- Email: [email protected]
Distribution of corticocortical and thalamocortical synapses on identified motor cortical neurons in the cat: Golgi, electron microscopic and degeneration study
Brain Research, 345 (1985) 87-101 Elsevier
87
BRE 11060
Distribution of Corticocortical and Thalamocortical Synapses on Identified Motor Cortical Neurons in the Cat: Golgi, Electron Microscopic and Degeneration Study M. ICHIKAWA** K. ARISSIAN and H. ASANUMA The Rockefeller University, New York, NY 10021 (U.S.A.) (Accepted January 8th, 1985) Key words: motor cortex - - association fiber - - thalamocortical fiber - - stellate cell - - PT cell
Details of the terminal connections of corticocortical and thalamocortical fibers on pyramidal and stellate neurons in the cat motor cortex were studied using the electron microscope in combination with the Golgi and axonal degeneration techniques. Corticocortical terminals were examined in 23 identified neurons of which 11 were pyramidal and 12 were stellate. Stellate neurons located in layer III received many degenerating terminals (average 8.4 + 2.2 per unit length of dendrite (ULD)) and the majority of these (95%) were found on the proximal dendrites or on the cell bodies. The pyramidal neurons received fewer degenerating terminals (average 2.1 + 0.27/ULD) and these were located on more distal dendritic shafts or on dendritic spines. The majority of these synapses were of the asymmetric type. Thalamocortical terminals were examined in 9 pyramidal and 9 stellate neurons. Pyramidal neurons received many terminals (average 6.0 + 1.23/ULD) and these were found on the basal as well as the apical dendrites and on dendritic spines. Stellate neurons received fewer terminals (average 4.2 _+0.64/ULD) and were located primarily on proximal dendritic shafts. The majority of these synapses were of the asymmetric type. The functional role of these synapses is discussed in relation to the physiological results reported in the preceding paper. INTRODUCTION
to the terminals of thalamocortical afferents. Recent-
In the preceding study, Kosar et a132 reported that microstimulation of cat somatic sensory cortex (area
ly, Sloper and Powel122 studied other afferents to the monkey motor cortex, including the corticocortical input from the somatic sensory cortex, but the em-
2) produced monosynaptic EPSPs within n e u r o n s located in the superficial layers (II and III) of the motor cortex, but not within those in the deep layers (V and VI). Injection of horseradish peroxidase ( H R P ) into
phasis was on the postsynaptic profiles of termination sites, i.e. whether the fibers terminated on the den-
neurons receiving monosynaptic input from the sensory cortex suggested that these were primarily stellate neurons located in the superficial layers. The terminal sites of afferent fibers in the neocortex have been studied by many investigators at the ultrastructural level ~,5,7,1t,15.19.2s. However, most of these stud-
spines and 20% on shafts. Although they did not identify the postsynaptic neurons, they suggested, from various ultrastructural configurations, that these spines belonged to pyramidal n e u r o n s and the shafts belonged to stellate neurons. If pyramidal neurons receive 80% of the corticocortical synaptic input, then one would think that sensory cortex stimulation should elicit monosynaptic EPSPs mostly within pyramidal neurons. Since the pyramidal neurons are, in general, larger than stellate neurons, they should be easier to impale with an electrode and inject HRP. However, Kosar et al. 12 were unable to
ies were carried out within the sensory or parietal cortices and only a few studies exist which examined the terminal sites within the motor cortex. Strick and Sterling24 and Sloper 21 examined synaptic termination within the m o n k e y motor cortex using the electron microscope, but their studies were restricted
dritic spines or shafts. They reported that about 80% of the corticocortical terminations occur on dendritic
* Present address: Department of Anatomy and Embryology, Tokyo Metropolitan Institute for Neurosciences, 2-6 Musashidai, Fuchu, Tokyo 183, Japan. Correspondence: H. Asanuma, The Rockefeller University, 1230York Avenue, New York, NY 10021, U.S.A. 0006-8993/85/$(}3.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
88 identify pyramidal neurons as the main recipients of corticocortical input. We were puzzled by this observation and started this experiment to resolve the discrepancy. Recent development of the electron microscopic technique made it possible to combine an ultrastructural study with a Golgi impregnation method ~.~4. 23.27. Utilizing this technique we sought to determine the sites as well as the density of the synapses that corticocortical and thalamocortical fibers make with identified motor cortical neurons. MATERIALS AND METHODS Six adult cats of either sex weighing 2.5-3.5 kg were used. Operations were carried out under Nembutal anesthesia (35 mg/kg). In 3 cats, the sensory cortex was exposed and the anterior bank of the ansate sulcus (area 2) was removed by suction. In the remaining 3 cats, kainic acid (2.5/~g dissolved in 1.0 ~tl of 0.9% saline) was injected into the ventrolateral nucleus of the thalamus (VL) stereotaxically (A 11.0, L 4.5, H 1.5) guided by the atlas of Jasper and Ajmone-Marsan 9. After 5 days survival, the animals were reanesthetized and perfused with saline followed by a mixture of 2.0% glutaraldehyde and 1.5-2.0% paraformatdehyde in 0.1 M phosphate buffer solution. Subsequently, the sensory and motor cortices were removed and in the latter group of animals, a thalamic block containing VL was also removed. The motor cortex was cut into sections of 2-3 mm thickness and these were immersed in a rapid Golgi solution (3.0% potassium dichromate plus 0.2% osmium tetroxide) for 3-5 days. The sections were then transferred into a silver nitrate solution (0.75%) after washing with distilled water and left immersed for 24 h. The tissue was then dehydrated in a series of graded alcohols, embedded in celloidine, cut serially at 80-100 ~tm thickness and mounted on slides. The lesioned tissues (sensory cortex and thalamus were frozen and sectioned at 40ktm, and stained with the Klfiver and Barrera method 13.
Electron microscopy (EM) Well impregnated neurons were selected from the motor cortical area posterotateral to the cruciate sulcus where association fibers from area 2 project most densely26,29. Small pieces of the sections containing
the selected cells were re-fixed with 1.0~ OsCL stained "en bloc" with uranyl acetate and re-embedded in Epon 812. The Gotgl image ot the selected neurons was photographed and/or sketched using a camera lucida. The tissue was then cut into series of a semithin section (0.5-1.0/~m in thickness) followed by 6 ultrathin sections of 60-100 nm thickness using a diamond knife. The semithin sections were mounted on glass slides and stained with toluidine blue. The locations of the impregnated processes in these sections were sketched using a camera tucida and these were used in locating EM images obtained from the adjacent ultrathin sections. The ultrathin sections were mounted on one hole copper grids, stained with lead citrate and scanned and photographed at 10-60 × 103 magnification using the electron microscope (Hitachi HU-11C or J E O L JEM I00 CX). In our procedure, nearly all ultrathin sections could be recovered with only an occasional loss of one or two sections between two semithin sections. An average of 40 semithin sections and 250 ultrathin sections were cut and examined per neuron covering an area of 60-70#m in width. In this way we were able to examine almost all parts of small neurons but only proximal dendrites and somas of large pyramidal neurons except for the parts included in semithin sections. Since the extent of areas examined were different for each cell, the density of degenerating terminals between different cells was compared by the number of terminals per unit length of dendrites. This was done by measuring the length of dendrites examined with EM utilizing the camera lucida drawings and dividing the total number of degenerating terminals by the length. Therefore: DT ULD
Total DT Total length of dendrite (mm)
where DT is the number of degenerating terminals and ULD the unit length of dendrite. By this way the difference of the density of degenerating terminals between the cells could be compared objectively because the ratio of the area included in semithin sections is the same irrespective of the difference in the total areas examined. RESULTS The cortical lesions were primarily limited to the
89 anterior bank of the ansate sulcus (area 2) in all 3 cats as shown in Fig. 1A. Although there was some restricted damage to the underlying white matter, it is most likely that only those fibers entering or leaving the lesioned cortex, i.e. area 2, were affected. The center of the kainic acid injection in the thalamus was characterized by a complete loss of neuronal perikarya and an increase in density of glial cells. Surrounding this area, there was a zone of decreased neuronal density extending about 0.5 mm in width. The shape of the lesioned areas was eliptical and the diameter ranged from 3.0 to 6.0 mm as shown in Fig. lB. The lesioned area, thus included not only the ventrolateral nucleus, but also the ventroanterior nucleus, dorsal part of the subthalamic area and a portion of the thalamic reticular nucleus.
