THE JOURNAL OF COMPARATIW NEUROLOGY 300:47-60 (1990)

Identification of Neurons producing Long-TermPotentiationin the Cat Motor Cortex IntracelldarRecordings andLakhg ASAF KELLER, ATSUSHI WKI, AND HIROSHI ASANUMA The Rockefeller University, New York, New York 10021

ABSTRACT Intracellular, in vivo recordings were used to identify and subsequently to label neurons in the cat motor cortex in which long-term potentiation (LTP) was induced. Thirty-nine motor cortical neurons that produced excitatory postsynaptic potentials (EPSPs) in response to microstimulation in areas 1-2 (SI) or in area 5a (SIII) were studied. Amplitudes of EPSPs produced in response to test stimulation (1 Hz) were recorded before and after tetanic stimulation (200 Hz, 20 seconds). In 25/39 cells (64%), EPSP amplitudes were significantly increased following the tetanic stimulation (65 ? 51% average increase), and remained at the potentiated level as long as stable recordings could be maintained (20 ? 18 minutes, maximum = 90 minutes). LTP was induced exclusively in cells that produced monosynpatic EPSPs in response to area 1-2 or area 5a stimulation. Of the 39 analyzed cells, 13 were labeled by intracellular injections of 5% biocytin. Neurons in which LTP was induced included both pyramidal and nonpyramidal cells and were located exclusively in layers I1 or I11 of the motor cortex; cells in deeper cortical layers were not potentiated. These findings indicate that various corticocortical inputs can increase the efficacy of synaptic transmission in a subset of motor cortical neurons. We propose that this plasticity in synaptic transmission constitutes one of the bases of motor learning and memory. Key words: plasticity, motor learning and memory, corticocortical, sensorimotor integration, biocytin

The neuronal mechanisms underlying the acquisition be a cellular basis for certain types of memory (Berger and and retention of motor skills are still unknown. Available Sclabassi, '88). LTP has been demonstrated and studied mainly in evidence indicates that motor learning does not involve hippocampal structures (Corkin, '651, which are related to hippocampal structures, where its molecular and biophysicognitive mnemonic processes (e.g., Squire, '82). Some cal properties have been worked out in detail (Davies et al., insight into the neuronal circuitry governing motor '89; Kauer et al., '88). More recently, LTP has been shown learning was provided by Woody and coworkers (e.g., to occur also in neocortical structures (Artola and Singer, Woody, '86), who observed an increase in the excitability of '87; Berry et al., '89; Kimura et al., '89; Komatsu et al., '88). neurons in the motor cortex of cats trained by classical We have recently demonstrated that LTP can be induced in Pavlovian conditioning. These findings suggest that the neurons in the motor cortex by brief tetanic stimulation in motor cortex is one of the sites where motor learning takes the somatosensory cortex (area 2) (Iriki et al., '89; Sakamot0 et al., '87). The purpose of the present study is to place. Ramon y Cajal ('11)is credited with first proposing that expand these findings and to determine whether LTP can learning and memory result from plasticity in synaptic be induced also by activation of other afferent inputs to the transmission. The discovery of long-term potentiation motor cortex. Furthermore, the combined electrophysiolog(LTP),the phenomenon of long-lasting increase in synaptic ical and anatomical approach employed in the present study efficacy following brief repetitive synaptic activation (Bliss enables one to delineate neural circuitry related to plasticand L@mo,'73), provided a model for studying neuronal ity in the motor cortex. mechanisms of synaptic plasticity (see Kauer et al., '88). Although not directly demonstrated, LTP is considered to Accepted July 4,1990

