells in layer IV were examined with the electron microscope, and portions of their dendrites were reconstructed from serial thin sections. These cells are of the nonspiny, multipolar or bitufted varieties, and some of them have beaded dendrites. The labeled cell bodies and their reconstructed dendrites were postsynaptic at asymmetrical synapses with thalamocortical axon terminals labeled by lesion-induced degeneration and with unlabeled axon terminals. Each cell also received symmetrical synapses from GABAergic axon terminals and from unlabeled axon terminals. Our results indicate that GABAergic cell bodies and processes receive synapses from thalamocortical axon terminals but that different cells display marked differences in the proportion of thalamocortical and other synapses they receive. These results indicate that GABAergic cells form a heterogeneous population with respect to their morphologies and patterns of synaptic inputs. The synaptic sequences revealed here for GABAergic neurons represent an anatomical substrate for various inhibitory processes known to occur within the cerebral cortex.

The shape of dendritic spines in mouse Sm1 cortex, which synapse with degenerating thalamocortical axon terminals, differs significantly from that of adjacent spines along the same spiny dendrites, which synapse with intact axon terminals. It is concluded that this morphological difference results from focal alterations in the heads of spines imposed by the degeneration of their presynaptic afferents.

Barrels in the posteromedial barrel subfield (PMBSF) in layer IV of mouse somatosensory cortex are consistently identifiable and somatotopically related to the large whiskers on the animal's snout32,38. This is a study of the ultrastructure - in particular the synaptic connections - of mouse PMBSF barrels. A technique was developed which enabled the precise selection for electron microscopy of specific regions of barrels identified in the light microscope. Barrel "sides" containing a high density of neuronal cell bodies and myelinated axons, contrasted with barrel "hollows" in which these elements were relatively sparse. The types and frequencies of synapses were examined in series of thin sections through sides and/or hollows of barrels B2, C3, C7, D4, E4, E5, and E8 from 6 mice. In all, 3042 synapses from 20 fields, each at least 10 mum x 10 mum x 4.8 mum in size, were classified. Four distinct kinds of presynaptic terminal were identified: (1) darkly, and (2) lightly stained, asymmetrically synapsing terminals (AD, AL respectively), and (3) darkly, and (4) lightly stained, symmetrically synapsing terminals (SD, SL respectively). Synapses of these terminals were distributed in roughly similar proportions throughout the neuropil of barrel sides and hollows. Thus in all barrel regions approximately 92% of the synapses in neuropil were made by AD terminals, 1.0% by AL, 2.4% by SD and 4.0% by SL terminals.

The objective of this study was to examine synaptic relationships among 3 neuronal elements in the cerebral cortex: thalamocortical afferents (TC), corticothalamic projection cells (CT), and GABAergic neurons. TC axon terminals in the barrel cortex of the mouse were labeled by lesion induced degeneration; local axon collaterals belonging to CT cells were labeled by the retrograde transport of horseradish peroxidase; and GABAergic neurons were identified using immunocytochemistry. CT and GABAergic neurons form synapses with each other and both receive synapses from TC afferents. These findings indicate the existence in the cerebral cortex of a triadic circuit involving afferent input both to projection and to local inhibitory neurons, and reciprocal synaptic interactions among these neuronal populations.

Electrolytic lesions were made of the nucleus ventralis posterior pars lateralis thalami and of the nucleus posterior thalami in the male CD/1 mice to label thalamocortical axon terminals in mouse SmI cortex. Pyramidal cells in layers II through V of SmI cortex were labeled by the retrograde transport of horseradish peroxidase injected into ipsilateral MsI. Numerous pyramidal cells, particularly in the more superficial layers of SmI cortex, were filled with reaction product so that even their dendritic spines and local axon collaterals were clearly visible. Six well-filled pyramidal cells with somata in layers III and IV were serial thin-sectioned, and portions of their dendrites in layer IV were examined with the electron microscope to determine the distribution of their thalamocortical and other synapses. It was found that, in general, different dendrites of a single pyramidal cell formed similar proportions of thalamocortical synapses and that the six pyramidal cells, as a group, also formed similar proportions of thalamocortical synapses with their dendrites in layer IV. In contrast, when the thalamocortical connectivity of the six cells was considered as a function of their depths within the cortex, a clear trend was seen for the proportion of thalamocortical synapses to increase with increasing proximity of the cell body to layer IV. A hypothesis based on the timing of developmental events is proposed to account for this observation.

Golgi-impregnated and -deimpregnated neurons having somata in layer IV of mouse posteromedial barrel subfield (PMBSF) cortex were identified with the light microscope and then extensive portions of them were examined with the electron microscope. Dendrites of nine nonspiny multipolar cells and eight of their cell bodies were reconstructed from serial thin sections to determine the numbers and types of symmetrical, asymmetrical, and thalamocortical synapses they formed. Results of this analysis show that cells of the same general morphological class may form widely different patterns of synaptic connections: some nonspiny multipolar cells had dendrites that formed a high proportion of their synapses with thalamocortical axon terminals, whereas dendrites belonging to other cells formed only very small proportions of thalamocortical synapses. A similar diversity characterized the synaptic connections of cell bodies: some formed more symmetrical than asymmetrical synapses, others the reverse. Some formed high proportions of thalamocortical synapses, others much less. Comparisons of thalamocortical synaptic input to cell bodies and dendrites showed that one cell formed about the same proportions of thalamocortical synapses with its cell body as with its dendrites. For two other cells the proportions of thalamocortical synapses formed with their somata was about double that formed with their dendrites. The remaining five cell bodies examined formed far higher proportions of thalamocortical synapses than did their dendrites. That different nonspiny multipolar cells form such contrasting synaptic patterns suggests that included within this morphological classification are cells which are likely to have very different functional roles.