Classification of cell types The rapid Golgi method used in this study was successful in staining a variety of neuronal types as reported elsewherem,14, TM. However, we have selected only typical pyramidal cells and stellate cells for examination since the preceding electrophysiological study 12 suggested a difference in associational inputs upon these two types of cells. The pyramidal cells were characterized by a triangular or conical cell body, a distinct ascending apical dendrite, and several basal dendrites extending horizontally from the cell body as well as by a relatively straight axon arising from the base of the cell body or the basal dendrite. These cells were located in layers IIl and V. The stellate cells selected were those having round somata and radiating dendrites with no particular orientation and sparse or no dendritic spines. Although it is known that there are spiny stellate cells which possess abundant dendritic spines, we did not observe these cells in our preparations. One possibility is that these spiny stellate cells are known to be located in layer IVlO.~4,1~which is virtually absent in the motor cortex. Fig. 2 shows examples of Golgi stained pyramidal and stellate cells drawn with a camera lucida. Altogether 21 stellate cells and 20 pyramidal cells were examined using the electron microscope. Golgi impregnation resulted in electron-dense precipitates which filled the cytoplasm, but in our preparation, most of the precipitates were washed away from the cytoplasm during lead citrate staining. However, the vacant spaces which remained after
the staining served as reliable indicators of the impregnated cells as did the presence of the precipitate, examples of which are shown in Figs. 3 and 4. Utilizing these positive or negative images of impregnated cells, we have examined the terminal sites of the degenerating fibers produced by area 2 or thalamic ablation. The degenerating terminals in our samples appeared to be at various stages of degeneration, but they were commonly characterized by electrondense cytoplasm with markedly distorted or absent organelles (Figs. 3 and 4). Synaptic contact between Golgi impregnated cells and degenerating terminals could be recognized by the presence of a synaptic cleft and by the thickening of the postsynaptic membrane (Figs. 3 and 4). Since.the sketches drawn from the nearby semithin sections were available, it was not difficult to recognize the location of these degenerating terminals in relation to the cell body. However, because of the artifact which resulted from degenerative changes as well as from the removal of silver precipitates, it was often difficult to assess the type of synapses. Therefore, our effort was primarily concentrated on identifying the location of synapses, but general information could be obtained by examining degenerating terminals synapsing upon unlabelled postsynaptic neurons.
Association fibers Random examination of degenerating terminals in the pericruciate gyrus revealed that the distribution of these terminals was the greatest in the area posterolateral to the cruciate sulcus. The number of degenerating terminals per 100 ~tm square in layer III ranged from 8 to 20. Within the same area, there were cortical patches which were relatively rich with degenerating terminals intermixed with less dense patches. Although an accurate assessment of the difference in density of degenerating terminals between these patches and the size of the patches was difficult to make, it was our general impression that the number of degenerating terminals in some patches was twice that in the neighboring patches. However, within the region analysed, there were no patches which were void of degenerating terminals. The sites of synapses of corticocortical fibers were examined using 11 pyramidal cells and 12 stellate cells randomly selected from the same area. On all of these neurons there were synapses which had degen-
90
as
cat 1
cs
as
cs
ABC
A lrnm
A cat 4
5
A 6
j
5mm
B Fig. 1. Locations of lesions in the sensory cortex and the thalamus. A: parasagittal sections of the cortex through the sensory and motor cortices. Arrows in the left figurines show the levels of sections illustrated on the right. Hatched areas, extent of the lesions, as. ansate sulcus; cs, cruciate sulcus. B: parasagittal sections of the thalamus at the levels of L 3.7.4.5 and 5.6. The drawings are based on the atlas by Berman 3. Dotted areas, extent of the lesions. AV, anteroventrat nucleus; CA, caudate nucleus; IC. internal capsule: V A . ventroanterior nucleus; VL, ventrolateral nucleus; OT, optic tract; ZI, zona incerta, FF, nucleus of the fields of Forel. Further details in text.
91
\
II
c
III
A
J
4c
"M
V loopm Fig. 2. Camera lucida drawing of Golgi impregnated cells in the motor cortex. Roman numerals in the left show the layers. Two pyramidal (A, B) and two stellate type cells (C, D) are shown. Note that the apical dendrites of the pyramidal cells were cut off. Arrows and small letters indicate the sites of EM pictures shown in Figs. 3 and 4. erating profiles on the presynaptic side. The locations of these terminals on the pyramidal n e u r o n s are shown in Fig. 5A. The cell bodies of 7 of these neurons were located in layer III and 4 were in layer V. The thickened portions of the dendrites indicate the regions which were examined with the electron mi-
croscope and arrows indicate the sites of the degenerating terminals. Altogether 24 degenerating terminals were found on these 11 n e u r o n s of which 12 made contacts with dendritic shafts and 12 with dendritic spines. No degenerating terminals contacted the cell bodies. In our samples, all of the pyramidal
92
Fig. 3. Electron micrographs of degenerating terminals of thalamocortical fibers on Golgl impregnated cells, a: terminal on dendritic spine of pyramidal cell. x25.000, b: higher magnification of the terminal shown in a. x60. D00. c-f: serial electron micrographs of terminals on dendritic shaft of pyramidal cell. Synaptic contact is observed in e and f. x60.000. DT. degenerating terminals: D. dendritic shaft; SP, dendritic spine: M, mitochondria: NT. normal terminal.
93
Fig. 4. Electron micrographs of degenerating terminals of corticocortical fibers on Golgi impregnated cells. Small letters correspond to small letters in Fig. 2. a: a terminal on the dendritic shafts of a stellate type cell. ×25,000. b: higher magnification of the terminal shown in a. ×60,000. c: terminal on cell body of a stellate type cell. x60,000. DT, degenerating terminal; PE, perikaryon; N, nucleus; D, dendritic shaft; M, mitochondria. Further details in text.
94 neurons received only a few corticocortical synapses averaging 1.8 synapses/ULD on layer III neurons and 2.5/ULD on layer V neurons. The number and distribution of corticocortical synapses on stellate neurons were different from those on pyramidal neurons. As shown in Fig. 4b, all of the stellate neurons located in the superficial layers received about 3 times as many synapses as the pyramidal neurons. The minimum density of degenerating terminals on a stellate neuron was 2.4/ULD the maximum was 12.0/ULD and the average was 8.4 + 2.20 (S.E.D.). These terminals were located primarily on cell bodies and dendritic shafts near the cell body (95%) and only a few were on the spines. A stellate cell located in layer V received only 3.1 degenerating terminals/ULD Fig. (5B, g). As shown in Table II, a total of 89 degenerating corticocortical terminals were found on identified target cells and only 15 (17%) of these were on dendritic spines. This is a different ratio from the results reported by Sloper and Powel122 who found that in the m o n k e y motor cortex, 80% of the association terminals contacted dendritic spines and only 20% were on dendritic shafts. To examine whether our criterion of sampling the degenerating terminals was different from their criterion, we randomly selected 200 degenerating corticocortical terminals on unidentified neurons and classified the postsynaptic profile. We found, in agreement with Sloper and Powel122, that 156 (78%) of these profiles were dendritic spines. Of these 156 terminals, 153 made asymmetric synapses and 3 were difficult to determine. Of the 44 degenerating synapses found on dendritic shafts or cell bodies, 37 were asymmetric and 7 were difficult to determine. Examples of these synapses are shown in Fig. 6. The terminal sites of thalamic fibers were examined on 9 pyramidal cells and 9 stellate cells from the same region of the cortex in which the thalamic input occurred. Fig. 7A summarizes the locations of degenerating thalamocortical terminals on pyramidal cells in layers III and V. A total of 61 degenerating terminals were identified and many of these were on the proximal dendrites. These pyramidal neurons received a rather massive thalamocortical input except for cells f and i, which were located in upper layer III and in layer V. The maximum number of degenerating terminals for a cell was 10.0/ULD (cell c) and the
TABLE I Number of degenerating terminals per unit length (1.0 ram) oF dendrites in each identified cell (mean + S. E. M. )
n, number of cells. No. of degenerating terminals Pyramidal cells" Stellate type cells
Corticocortical terminals Thalamocorticalterminals
2.1 _+ 0.27 (n = 11) 6.0 + 1.23 (n = 9)
S.4 _+ 2.20 (n = 12) 4.2 _+ 0.64 (n = 9)
average was 6.0 + 1.23 (Table I). As shown in Table II, nearly half of the degenerating synapses were found on the dendritic shafts and the rest were on dendritic spines. No thalamic terminals were on the somata. There were fewer thalamocortical terminals on stellate neurons than on pyramidal neurons. As shown in Fig. 7B, the maximum number of degenerating terminals found on a stellate neuron was 5/ULD (cell a, b) and the average was 4.2 + 0.64. The majority of these contacted dendritic shafts and only 3 (11%) were found on dendritic spines (Table I). The results suggests that there are more thatamocortical synapses on pyramidal neurons than on stellate neurons. This is quite different from the associational terminals, most of which were found on stellate neurons. Altogether. 89 degenerating thalamocortical terminals were found on identified neurons. Of these. 36 (41%) made contact with dendritic spines, 50 (56%) with dendritic shafts and 3 (3%) with cell bodies. This distribution is different from that of previous reports 22.24 in which 90% of thalamocortical terminals were found to contact dendritic spines. As before, we randomly selected 100 degenerating thalamic terminals and found, in agreement with previous investigators, 80 made contact with spines and 20 with shafts or cell bodies. Of these. 75 were asymmetric synapses and 25 could not be identified. DISCUSSION In this study, less than half of the degenerating synapses of both association and thalamic fibers terminated on dendritic spines of identified-motor cortical neurons. In previous studies 222& the majority of degenerating synapses from both sources was found on the dendritic spines. This discrepancy was not due to
95
I
II
Ill
b ¢
,/ ¥
i
A
/ f
k
A I
III
¢
b
B Fig. 5. Locations of degenerating corticocortical terminals on Golgi impregnated cells. Arrows indicate sites of identified terminals. A: terminals on pyramidal cells. B: terminals on stellate cells. Thick lines indicate the portions of dendrites which were examined with electron microscope. Further details in text.