Q

1990 WILEY-LISS, INC.

A. KELLER ET AL.

48

MMXFUALSANDMETHODS Electrophysiology Experiments were performed on 35 adult cats of both sexes (weight 2.5-3.0 kg), using Nembutal anesthesia (initial dose 35 mg/kg, i.p.; maintenance dose 2 mg/kg/h, i.v.1. Arterial blood pressure, respiration rate, pupil size, and body temperature were monitored throughout the experiments. The left motor and somatosensory cortical areas were exposed through a craniotomy, and a closed chamber was installed over the exposed cortical surface (Thompson et al., '70). An array of five to ten platinum-in-glass stimulating electrodes was inserted into the periansate region at depths ranging from 1 to 2 mm. Glass micropipettes filled with 3 M KC1, or with 5% biocytin (Sigma, USA) dissolved in 2.5 M KCl and 0.05 M Tris (pH 7.4,37"C) were used for intracellular recordings from neurons in the motor cortex (area 47). The resistance of the microelectrodes used ranged from 50 to 70 MR. The resistance of biocytin-filled microelectrodes was typically 5 to 10%higher than comparable electrodes filled with 3 M KC1. Intracortical microstimulation (ICMS; 30 FA, 0.2 ms, 1 Hz) was delivered through all the stimulating electrodes while advancing the recording microelectrode through the depths of the motor cortex. When an impaled neuron responded to the ICMS with excitatory postsynaptic potentials (EPSPs), the stimulating electrode responsible for producing the EPSP was determined. After at least 3 minutes of control recordings, tetanic stimulation (200 Hz, 20 seconds) was delivered through the responsible stimulating electrode. Immediately following the tetanic stimulation, recordings of EPSPs in response to the test ICMS (1 Hz) were resumed. Occasionally, hyperpolarizing current (0.5 to 2.0 nA) was used to stabilize the recordings; no hyperpolarizing current was passed during the tetanic stimulation. Intracellular recordings of EPSPs produced in response to test stimuli were averaged (ten sweeps) every 30 to 60 seconds and stored on a n IBM/PC averager (developed by B. Stromquist, C. Pavlides, and J. Zelano, manuscript in preparation). Injections of biocytin were accomplished by passing hyperpolarizing current through the recording electrode. Preliminary studies established that the most effective and reproducible method for biocytin injection was to deliver hyperpolarizing, rectangular pulses (3 nA, 5 Hz, 100 ms) for 1to 5 minutes. The location of neurons recorded with microelectrodes filled with 3 M KCl was determined by producing small electrolytic lesions at the end of the recordings. These lesions were produced by passing DC current (10 p A , 10 seconds) through a tungsten-in-glass electrode inserted into the general vicinity of the cell, identified from photographs of the cortical surface and depth readings from the micromanipulator. At the end of the experiments, the stimulating sites were similarly labeled by passing DC current (10 FA, 20 seconds) through the stimulating electrodes.

Histology The cats were perfused under Nembutal anesthesia by transcardial infusion of 350 cc of buffered saline followed by a fixative containing 4% paraformaldehyde, 0.1%glutaraldehyde, and 0.2%picric acid. The brains were removed and stored in the same fixative solution overnight at 4°C. Blocks of tissue containing area 4y were cut into 50 pm thick

TABLE 1. Biocytin Histochemistry 5 x 10 min PBS' 45 min 0.6% avldin Wedor Labs) and 0.06%Tntnn-X UIPBS 3 x l0minPBS 45 rnin0.125%biotinylatd-HRP (Vector Labs) and 0.06%Tnton-X in PBS 1 x 10 min PBS 2 x 10min TBS* 1 x 15minO,5%CoCI,inTBS,inthedark 1 x 5 min TBS 2 x l0minPBs 20 min 0.05% D M 3in PBS, in the dark 20 rnin 0.05%DAB + 0.01%H20, + 0.07%imidazole + 0.02% NiC1, in PBS, in the dark 5 x 10minPBS 'PBS, phosphatebuffered saline, 0.1 M, pH 7.4. 'TBS, tm-bufferedsaline, 0.05 M, pH 7.4. 'DAB, 3,VDiaminobenzidine. All steps performed at rmm temperature.