96
Fig. 6. Examples of degenerating corticocortical synapses, a: asymmetric synapse on a dendritic spine, b: asymmetric synapse on dcndritic shaft and a spine, c: asymmetric synapse on cell body. Postsynaptic thickening is not as marked as those shown in a and b. d: synapse of unidentified type on dendritic shaft, DT, degenerating terminals; SP, dendritic spine; SH, dendritic shaft. Magnification. x 50,000.
o u r failure of i d e n t i f y i n g spines b e c a u s e in the control s u r v e y in which t h e t e r m i n a l sites w e r e not restricted to i d e n t i f i e d n e u r o n s , t h e ratio o f synapses of
spines was similar to that of the p r e v i o u s investigators. O n e possible e x p l a n a t i o n for the d i s c r e p a n c y is that the r a p i d G o l g i staining did not stain all the d e n -
97
I:00 m
I
H
f
h I
A I
II
~
f
e
i
B Fig. 7. Locations of degenerating thalamocortical terminals on Golgi impregnated cells. The format is the same with that of Fig. 5. A: pyramidal cells. B: stellate type cells. dritic spines. However, this is not likely because as shown in Fig. 2, spines were present on the pyramidal n e u r o n s we selected in as great an a b u n d a n c e as
those reported by the previous investigators 10,1a. The most likely source of the discrepancy is that the dendrites of the identified n e u r o n s that we examined
98 ]'ABLE 1I Number of degenerating terminals synapsing on d((ferentpostsynaptic sites No. of cells
No. of deg.
No. of synapses Cell body (%)
Dendritic spines (%)
Dendritic shafts (%)
Corticocortical fibers pyramidal cell stellate type cell
11 12
24 65
0 (0) 12 (18)
12 (50) 3 (5)
12 ~501 5(I ~77j
Thalamocortical fibers pyramidal cell stellate type cell
9 9
61 28
0 (0) 3 (11)
33 (54) 3 (11)
28 {46) 22 (78)
were located rather close to the cell body because of obvious technical difficulties (see Materials and Methods). Since the terminals of VL fibers are concentrated in layer 11122,24 and those of association fibers are largely found in the superficial layers 22, it is likely that our sample missed a considerable fraction of the degenerating terminals located on spines of distal dendrites of deep pyramidal cells. On the other hand, the dendrites of the stellate cells do not extend for such a great distance and therefore, it was easier to examine their dendritic arborizations more thoroughly. Although our sampling methods do not allow quantitative statements to be made regarding synaptic density, we feel that the ratios we obtained concerning the relative distributions of degenerating terminals on the two types of neurons are reasonably accurate and do not reflect a sampling bias other than that already mentioned in the preceding paragraph. This is because: (1) comparable numbers of each type of neuron were selected for analysis; (2) comparable lengths of dendrites were surveyed for each neuron; (3) the portions of dendrites examined in the electron microscope were evenly spaced (between our semithin sections); and (4) comparable numbers ( - 90 each) of degenerating synapses were identified for each paradigm. Corticocortical projections
Our results revealed that the stellate cells in the superficial layers of the motor cortex received a substantial input from association fibers. These synaptic terminals were located mostly on the proximal dendritic shafts and some were on the cell bodies. Very few were found on dendritic spines. This observation
is in agreement with a previous study 22 in which the authors suggested that the dendritic shafts receiving associational terminals in the motor cortex were for the most part dendrites of stelIate neurons. A random sampling of these degenerating terminals which contacted unidentified dendritic shafts indicated that almost all of these consisted of asymmetric membrane specializations. This observation suggests that the majority of association fibers making contact with stellate neurons in the motor cortex form asymmetric synapses. Asymmetric membrane specializations are often assumed to be indicative of excitatory synapses and Kosar et al. 12 have shown that microstimulation within area 2 of the sensory cortex generated monosynaptic EPSPs exclusively in neurons located in the superficial layers of the motor cortex: monosynaptic IPSPs were never recorded. Together these results strongly suggest that the asymmetric synapses in the corticocortical projection function as excitatory synapses although we could not identify the type of vesicles in the presynaptic terminals. As shown in Fig. 5, association fibers made contacts not only with stellate cells, but also with pyramidal cells in layers III and V. However. these terminals must represent only a fraction of the total degenerating terminals synapsing on these neurons. This is because in our sample of degenerating terminals synapsing with pyramidal cells, only 17% made axospinous contacts yet in the control survey. 80% of the degenerating terminals were axospinous. Therefore, it is likely that the majority of association terminals synapsed with spines of pyramidal cell dendrites located further distally, beyond the range of our sample. A question then arises as to why Kosar et al. 12 could not record monosynaptic corticocorticat EPSPs
99 from the pyramidal neurons located either in the superficial or in the deep layers. Since the pyramidal cells are larger than the stellate cells, they should be easier to impale with electrodes and to record EPSPs. However, Kosar et al. J2 reported that when the intracellular recordings were stable and the membrane potentials were large suggesting that they were recording from large pyramidal cells, they were unable to record short-latency cortically activated EPSPs or IPSPs. Several reasons could be considered to account for this observation. The first is that association terminals are aggregated within the cortex and make contacts only with certain groups of pyramidal cells, and that Kosar et al.L2 penetrated cells outside of these aggregates. In our serial sections, we noticed that there were strips which contained dense degenerating terminals as well as strips which were less densely populated by these animals. This is, however, unlikely to be the explanation since there were no strips within the sampled area which were devoid of degenerating terminals. Furthermore, as shown in Fig. 5, all of our randomly selected pyramidal neurons received some association input. Therefore, the possibility that Kosar et a1.12 selected samples from outside of such aggregates is small. The second possibility is that the terminals synapsing on the spines belong to small diameter axons. Then the postsynaptic potentials generated by these synapses would appear too late to be recognized as monosynaptic potentials. The third possibility is that because of their specific shape, spines are electrically isolated from the cell body. The spine forms a clavate head and the axon contacts only the head of the spine, not the stem 20. These are attached to the dendritic shaft by a thin stem which is a tenuous connector with high electrical resistance. Because of this configuration, Palay and Chan-Palay 17 suggested that the spines are specialized for the transmission of the information from a large number of similar inputs in which the individual signal counts for very little to the postsynaptic neurons. If this is the case, it is possible that the synaptic potentials generated by microstimulation of the sensory cortex were too small to be detected by averaging a small number of trials, i.e. only 20 superimpositions 12. The above possibility, however, is unlikely because of recent calculations by Turner and Schwarzkroin 25 on the electrical properties of dendritic spines of hippocampal pyramidal cells. They concluded that
the input resistance of a spine is much greater than the resistance at the neck of the spine and therefore the attenuation across the neck is negligible. Instead the attenuation of the synaptic potential takes place at the distal dendritic shaft because of its high resistance. Therefore, the fourth possibility is that because of the high resistance of the dendritic shaft and the resulting short length constant of the shaft, the EPSPs at the spine became too small to be detected at the soma. In this model, however, the synaptic currents at the dendrites should produce field potentials as discussed by Kosar et al. 12 even if the current did not reach the cell body. Therefore, area 2 stimulation should have produced negative field potentials in layer III where the majority of terminals synapse on the dendritic spines. Yet, Kosar et al. ~2 did not record these negative loci. Therefore, the fifth possibility is that the membrane characteristics of a spine is different from that of the soma and the input resistance of spines is smaller than they calculated25. If this is the case, the high resistance of the dendritic shaft prevents the entry of synaptic current and the current flows out through the membrane of the spine. Since the axis of the spine is directed randomly, the outgoing currents as a whole do not generate field potentials. This model is in agreement with the hypothesis proposed by HorwitzS. He calculated the electrical properties of the spine and concluded that the synaptic activity at a spine head can generate a large electrical field along the shaft of the spine. Then he proposed that this electric field functions by inducing electrophoretic migration of charged metabolites into the spine. At present we do not know which of the above possibilities is correct nor the functional role of the synapses on the spines, but it seems clear from the physiological data 1: that axospinous synapses are not participating in the rapid transmission of impulses to the pyramidal neurons in the motor cortex. However, it would be interesting to note that following chronic lesion of VL, pattern of the corticocortical projections changes significantly in parallel with physiological changes 2 which were reported in the preceding paper.
Thalamocortical projections It has been reported that 90% of thalamic terminals synapse on dendritic spines, most likely of py-
r a m i d a l n e u r o n s and t h e s e synapses h a v e b e e n sug-
er p o p u l o u s synapses with shafts of basal d e n d r i t e s
gested to be the m a j o r t h a l a m i c i n p u t to the p y r a m i -
a c c o u n t i n g for the fast rising m o n o s y n a p t i c E P S P s .
dal n e u r o n s 22,24. T h e n t h a l a m i c s t i m u l a t i o n s h o u l d
Since the nucleus V L sends massive fibers to the mo-
not p r o d u c e s h o r t - l a t e n c y m o n o s y n a p t i c e x c i t a t i o n
tor cortex, it is possible that only 10% of the total
of p y r a m i d a l n e u r o n s in the m o t o r c o r t e x b e c a u s e of
synapses c o n t a c t i n g d e n d r i t i c shafts are sufficent to
the r e a s o n s a l r e a d y discussed. H o w e v e r , it has b e e n
excite these n e u r o n s e v e n if the synapses at the
s h o w n that s o m e t h a l a m i c fibers t e r m i n a t e on t h e
spines are i n e f f e c t i v e for the g e n e r a t i o n of EPSPs.
basal d e n d r i t e s 24 a n d t h a l a m i c s t i m u l a t i o n excites pyr a m i d a l n e u r o n s m o n o s y n a p t i c a l l y and p o l y s y n a p -
ACKNOWLEDGEMENTS
ticaily 1. K o s a r et al. 12 c o n f i r m e d the results and furt h e r d e m o n s t r a t e d that the t i m e f r o m the start to the
T h e a u t h o r s w o u l d like to express t h e i r thanks to
p e a k of the E P S P was r a t h e r short. T h e results sug-
Drs. B r e n t B. and C h i s a t o A. Stanfield o f Salk Insti-
gested that the site of the synapses was close to the
tute for their c o m m e n t s on the m a n u s c r i p t . T h e i r
s o m a b e c a u s e t h e s e fast rising p o t e n t i a l s w e r e unlike-
t h a n k s are also d u e to Ms. M. E. G e n t h e r for h e r edi-
ly to be g e n e r a t e d by synapses at the distal d e n d r i t i c
ting o f the m a n u s c r i p t . This r e s e a r c h was s u p p o r t e d
spines. O u r results with i d e n t i f i e d p o s t s y n a p t i c n e u -
by the N I H G r a n t s NS-18581 and NS-10705.
rons r e v e a l e d that t h a l a m o c o r t i c a l fibers m a d e rath-
REFERENCES 1 Amassian, V. E. and Weiner. H.. Monosynaptic and polysynaptic activation of pyramidal tract neurons by thalamic stimulation. In D. P. Purpura and M. D. Yahr (Eds.) The Thalamus, Columbia University Press. New York. 1966. pp. 255-282. 2 Asanuma, H., Kosar, E.. Tsukahara. N. and Robinson. H., Modification of the projection from the sensory cortex to the motor cortex following the elimination of thalamic projections to the motor cortex. Brain Research, 345 (1985) 79-86. 3 Berman, A. L., The Stereotaxic Atlas of the Cat. Madison University Wisconsin Press. 1968. 4 Cipolloni, P. B. and Peters. A.. The termination of caltosal fibers in the auditory cortex of the rat. A combined Golgielectron microscope and degeneration study, J. NeurocytoL, 12 (1983) 713-726. 5 Colonnier, M. and Rossignol. S.. Heterogeneity of the cerebral cortex. In H. H. Jasper, A. A. Ward and A. Pope (Eds.), Basic Mechanisms of the Epilepsies, London. Churchill, 1969, pp. 29-40. 6 Fairen, A., Peters, A. and Saldanha. J., A new procedure for examining Golgi impregnated neurons by light and electron microscopy. J. Neurocytol., 6 (1977) 311-337. 7 Garey, L . J . and Powell, T. P. S.. Experimental study of the termination of the lateral geniculo-cortical pathway in the cat and monkey, Proc R. Soc. London Ser. B.. 179 (1971) 41-63. 8 Horwitz, B., Electrophoretic migration due to postsynaptic potential gradients: theory and application to autonomic ganglion neurons and to dendritic spines. Neuroscience. 12 (1984) 887-905. 9 Jasper, H. H. and Ajmone-Marsan. C. A.. Stereotaxic Atlas of the Diencephalon of the Cat. National Research Council of Canada. Ottawa. 1954. 10 Jones, E. G., Varieties and distribution of non-pyramidal cells in the somatic sensory cortex of the squirrel monkey, J. Comp. Neurol., 160 (1975)205-268
11 Jones. E G. and Powell, T. P. S.. Electron microscopy of the somatic sensory cortex of the cat. II. The fine structure of layers I and II. Phil. Trans. R. Soc. London Ser. B.. 257 (1970) 12-21. 12 Kosar, E.. Waters. R. S., Tsukahara, N. and Asanuma. H.. Anatomical and physiological properties of the projection from the sensory cortex to the motor cortex in normal cats: the difference between corticocorticat and thalamocortieal projections. Brain Research. 345 (1985) 68-78. 13 Kluver. H. and Barrera. E.. A method for the combined staining of cells and fibers in the nervous system. L Neuropathol. Exp. Neurol. 12 (1953) 400-403. 14 LeVay, S., Synaptic patterns in the visual cortex of the cat and monkey. Electron microscopy of Golgi preparations, J. Comp. Neurol., 150(1973)53-86. 15 Lund. J. S. and Lund. R. D.. Termination of callosal fibers m the paravisual cortex of the rat. Brain Research. 17 (1970) 25-45. 16 O'Leary, J. L.. A structural analysis of lateral geniculate nucleus of the cat, J. Comp. Neurol.. 73 (1940) 405-430. 17 Palay, S. and Chan-Palay, V.. General morphology of neurons and neuroglia. Handbook of Physiology, 'The Nervous System' Vol. 1. Am. Physiol. Soc.. Bethesda MD. 1977, pp. 5-37. 18 Peters, A., Stellate cells of the rat parietal cortex. J. Comp. Neurol.. 141 (1971) 345-373. t9 Peters. A. and Feldman. M. L.. The projection of the lateral genicotate nucleus to area 17 of the rat cerebral cortex. IV. Terminations upon spiny dendrites, J. Neurocytol.. 6 (1977) 660-689. 20 Peters. A. and Kaiserman-Abramof. I. R.. The small pyramidal neuron of the rat cerebral cortex. The perikaryon. dendrites and spines, Am. J. Anat., 127 (1970)321-355. 21 Sloper. J. J., An electron microscope study of the termination of afferent connections to the primate motor cortex. J. Neurocytol., 2 (1973) 361-368. 22 Sloper, J. J. and Powell, T. P. S.. An experimental electron microscopic study of afferent connections to the primate motor and somatic sensory cortices, Phil. Trans. R. Soc.
101
London Set. B., 285 (1979) 199-224. 23 Somogyi, P., The study of Golgi stained cells and of experimental degeneration under the electron microscope: a direct method for the identification in the visual cortex of three successive links in a neuron chain, Neuroscience, 3 (1978) 167-180. 24 Strick, P. L. and Sterling, P., Synaptic termination of afferents from the ventro-lateral nucleus of the thalamus in the cat motor cortex. A light and electron microscope study, J. Comp. Neurol., 153 (1974) 77-106. 25 Turner, D. A. and Schwartzkroin, P. A., Electrical characteristics of dendrites and dendritic spines in intracellularly stained CA3 and dentate hippocampal neurons, J. Neurosci., 3 (1983) 2381-2404. 26 Waters, R. S., Favorov, O. and Asanuma, H., Physiological properties and pattern of projection of cortico-cortical
connections from the anterior bank of the ansate sulcus to the motor cortex area 4 in the cat, Exp. Brain Res., 45 (1982) 403-412. 27 White, E. L., Identified neurons in mouse SmI cortex wl~ich are postsynaptic to thalamocortical axon terminals: a combined Golgi-electron microscopic and degeneration study. J. Comp. Neurol., 181 (1978) 627-662. 28 White, E. L., Hersch, S. M. and Rock, M. P., Synaptic sequences in mouse SmI cortex involving pyramidal cells labelled by retrograde filling with horseradish peroxidase, Neurosci. Lett., 14 (1980) 149-154. 29 Yumiya, H. and Ghez, C., Specialized subregions in the cat motor cortex: anatomical demonstration of differential projections of rostral and caudal sectors, Exp. Brain Res., 53 (1984) 259-276.