sagittal sections using a vibratome; in several cases frozen sections were obtained from tissue blocks saturated with 20% sucrose. The sections were then processed to reveal biocytin-labeled cells using the avidin-biotin horseradish peroxidase (ABC) method of Hsu et al. ('81). In earlier experiments, the sections were processed with the ABC kit produced by Vector Labs (USA). However, this procedure necessitated the use of high concentrations of detergent ( > 0.2% Triton X) to achieve adequate labeling. Since the use of such high concentrations of detergent considerably damages the tissue, rendering it unsuitable for electron microscopy, and because the ultrastructural details and the synaptic relationships of the biocytinlabeled cells are important for our future studies, we developed the protocol presented in Table 1. With this protocol, adequate labeling could be obtained when Triton-X concentrations as low as 0.06% were used. Sections containing labeled cells were postfixed in 0.2% OSO, and examined with a light microscope. Several sections were processed for transmission electron microscopy as previously described (Keller and White, '87). The labeled cells were photographed and drawn with the aid of a drawing tube attached to a light microscope. Sections immediately adjacent to those containing labeled cells were Nissl-stained to identify the boundaries of cytoarchitechtonic areas and of cortical layers. Blocks of tissue containing recording and stimulating sites labeled by electrolytic lesions were placed overnight in a fixative solution containing 20% sucrose. These blocks were cut in the sagittal plane into 25 pm sections on a freezing microtome. The sections were Nissl-stained, and camera lucida drawings of the lesion sites were obtained with a light microscope.

RESULTS InductionofLTP Stable intracellular recordings were obtained from more than 800 neurons in the motor cortex. Approximately 10% of these cells responded with EPSPs to ICMS in area 5a or in area 1-2. None of the cells responded to activation of stimulating electrodes in area 5b. The locations of the stimulating electrodes were identified in Nissl-stained sections containing sites of electrolytic lesions. Area 5a was distinguished by the presence in layer V of a row of very large cells (40-50 Fm in diameter) that were not present in adjacent cortical areas. This feature characterizes area praeparietalis gigantopyramidalis of Hassler and MuhsClement, ('641, corresponding to the third somatosensory area (SIII) described by Tanji et al. ('78). The row of large

LTP-PRODUCING NEURONS IN CAT MOTOR CORTEX pyramids ended abruptly at the border between area 5a and the more caudal area 5b, and at the border between 5a and the more rostral area 2 (see Hassler and Muhs-Clement, '64). In comparison, the transition from area 2 and the more rostral area 1 was not as distinct, and therefore this region will be collectively referred to as area 1-2. However, all the lesion sites in area 1-2 were located in the more caudal end of this region (i.e., immediately rostral to area 5a), suggesting that these stimulating sites were restricted to area 2. All stimulating sites in area 1-2 and in area 5a were located in the superficial layers of the cortex (layers 11-IV). In no instance were stimulating sites found in, or near the subcortical white matter. This finding and previous data on the effective current spread produced by ICMS (Asanuma et al., '76) suggest that the microstimulation did not directly activate fibers in the white matter. Thirty-nine neurons that satisfied the following criteria were accepted for analysis: 1) they responded with clear EPSPs to stimulation of one or more electrodes in areas 1-2 or 5a; 2) the latency and rise time of these EPSPs remained constant throughout the recording period; 3) the membrane potential was at least -50 mV and was stable throughout the recording period; and 4) the recording period lasted at least 10 minutes. Since intracellular recordings were obtained with KC1-filled electrodes, it is possible that some of the synaptic potentials recorded were not EPSPs, but inhibitory posysynaptic potentials (IPSPs) which reversed in polarity because of infusion of chloride ions from the recording electrode. Although this possibility cannot be excluded, it is highly unlikely because short latency synaptic potentials in motor cortical neurons, produced by stimulation of the somatosensory cortex, have been demonstrated to be exclusivelyexcitatory (Kosar et al., '85). Furthermore, both the latencies and the rise times of the synaptic potentials analyzed remained stable throughout the recording period, even after passing hyperpolarizing current through the recording electrodes, indicating that these potentials were not affected by diffusion of electrolytes from the recording electrodes. Data on the 39 neurons accepted for analysis are presented in Table 2. Fig. 1 illustrates a representative example of LTP induced in a motor cortical neuron following tetanic stimulation in area 5a. EPSP amplitudes recorded from this cell !cell #2; Table 2) were stable during 3 minutes of control recordings. Immediately following the tetanic stimulation, EPSP amplitudes decreased, and then gradually increased and stabilized after 4 minutes at the potentiated level (192% relative to control; P < 0.001, two-tailed MannWhitney U test). EPSP amplitudes remained stable at this potentiated level for more than 25 minutes, until the recording electrode went out of the cell. Of the 39 cells accepted for analysis, 25 cells (64%) exhibited LTP similar to the one depicted in the example in Fig. 1. In each of the 25 cells, EPSP amplitudes decreased immediately following the tetanic stimulation, and stabilized within 2 to 4 minutes at a potentiated level. Cells were considered to have produced LTP only if EPSP amplitudes remained stable at the potentiated level for the duration of the posttetanus recording period. The degree of potentiation was calculated by comparing the mean amplitudes of pretetanus EPSPs to those of posttetanus EPSPs measured at the stabilized potentiated level. A two-tailed MannWhitney U test, corrected for ties, was employed to determine the significance of the increase in EPSP amplitudes (Table 2). Posttetanic EPSP amplitudes were 117 to 314% relative to

49 TABLE 2. Data on the 39 Analyzed Motor Cortical Neurons

Area'

Posttetanus EPSP amplitudes relative to control amplitudes (%I2

Membrane potential (mv)

Recording time (minI3

Cell no.

Layer morphology

1

11 Pyramid

5a

118'*

60

40

7 ?

5a 5a

I1 Nonpyranud I11 Pyramid I1 Pyramid I1 11 111

5a

192** 127'* 124*

50 65 70

25 19 17

5a

125.

SO

10

5a

128*"

60

5

1-2 1-2 1-2 1-2

151"* 164** 203** 158**

75 80 65 50

90 56 33 14

1-2 1-2 1-2

297'* 180** 189*'

50 50 65

14 14 13

1-2

139*

50

10

1-2

314"'

55

10

1-2

191**

50

9

1-2

55 60 50 50 50 55 50 65 55

9 9 9 8 8 8

1-2 1-2

120* 117. 153* 122* 182** 149* 204*" 133' 142**

5a

PTPd

50

38

5a 5a

PTP PTP

60 55

20 13

1-2

NS NS NS NS NS NS NS NS

70 60 60 65 50 50 60 55

62 46 26 23 14 19

NS NS NS

60

12 7

2 3 4 5 6

7 8 9 10

11 12 13

14 15 16 17 18 19 20 21 22 23 24

25 26 27 28

29 30 31

32 33 34 35 36 37 38 39

111

Nonpyramid I11 11 111 Nonpyramid I11 Pyramid I1 Pyramid I11 Pyramid ? I11 I11

I1 111 111

I11 I11 I11 Pyramid I1 Pyramid ? 11

V V 111 In

1-2

1-2 1-2 1-2 1-2 1-2

1-2 1-2 1-2

1-2

I1 V I11 Nonpyramid

1-2 1-2

?

1-2

v V

1-2 1-2

1-2

50 60

7

7 5

1s 13

7

'Stmdation area.1-2, primary somatosensorycortex; 5% somatosensory association cortex. 'Percent change in average EPSP amplitudes followingtetanic stimulation. *P< 0.01. **P< 0.001. NS, not s i g n i h t (two-tailed,Mann-Whitney U test). 3Posttetanusremrdingperiod. 4Posttetanic potentiation.

control amplitudes (mean 2 SD = 164.9 2 51.0%, n = 25). LTP was induced following tetanic stimulation in either area 5a (6/9 cells, 67%) or area 1-2 (19/30 cells, 63%).There was no correlation between the amplitude of pretetanus EPSPs or the degree of potentiation and the stimulating sites. Intracellular recordings obtained from the cells accepted for analysis lasted 19.5 -C 18.0 min (mean & SD, n = 39); 17 of these cells were recorded from for more than 20 minutes (maximum, 96 minutes). Control (i.e., pretetanus) recordings lasted at least 3 minutes, during which EPSP amplitudes remained stable. In several instances, control recordings obtained for more than 20 minutes established that EPSP amplitudes did not change after the first 3 minutes of recordings. Whenever possible, input resistance was measured using a bridge circuit built in the amplifier;