87
BRE 11060
Distribution of Corticocortical and Thalamocortical Synapses on Identified Motor Cortical Neurons in the Cat: Golgi, Electron Microscopic and Degeneration Study M. ICHIKAWA** K. ARISSIAN and H. ASANUMA The Rockefeller University, New York, NY 10021 (U.S.A.) (Accepted January 8th, 1985) Key words: motor cortex - - association fiber - - thalamocortical fiber - - stellate cell - - PT cell
Details of the terminal connections of corticocortical and thalamocortical fibers on pyramidal and stellate neurons in the cat motor cortex were studied using the electron microscope in combination with the Golgi and axonal degeneration techniques. Corticocortical terminals were examined in 23 identified neurons of which 11 were pyramidal and 12 were stellate. Stellate neurons located in layer III received many degenerating terminals (average 8.4 + 2.2 per unit length of dendrite (ULD)) and the majority of these (95%) were found on the proximal dendrites or on the cell bodies. The pyramidal neurons received fewer degenerating terminals (average 2.1 + 0.27/ULD) and these were located on more distal dendritic shafts or on dendritic spines. The majority of these synapses were of the asymmetric type. Thalamocortical terminals were examined in 9 pyramidal and 9 stellate neurons. Pyramidal neurons received many terminals (average 6.0 + 1.23/ULD) and these were found on the basal as well as the apical dendrites and on dendritic spines. Stellate neurons received fewer terminals (average 4.2 _+0.64/ULD) and were located primarily on proximal dendritic shafts. The majority of these synapses were of the asymmetric type. The functional role of these synapses is discussed in relation to the physiological results reported in the preceding paper. INTRODUCTION
to the terminals of thalamocortical afferents. Recent-
In the preceding study, Kosar et a132 reported that microstimulation of cat somatic sensory cortex (area
ly, Sloper and Powel122 studied other afferents to the monkey motor cortex, including the corticocortical input from the somatic sensory cortex, but the em-
2) produced monosynaptic EPSPs within n e u r o n s located in the superficial layers (II and III) of the motor cortex, but not within those in the deep layers (V and VI). Injection of horseradish peroxidase ( H R P ) into
phasis was on the postsynaptic profiles of termination sites, i.e. whether the fibers terminated on the den-
neurons receiving monosynaptic input from the sensory cortex suggested that these were primarily stellate neurons located in the superficial layers. The terminal sites of afferent fibers in the neocortex have been studied by many investigators at the ultrastructural level ~,5,7,1t,15.19.2s. However, most of these stud-
spines and 20% on shafts. Although they did not identify the postsynaptic neurons, they suggested, from various ultrastructural configurations, that these spines belonged to pyramidal n e u r o n s and the shafts belonged to stellate neurons. If pyramidal neurons receive 80% of the corticocortical synaptic input, then one would think that sensory cortex stimulation should elicit monosynaptic EPSPs mostly within pyramidal neurons. Since the pyramidal neurons are, in general, larger than stellate neurons, they should be easier to impale with an electrode and inject HRP. However, Kosar et al. 12 were unable to
ies were carried out within the sensory or parietal cortices and only a few studies exist which examined the terminal sites within the motor cortex. Strick and Sterling24 and Sloper 21 examined synaptic termination within the m o n k e y motor cortex using the electron microscope, but their studies were restricted
dritic spines or shafts. They reported that about 80% of the corticocortical terminations occur on dendritic
* Present address: Department of Anatomy and Embryology, Tokyo Metropolitan Institute for Neurosciences, 2-6 Musashidai, Fuchu, Tokyo 183, Japan. Correspondence: H. Asanuma, The Rockefeller University, 1230York Avenue, New York, NY 10021, U.S.A. 0006-8993/85/$(}3.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
88 identify pyramidal neurons as the main recipients of corticocortical input. We were puzzled by this observation and started this experiment to resolve the discrepancy. Recent development of the electron microscopic technique made it possible to combine an ultrastructural study with a Golgi impregnation method ~.~4. 23.27. Utilizing this technique we sought to determine the sites as well as the density of the synapses that corticocortical and thalamocortical fibers make with identified motor cortical neurons. MATERIALS AND METHODS Six adult cats of either sex weighing 2.5-3.5 kg were used. Operations were carried out under Nembutal anesthesia (35 mg/kg). In 3 cats, the sensory cortex was exposed and the anterior bank of the ansate sulcus (area 2) was removed by suction. In the remaining 3 cats, kainic acid (2.5/~g dissolved in 1.0 ~tl of 0.9% saline) was injected into the ventrolateral nucleus of the thalamus (VL) stereotaxically (A 11.0, L 4.5, H 1.5) guided by the atlas of Jasper and Ajmone-Marsan 9. After 5 days survival, the animals were reanesthetized and perfused with saline followed by a mixture of 2.0% glutaraldehyde and 1.5-2.0% paraformatdehyde in 0.1 M phosphate buffer solution. Subsequently, the sensory and motor cortices were removed and in the latter group of animals, a thalamic block containing VL was also removed. The motor cortex was cut into sections of 2-3 mm thickness and these were immersed in a rapid Golgi solution (3.0% potassium dichromate plus 0.2% osmium tetroxide) for 3-5 days. The sections were then transferred into a silver nitrate solution (0.75%) after washing with distilled water and left immersed for 24 h. The tissue was then dehydrated in a series of graded alcohols, embedded in celloidine, cut serially at 80-100 ~tm thickness and mounted on slides. The lesioned tissues (sensory cortex and thalamus were frozen and sectioned at 40ktm, and stained with the Klfiver and Barrera method 13.
Electron microscopy (EM) Well impregnated neurons were selected from the motor cortical area posterotateral to the cruciate sulcus where association fibers from area 2 project most densely26,29. Small pieces of the sections containing
the selected cells were re-fixed with 1.0~ OsCL stained "en bloc" with uranyl acetate and re-embedded in Epon 812. The Gotgl image ot the selected neurons was photographed and/or sketched using a camera lucida. The tissue was then cut into series of a semithin section (0.5-1.0/~m in thickness) followed by 6 ultrathin sections of 60-100 nm thickness using a diamond knife. The semithin sections were mounted on glass slides and stained with toluidine blue. The locations of the impregnated processes in these sections were sketched using a camera tucida and these were used in locating EM images obtained from the adjacent ultrathin sections. The ultrathin sections were mounted on one hole copper grids, stained with lead citrate and scanned and photographed at 10-60 × 103 magnification using the electron microscope (Hitachi HU-11C or J E O L JEM I00 CX). In our procedure, nearly all ultrathin sections could be recovered with only an occasional loss of one or two sections between two semithin sections. An average of 40 semithin sections and 250 ultrathin sections were cut and examined per neuron covering an area of 60-70#m in width. In this way we were able to examine almost all parts of small neurons but only proximal dendrites and somas of large pyramidal neurons except for the parts included in semithin sections. Since the extent of areas examined were different for each cell, the density of degenerating terminals between different cells was compared by the number of terminals per unit length of dendrites. This was done by measuring the length of dendrites examined with EM utilizing the camera lucida drawings and dividing the total number of degenerating terminals by the length. Therefore: DT ULD
Total DT Total length of dendrite (mm)
where DT is the number of degenerating terminals and ULD the unit length of dendrite. By this way the difference of the density of degenerating terminals between the cells could be compared objectively because the ratio of the area included in semithin sections is the same irrespective of the difference in the total areas examined. RESULTS The cortical lesions were primarily limited to the
89 anterior bank of the ansate sulcus (area 2) in all 3 cats as shown in Fig. 1A. Although there was some restricted damage to the underlying white matter, it is most likely that only those fibers entering or leaving the lesioned cortex, i.e. area 2, were affected. The center of the kainic acid injection in the thalamus was characterized by a complete loss of neuronal perikarya and an increase in density of glial cells. Surrounding this area, there was a zone of decreased neuronal density extending about 0.5 mm in width. The shape of the lesioned areas was eliptical and the diameter ranged from 3.0 to 6.0 mm as shown in Fig. lB. The lesioned area, thus included not only the ventrolateral nucleus, but also the ventroanterior nucleus, dorsal part of the subthalamic area and a portion of the thalamic reticular nucleus.