A. KELLER ET AL.

50

0

0

0

0

0

5

10

15

20

25

30

Time (min) Fig. 1. Time course of the induction of LTP in a motor cortical neuron (cell #2, Table 2). The amplitudes of EPSPs produced by test stimulation (30 PA, 1 Hz) in area 5a were recorded before, and after tetanic stimulation (200 Hz, 20 seconds). No hyperpolarizing current

was passed throughout the recording. Representative intracellular recordings of EPSPs before (A)and after (B)the tetanic stimulation are shown above.

the input resistance of these cells ranged from 12 to 23 M a (n = 15). In 13 cases, the input resistance was measured again following the tetanic stimulation; in no instance was a change in resistance observed. Three of the cells responded to stimulation through more than one stimulating electrode. Cell #3 responded with EPSPs to stimulation through an electrode in area 5a as well as an electrode in area 1-2; cells #11 and #13 responded to two different electrodes in area 1-2. In all these instances, tetanic stimulation was delivered through only one of the electrodes, and the second electrode was used for control stimulation. In all of these cells, EPSPs elicited through the tetanized electrodes, but not by the control electrodes, were potentiated. In 14 of the 39 neurons accepted for analysis LTP was not induced. Three of these cells (cells #26-28) produced

EPSPs in response to area 5a stimulation. Fig. 2 illustrates, the effects of tetanic stimulation on the EPSPs recorded from one of these cells (cell #26). Three trains of tetanic stimuli were delivered, with approximately 10 minute intervals. Immediately following each tetanic stimulation, EPSP amplitudes decreased, and then increased to a level of approximately 150% relative to control amplitudes. EPSP amplitudes remained at the potentiated level for about 2 minutes and then returned to pretetanic levels. Cells #27 and #28 responded with similar, short-lasting posttetanic potentiation (PTP) to several trains of tetanic stimuli. The other nonpotentiated cells (cell #29-39) produced EPSPs in response to area 1-2 stimulation. In some of these cells, two tetanic stimulations were delivered, the first after 3 minutes of control recordings and the second 5-10 minutes later; the tetanic stimulation did not induce a significant

LTP-PRODUCING NEURONS IN CAT MOTOR CORTEX

.

5-

51

0

4.5-

0

0 . 4-

5' E

~

0

0

. 0 .

0

.

3.5.

0 .

Area2

0 Area 5

0

m e 0.

v

2.5.

0 .

2.

0

1.5.

0

0,

0.0 O.

1.

0.50

2

1

3

4

5

6

7

Latency (ms)

Fig. 2. Posttetanic potentiation (PTP) induced in a pyramidal neuron in layer I I of the motor cortex (cell #26, Table 2). Following each of the three tetanic stimuli, EPSP amplitudes increased, and returned within 2 minutes to control levels. This cell responded with long-latency,polysynaptic EPSPs to stimulation in area 5a.

increase in mean EPSP amplitudes (Table 2). Neither did the tetanic stimulation affect the variability of EPSP amplitudes in these cells: the standard deviations of the mean EPSP amplitudes before and after tetanic stimulation were nearly identical.

Fig. 3. Histograms of latencies of EPSPs recorded from the 39 analyzed neurons. Histograms were constructed using 0.5 mV bins.

3501 300.