Classification of cell types The rapid Golgi method used in this study was successful in staining a variety of neuronal types as reported elsewherem,14, TM. However, we have selected only typical pyramidal cells and stellate cells for examination since the preceding electrophysiological study 12 suggested a difference in associational inputs upon these two types of cells. The pyramidal cells were characterized by a triangular or conical cell body, a distinct ascending apical dendrite, and several basal dendrites extending horizontally from the cell body as well as by a relatively straight axon arising from the base of the cell body or the basal dendrite. These cells were located in layers IIl and V. The stellate cells selected were those having round somata and radiating dendrites with no particular orientation and sparse or no dendritic spines. Although it is known that there are spiny stellate cells which possess abundant dendritic spines, we did not observe these cells in our preparations. One possibility is that these spiny stellate cells are known to be located in layer IVlO.~4,1~which is virtually absent in the motor cortex. Fig. 2 shows examples of Golgi stained pyramidal and stellate cells drawn with a camera lucida. Altogether 21 stellate cells and 20 pyramidal cells were examined using the electron microscope. Golgi impregnation resulted in electron-dense precipitates which filled the cytoplasm, but in our preparation, most of the precipitates were washed away from the cytoplasm during lead citrate staining. However, the vacant spaces which remained after
the staining served as reliable indicators of the impregnated cells as did the presence of the precipitate, examples of which are shown in Figs. 3 and 4. Utilizing these positive or negative images of impregnated cells, we have examined the terminal sites of the degenerating fibers produced by area 2 or thalamic ablation. The degenerating terminals in our samples appeared to be at various stages of degeneration, but they were commonly characterized by electrondense cytoplasm with markedly distorted or absent organelles (Figs. 3 and 4). Synaptic contact between Golgi impregnated cells and degenerating terminals could be recognized by the presence of a synaptic cleft and by the thickening of the postsynaptic membrane (Figs. 3 and 4). Since.the sketches drawn from the nearby semithin sections were available, it was not difficult to recognize the location of these degenerating terminals in relation to the cell body. However, because of the artifact which resulted from degenerative changes as well as from the removal of silver precipitates, it was often difficult to assess the type of synapses. Therefore, our effort was primarily concentrated on identifying the location of synapses, but general information could be obtained by examining degenerating terminals synapsing upon unlabelled postsynaptic neurons.
Association fibers Random examination of degenerating terminals in the pericruciate gyrus revealed that the distribution of these terminals was the greatest in the area posterolateral to the cruciate sulcus. The number of degenerating terminals per 100 ~tm square in layer III ranged from 8 to 20. Within the same area, there were cortical patches which were relatively rich with degenerating terminals intermixed with less dense patches. Although an accurate assessment of the difference in density of degenerating terminals between these patches and the size of the patches was difficult to make, it was our general impression that the number of degenerating terminals in some patches was twice that in the neighboring patches. However, within the region analysed, there were no patches which were void of degenerating terminals. The sites of synapses of corticocortical fibers were examined using 11 pyramidal cells and 12 stellate cells randomly selected from the same area. On all of these neurons there were synapses which had degen-
90
as
cat 1
cs
as
cs
ABC
A lrnm
A cat 4
5
A 6
j
5mm
B Fig. 1. Locations of lesions in the sensory cortex and the thalamus. A: parasagittal sections of the cortex through the sensory and motor cortices. Arrows in the left figurines show the levels of sections illustrated on the right. Hatched areas, extent of the lesions, as. ansate sulcus; cs, cruciate sulcus. B: parasagittal sections of the thalamus at the levels of L 3.7.4.5 and 5.6. The drawings are based on the atlas by Berman 3. Dotted areas, extent of the lesions. AV, anteroventrat nucleus; CA, caudate nucleus; IC. internal capsule: V A . ventroanterior nucleus; VL, ventrolateral nucleus; OT, optic tract; ZI, zona incerta, FF, nucleus of the fields of Forel. Further details in text.
91
\
II
c
III
A
J
4c
"M
V loopm Fig. 2. Camera lucida drawing of Golgi impregnated cells in the motor cortex. Roman numerals in the left show the layers. Two pyramidal (A, B) and two stellate type cells (C, D) are shown. Note that the apical dendrites of the pyramidal cells were cut off. Arrows and small letters indicate the sites of EM pictures shown in Figs. 3 and 4. erating profiles on the presynaptic side. The locations of these terminals on the pyramidal n e u r o n s are shown in Fig. 5A. The cell bodies of 7 of these neurons were located in layer III and 4 were in layer V. The thickened portions of the dendrites indicate the regions which were examined with the electron mi-
croscope and arrows indicate the sites of the degenerating terminals. Altogether 24 degenerating terminals were found on these 11 n e u r o n s of which 12 made contacts with dendritic shafts and 12 with dendritic spines. No degenerating terminals contacted the cell bodies. In our samples, all of the pyramidal
92
Fig. 3. Electron micrographs of degenerating terminals of thalamocortical fibers on Golgl impregnated cells, a: terminal on dendritic spine of pyramidal cell. x25.000, b: higher magnification of the terminal shown in a. x60. D00. c-f: serial electron micrographs of terminals on dendritic shaft of pyramidal cell. Synaptic contact is observed in e and f. x60.000. DT. degenerating terminals: D. dendritic shaft; SP, dendritic spine: M, mitochondria: NT. normal terminal.
93
Fig. 4. Electron micrographs of degenerating terminals of corticocortical fibers on Golgi impregnated cells. Small letters correspond to small letters in Fig. 2. a: a terminal on the dendritic shafts of a stellate type cell. ×25,000. b: higher magnification of the terminal shown in a. ×60,000. c: terminal on cell body of a stellate type cell. x60,000. DT, degenerating terminal; PE, perikaryon; N, nucleus; D, dendritic shaft; M, mitochondria. Further details in text.
94 neurons received only a few corticocortical synapses averaging 1.8 synapses/ULD on layer III neurons and 2.5/ULD on layer V neurons. The number and distribution of corticocortical synapses on stellate neurons were different from those on pyramidal neurons. As shown in Fig. 4b, all of the stellate neurons located in the superficial layers received about 3 times as many synapses as the pyramidal neurons. The minimum density of degenerating terminals on a stellate neuron was 2.4/ULD the maximum was 12.0/ULD and the average was 8.4 + 2.20 (S.E.D.). These terminals were located primarily on cell bodies and dendritic shafts near the cell body (95%) and only a few were on the spines. A stellate cell located in layer V received only 3.1 degenerating terminals/ULD Fig. (5B, g). As shown in Table II, a total of 89 degenerating corticocortical terminals were found on identified target cells and only 15 (17%) of these were on dendritic spines. This is a different ratio from the results reported by Sloper and Powel122 who found that in the m o n k e y motor cortex, 80% of the association terminals contacted dendritic spines and only 20% were on dendritic shafts. To examine whether our criterion of sampling the degenerating terminals was different from their criterion, we randomly selected 200 degenerating corticocortical terminals on unidentified neurons and classified the postsynaptic profile. We found, in agreement with Sloper and Powel122, that 156 (78%) of these profiles were dendritic spines. Of these 156 terminals, 153 made asymmetric synapses and 3 were difficult to determine. Of the 44 degenerating synapses found on dendritic shafts or cell bodies, 37 were asymmetric and 7 were difficult to determine. Examples of these synapses are shown in Fig. 6. The terminal sites of thalamic fibers were examined on 9 pyramidal cells and 9 stellate cells from the same region of the cortex in which the thalamic input occurred. Fig. 7A summarizes the locations of degenerating thalamocortical terminals on pyramidal cells in layers III and V. A total of 61 degenerating terminals were identified and many of these were on the proximal dendrites. These pyramidal neurons received a rather massive thalamocortical input except for cells f and i, which were located in upper layer III and in layer V. The maximum number of degenerating terminals for a cell was 10.0/ULD (cell c) and the
TABLE I Number of degenerating terminals per unit length (1.0 ram) oF dendrites in each identified cell (mean + S. E. M. )
n, number of cells. No. of degenerating terminals Pyramidal cells" Stellate type cells
Corticocortical terminals Thalamocorticalterminals
2.1 _+ 0.27 (n = 11) 6.0 + 1.23 (n = 9)
S.4 _+ 2.20 (n = 12) 4.2 _+ 0.64 (n = 9)
average was 6.0 + 1.23 (Table I). As shown in Table II, nearly half of the degenerating synapses were found on the dendritic shafts and the rest were on dendritic spines. No thalamic terminals were on the somata. There were fewer thalamocortical terminals on stellate neurons than on pyramidal neurons. As shown in Fig. 7B, the maximum number of degenerating terminals found on a stellate neuron was 5/ULD (cell a, b) and the average was 4.2 + 0.64. The majority of these contacted dendritic shafts and only 3 (11%) were found on dendritic spines (Table I). The results suggests that there are more thatamocortical synapses on pyramidal neurons than on stellate neurons. This is quite different from the associational terminals, most of which were found on stellate neurons. Altogether. 89 degenerating thalamocortical terminals were found on identified neurons. Of these. 36 (41%) made contact with dendritic spines, 50 (56%) with dendritic shafts and 3 (3%) with cell bodies. This distribution is different from that of previous reports 22.24 in which 90% of thalamocortical terminals were found to contact dendritic spines. As before, we randomly selected 100 degenerating thalamic terminals and found, in agreement with previous investigators, 80 made contact with spines and 20 with shafts or cell bodies. Of these. 75 were asymmetric synapses and 25 could not be identified. DISCUSSION In this study, less than half of the degenerating synapses of both association and thalamic fibers terminated on dendritic spines of identified-motor cortical neurons. In previous studies 222& the majority of degenerating synapses from both sources was found on the dendritic spines. This discrepancy was not due to
95
I
II
Ill
b ¢
,/ ¥
i
A
/ f
k
A I
III
¢
b
B Fig. 5. Locations of degenerating corticocortical terminals on Golgi impregnated cells. Arrows indicate sites of identified terminals. A: terminals on pyramidal cells. B: terminals on stellate cells. Thick lines indicate the portions of dendrites which were examined with electron microscope. Further details in text.
96
Fig. 6. Examples of degenerating corticocortical synapses, a: asymmetric synapse on a dendritic spine, b: asymmetric synapse on dcndritic shaft and a spine, c: asymmetric synapse on cell body. Postsynaptic thickening is not as marked as those shown in a and b. d: synapse of unidentified type on dendritic shaft, DT, degenerating terminals; SP, dendritic spine; SH, dendritic shaft. Magnification. x 50,000.
o u r failure of i d e n t i f y i n g spines b e c a u s e in the control s u r v e y in which t h e t e r m i n a l sites w e r e not restricted to i d e n t i f i e d n e u r o n s , t h e ratio o f synapses of
spines was similar to that of the p r e v i o u s investigators. O n e possible e x p l a n a t i o n for the d i s c r e p a n c y is that the r a p i d G o l g i staining did not stain all the d e n -
97
I:00 m
I
H
f
h I
A I
II
~
f
e
i
B Fig. 7. Locations of degenerating thalamocortical terminals on Golgi impregnated cells. The format is the same with that of Fig. 5. A: pyramidal cells. B: stellate type cells. dritic spines. However, this is not likely because as shown in Fig. 2, spines were present on the pyramidal n e u r o n s we selected in as great an a b u n d a n c e as
those reported by the previous investigators 10,1a. The most likely source of the discrepancy is that the dendrites of the identified n e u r o n s that we examined
98 ]'ABLE 1I Number of degenerating terminals synapsing on d((ferentpostsynaptic sites No. of cells
No. of deg.
No. of synapses Cell body (%)
Dendritic spines (%)
Dendritic shafts (%)
Corticocortical fibers pyramidal cell stellate type cell
11 12
24 65
0 (0) 12 (18)
12 (50) 3 (5)
12 ~501 5(I ~77j
Thalamocortical fibers pyramidal cell stellate type cell
9 9
61 28
0 (0) 3 (11)
33 (54) 3 (11)
28 {46) 22 (78)
were located rather close to the cell body because of obvious technical difficulties (see Materials and Methods). Since the terminals of VL fibers are concentrated in layer 11122,24 and those of association fibers are largely found in the superficial layers 22, it is likely that our sample missed a considerable fraction of the degenerating terminals located on spines of distal dendrites of deep pyramidal cells. On the other hand, the dendrites of the stellate cells do not extend for such a great distance and therefore, it was easier to examine their dendritic arborizations more thoroughly. Although our sampling methods do not allow quantitative statements to be made regarding synaptic density, we feel that the ratios we obtained concerning the relative distributions of degenerating terminals on the two types of neurons are reasonably accurate and do not reflect a sampling bias other than that already mentioned in the preceding paragraph. This is because: (1) comparable numbers of each type of neuron were selected for analysis; (2) comparable lengths of dendrites were surveyed for each neuron; (3) the portions of dendrites examined in the electron microscope were evenly spaced (between our semithin sections); and (4) comparable numbers ( - 90 each) of degenerating synapses were identified for each paradigm. Corticocortical projections
Our results revealed that the stellate cells in the superficial layers of the motor cortex received a substantial input from association fibers. These synaptic terminals were located mostly on the proximal dendritic shafts and some were on the cell bodies. Very few were found on dendritic spines. This observation
is in agreement with a previous study 22 in which the authors suggested that the dendritic shafts receiving associational terminals in the motor cortex were for the most part dendrites of stelIate neurons. A random sampling of these degenerating terminals which contacted unidentified dendritic shafts indicated that almost all of these consisted of asymmetric membrane specializations. This observation suggests that the majority of association fibers making contact with stellate neurons in the motor cortex form asymmetric synapses. Asymmetric membrane specializations are often assumed to be indicative of excitatory synapses and Kosar et al. 12 have shown that microstimulation within area 2 of the sensory cortex generated monosynaptic EPSPs exclusively in neurons located in the superficial layers of the motor cortex: monosynaptic IPSPs were never recorded. Together these results strongly suggest that the asymmetric synapses in the corticocortical projection function as excitatory synapses although we could not identify the type of vesicles in the presynaptic terminals. As shown in Fig. 5, association fibers made contacts not only with stellate cells, but also with pyramidal cells in layers III and V. However. these terminals must represent only a fraction of the total degenerating terminals synapsing on these neurons. This is because in our sample of degenerating terminals synapsing with pyramidal cells, only 17% made axospinous contacts yet in the control survey. 80% of the degenerating terminals were axospinous. Therefore, it is likely that the majority of association terminals synapsed with spines of pyramidal cell dendrites located further distally, beyond the range of our sample. A question then arises as to why Kosar et al. 12 could not record monosynaptic corticocorticat EPSPs
99 from the pyramidal neurons located either in the superficial or in the deep layers. Since the pyramidal cells are larger than the stellate cells, they should be easier to impale with electrodes and to record EPSPs. However, Kosar et al. J2 reported that when the intracellular recordings were stable and the membrane potentials were large suggesting that they were recording from large pyramidal cells, they were unable to record short-latency cortically activated EPSPs or IPSPs. Several reasons could be considered to account for this observation. The first is that association terminals are aggregated within the cortex and make contacts only with certain groups of pyramidal cells, and that Kosar et al.L2 penetrated cells outside of these aggregates. In our serial sections, we noticed that there were strips which contained dense degenerating terminals as well as strips which were less densely populated by these animals. This is, however, unlikely to be the explanation since there were no strips within the sampled area which were devoid of degenerating terminals. Furthermore, as shown in Fig. 5, all of our randomly selected pyramidal neurons received some association input. Therefore, the possibility that Kosar et a1.12 selected samples from outside of such aggregates is small. The second possibility is that the terminals synapsing on the spines belong to small diameter axons. Then the postsynaptic potentials generated by these synapses would appear too late to be recognized as monosynaptic potentials. The third possibility is that because of their specific shape, spines are electrically isolated from the cell body. The spine forms a clavate head and the axon contacts only the head of the spine, not the stem 20. These are attached to the dendritic shaft by a thin stem which is a tenuous connector with high electrical resistance. Because of this configuration, Palay and Chan-Palay 17 suggested that the spines are specialized for the transmission of the information from a large number of similar inputs in which the individual signal counts for very little to the postsynaptic neurons. If this is the case, it is possible that the synaptic potentials generated by microstimulation of the sensory cortex were too small to be detected by averaging a small number of trials, i.e. only 20 superimpositions 12. The above possibility, however, is unlikely because of recent calculations by Turner and Schwarzkroin 25 on the electrical properties of dendritic spines of hippocampal pyramidal cells. They concluded that
the input resistance of a spine is much greater than the resistance at the neck of the spine and therefore the attenuation across the neck is negligible. Instead the attenuation of the synaptic potential takes place at the distal dendritic shaft because of its high resistance. Therefore, the fourth possibility is that because of the high resistance of the dendritic shaft and the resulting short length constant of the shaft, the EPSPs at the spine became too small to be detected at the soma. In this model, however, the synaptic currents at the dendrites should produce field potentials as discussed by Kosar et al. 12 even if the current did not reach the cell body. Therefore, area 2 stimulation should have produced negative field potentials in layer III where the majority of terminals synapse on the dendritic spines. Yet, Kosar et al. ~2 did not record these negative loci. Therefore, the fifth possibility is that the membrane characteristics of a spine is different from that of the soma and the input resistance of spines is smaller than they calculated25. If this is the case, the high resistance of the dendritic shaft prevents the entry of synaptic current and the current flows out through the membrane of the spine. Since the axis of the spine is directed randomly, the outgoing currents as a whole do not generate field potentials. This model is in agreement with the hypothesis proposed by HorwitzS. He calculated the electrical properties of the spine and concluded that the synaptic activity at a spine head can generate a large electrical field along the shaft of the spine. Then he proposed that this electric field functions by inducing electrophoretic migration of charged metabolites into the spine. At present we do not know which of the above possibilities is correct nor the functional role of the synapses on the spines, but it seems clear from the physiological data 1: that axospinous synapses are not participating in the rapid transmission of impulses to the pyramidal neurons in the motor cortex. However, it would be interesting to note that following chronic lesion of VL, pattern of the corticocortical projections changes significantly in parallel with physiological changes 2 which were reported in the preceding paper.
Thalamocortical projections It has been reported that 90% of thalamic terminals synapse on dendritic spines, most likely of py-
r a m i d a l n e u r o n s and t h e s e synapses h a v e b e e n sug-
er p o p u l o u s synapses with shafts of basal d e n d r i t e s
gested to be the m a j o r t h a l a m i c i n p u t to the p y r a m i -
a c c o u n t i n g for the fast rising m o n o s y n a p t i c E P S P s .
dal n e u r o n s 22,24. T h e n t h a l a m i c s t i m u l a t i o n s h o u l d
Since the nucleus V L sends massive fibers to the mo-
not p r o d u c e s h o r t - l a t e n c y m o n o s y n a p t i c e x c i t a t i o n
tor cortex, it is possible that only 10% of the total
of p y r a m i d a l n e u r o n s in the m o t o r c o r t e x b e c a u s e of
synapses c o n t a c t i n g d e n d r i t i c shafts are sufficent to
the r e a s o n s a l r e a d y discussed. H o w e v e r , it has b e e n
excite these n e u r o n s e v e n if the synapses at the
s h o w n that s o m e t h a l a m i c fibers t e r m i n a t e on t h e
spines are i n e f f e c t i v e for the g e n e r a t i o n of EPSPs.
basal d e n d r i t e s 24 a n d t h a l a m i c s t i m u l a t i o n excites pyr a m i d a l n e u r o n s m o n o s y n a p t i c a l l y and p o l y s y n a p -
ACKNOWLEDGEMENTS
ticaily 1. K o s a r et al. 12 c o n f i r m e d the results and furt h e r d e m o n s t r a t e d that the t i m e f r o m the start to the
T h e a u t h o r s w o u l d like to express t h e i r thanks to
p e a k of the E P S P was r a t h e r short. T h e results sug-
Drs. B r e n t B. and C h i s a t o A. Stanfield o f Salk Insti-
gested that the site of the synapses was close to the
tute for their c o m m e n t s on the m a n u s c r i p t . T h e i r
s o m a b e c a u s e t h e s e fast rising p o t e n t i a l s w e r e unlike-
t h a n k s are also d u e to Ms. M. E. G e n t h e r for h e r edi-
ly to be g e n e r a t e d by synapses at the distal d e n d r i t i c
ting o f the m a n u s c r i p t . This r e s e a r c h was s u p p o r t e d
spines. O u r results with i d e n t i f i e d p o s t s y n a p t i c n e u -
by the N I H G r a n t s NS-18581 and NS-10705.
rons r e v e a l e d that t h a l a m o c o r t i c a l fibers m a d e rath-
REFERENCES 1 Amassian, V. E. and Weiner. H.. Monosynaptic and polysynaptic activation of pyramidal tract neurons by thalamic stimulation. In D. P. Purpura and M. D. Yahr (Eds.) The Thalamus, Columbia University Press. New York. 1966. pp. 255-282. 2 Asanuma, H., Kosar, E.. Tsukahara. N. and Robinson. H., Modification of the projection from the sensory cortex to the motor cortex following the elimination of thalamic projections to the motor cortex. Brain Research, 345 (1985) 79-86. 3 Berman, A. L., The Stereotaxic Atlas of the Cat. Madison University Wisconsin Press. 1968. 4 Cipolloni, P. B. and Peters. A.. The termination of caltosal fibers in the auditory cortex of the rat. A combined Golgielectron microscope and degeneration study, J. NeurocytoL, 12 (1983) 713-726. 5 Colonnier, M. and Rossignol. S.. Heterogeneity of the cerebral cortex. In H. H. Jasper, A. A. Ward and A. Pope (Eds.), Basic Mechanisms of the Epilepsies, London. Churchill, 1969, pp. 29-40. 6 Fairen, A., Peters, A. and Saldanha. J., A new procedure for examining Golgi impregnated neurons by light and electron microscopy. J. Neurocytol., 6 (1977) 311-337. 7 Garey, L . J . and Powell, T. P. S.. Experimental study of the termination of the lateral geniculo-cortical pathway in the cat and monkey, Proc R. Soc. London Ser. B.. 179 (1971) 41-63. 8 Horwitz, B., Electrophoretic migration due to postsynaptic potential gradients: theory and application to autonomic ganglion neurons and to dendritic spines. Neuroscience. 12 (1984) 887-905. 9 Jasper, H. H. and Ajmone-Marsan. C. A.. Stereotaxic Atlas of the Diencephalon of the Cat. National Research Council of Canada. Ottawa. 1954. 10 Jones, E. G., Varieties and distribution of non-pyramidal cells in the somatic sensory cortex of the squirrel monkey, J. Comp. Neurol., 160 (1975)205-268
11 Jones. E G. and Powell, T. P. S.. Electron microscopy of the somatic sensory cortex of the cat. II. The fine structure of layers I and II. Phil. Trans. R. Soc. London Ser. B.. 257 (1970) 12-21. 12 Kosar, E.. Waters. R. S., Tsukahara, N. and Asanuma. H.. Anatomical and physiological properties of the projection from the sensory cortex to the motor cortex in normal cats: the difference between corticocorticat and thalamocortieal projections. Brain Research. 345 (1985) 68-78. 13 Kluver. H. and Barrera. E.. A method for the combined staining of cells and fibers in the nervous system. L Neuropathol. Exp. Neurol. 12 (1953) 400-403. 14 LeVay, S., Synaptic patterns in the visual cortex of the cat and monkey. Electron microscopy of Golgi preparations, J. Comp. Neurol., 150(1973)53-86. 15 Lund. J. S. and Lund. R. D.. Termination of callosal fibers m the paravisual cortex of the rat. Brain Research. 17 (1970) 25-45. 16 O'Leary, J. L.. A structural analysis of lateral geniculate nucleus of the cat, J. Comp. Neurol.. 73 (1940) 405-430. 17 Palay, S. and Chan-Palay, V.. General morphology of neurons and neuroglia. Handbook of Physiology, 'The Nervous System' Vol. 1. Am. Physiol. Soc.. Bethesda MD. 1977, pp. 5-37. 18 Peters, A., Stellate cells of the rat parietal cortex. J. Comp. Neurol.. 141 (1971) 345-373. t9 Peters. A. and Feldman. M. L.. The projection of the lateral genicotate nucleus to area 17 of the rat cerebral cortex. IV. Terminations upon spiny dendrites, J. Neurocytol.. 6 (1977) 660-689. 20 Peters. A. and Kaiserman-Abramof. I. R.. The small pyramidal neuron of the rat cerebral cortex. The perikaryon. dendrites and spines, Am. J. Anat., 127 (1970)321-355. 21 Sloper. J. J., An electron microscope study of the termination of afferent connections to the primate motor cortex. J. Neurocytol., 2 (1973) 361-368. 22 Sloper, J. J. and Powell, T. P. S.. An experimental electron microscopic study of afferent connections to the primate motor and somatic sensory cortices, Phil. Trans. R. Soc.
101
London Set. B., 285 (1979) 199-224. 23 Somogyi, P., The study of Golgi stained cells and of experimental degeneration under the electron microscope: a direct method for the identification in the visual cortex of three successive links in a neuron chain, Neuroscience, 3 (1978) 167-180. 24 Strick, P. L. and Sterling, P., Synaptic termination of afferents from the ventro-lateral nucleus of the thalamus in the cat motor cortex. A light and electron microscope study, J. Comp. Neurol., 153 (1974) 77-106. 25 Turner, D. A. and Schwartzkroin, P. A., Electrical characteristics of dendrites and dendritic spines in intracellularly stained CA3 and dentate hippocampal neurons, J. Neurosci., 3 (1983) 2381-2404. 26 Waters, R. S., Favorov, O. and Asanuma, H., Physiological properties and pattern of projection of cortico-cortical
connections from the anterior bank of the ansate sulcus to the motor cortex area 4 in the cat, Exp. Brain Res., 45 (1982) 403-412. 27 White, E. L., Identified neurons in mouse SmI cortex wl~ich are postsynaptic to thalamocortical axon terminals: a combined Golgi-electron microscopic and degeneration study. J. Comp. Neurol., 181 (1978) 627-662. 28 White, E. L., Hersch, S. M. and Rock, M. P., Synaptic sequences in mouse SmI cortex involving pyramidal cells labelled by retrograde filling with horseradish peroxidase, Neurosci. Lett., 14 (1980) 149-154. 29 Yumiya, H. and Ghez, C., Specialized subregions in the cat motor cortex: anatomical demonstration of differential projections of rostral and caudal sectors, Exp. Brain Res., 53 (1984) 259-276.