0 0

250. 0

Paramete~correlatedwithLWinduction In an attempt to clarify the conditions that influence the induction of LTP, several electrophysiological parameters . O 5 were examined in relation to the relative increase in EPSP amplitudes, Latency. The latencies of EPSPs recorded from the 39 analyzed cells are depicted in the histograms in Fig. 3. Twenty-one of the 30 cells (70%) that responded t o area 1-2 stimulation had EPSP latencies ranging from 1.2 to 2.2 ms. Previous results suggest that EPSPs having latencies within this range are elicited monosynaptically (Kosar et al., '85). Six of the nine cells (67%)responding to area 5a stimulation had EPSP latencies ranging from 1.2 to 2.8 ms, and the remaining three cells had latencies longer than 4.8 ms. Waters et al. ('82) have shown that 79% of area 5a neurons antidromically activated by area 4y stimulation had latencies ranging from 1.0 to 3.0 ms. Thus it is likely that area 5a-induced EPSPs having latencies shorter than 3.0 ms are elicited monosynaptically. In Fig. 4, the latencies of EPSPs recorded from the 39 analyzed cells are plotted against the posttetanus EPSP amplitudes. The amplitudes of EPSPs are expressed as a percent of the mean EPSP amplitudes. LTP was induced only in neurons in which the latencies of area 1-2 or area 5a-elicited EPSPs were 2.2 or 2.8 ms or less, respectively. Cells that responded with longer latency EPSPs were either not potentiated, or they potentiated only temporarily (PTP). Thus we conclude that LTP can be induced exclusively in short latency, presumably monosynaptic EPSPs. However, in two cells (cells #30 and #35) that responded to area 1-2 stimulation with short latency ( < 2.2 ms) EPSPs, LTP was not induced. Although these cells were not stained intracellularly, their laminar location could be approximated from histological examination of the lesions

I100 500 Area 2

Fig. 4. The relationship between the latencies of EPSPs and the relative increase in EPSP amplitudes. Long-term potentiation (LTP) was induced exclusively in neurons producing short-latency (monosynaptic) EPSPs.

made during the experiments (see Materials and Methods). This analysis revealed that these cells were located in deep layer V of the motor cortex. Four cells (cells #29, 31, 38, and 39; Table 2) recorded in layer V, which exhibited long-latencyEPSPs were also not potentiated. These finding is in agreement with our previous results (Iriki et al., '89) indicating that LTP can be induced exclusively in neurons in the superficial layers of the motor cortex. Time topeak. One of the factors related to the efficacy of neural transmission is the distance between the activated synapse and the soma of the postsynaptic cell. It has been proposed that the rise time of an EPSP [i.e., the time from the onset to the peak of the EPSP (TTP)] reflects the distance from the activated synapse to the recording electrode (Rall, '67). Assuming that the recording electrodes impaled cell bodies, and that the recorded neurons had similar membrane properties, the rise time of the recorded

A. KELLER ET AL. 3500

300-

Area 2 U Area 5

c=

0

250.

5

L

0

200-

0

. 0 .

0

O

.

0 0

05

1

15

2

25

3

35

4

45

5

Time to peak (ms) Fig. 5. Histograms of time to peak (TTP) of EPSPs. Only shortlatency EPSPs (area 2: < 2.2 ms; area 5a; < 3.0 ms) are included. Histograms were constructed using 0.5 ms bins.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Time to peak (ms) Area 5 (monosynaptic) p = 0.26; 0 Area 2 (monosynaptic) p = -0.22: 0

P > 0.5 P > 0.3

0 Area 2 (polysynaptic)

Fig. 6. The relationship between the time to peak (TTP) of EPSPs EPSPs may be indicative of the location of the activated and the relative increase in EPSP amplitudes. There was no correlation synapses relative to the somata of the cells being recorded. between the TTP of monosynaptic EPSPs and the potentiation of EPSP Histograms of TTP of the EPSPs are shown in Fig. 5; amplitudes (Spearman rank correlation). only the short-latency, presumably monosynaptic EPSPs were included in this analysis. TTP of short latency EPSPs induced by area 1-2 stimulation (0.6 to 2.7 ms; 1.5 k 0.7 affect membrane potential, amplitude, latency, or rise time ms) were, on average, shorter than TTP of short latency of EPSPs. EPSPs produced by area 5a stimulation (1.2 to 3.3 ms; Morphology of identified neurons. The 13 intracel2.3 2 0.8 ms) ( P < 0.1; two-tailed Mann-Whitney U test). lularly labeled neurons were classified according to comThis finding suggests that axon terminals belonging to monly accepted morphological criteria (Peters and Jones, fierents from area 5a form synapses on more distal ’84).All of the cells had somata in layers I1 or I11 of the dendritic segments, compared to synapses formed by affer- motor cortex. Nine of the cells were classified as pyramidal ents from area 1-2. neurons and four were identified as nonpyramidal cells. To determine whether the location of synapses is related Both classes of neurons responded to stimulation in area to the efficacy in LTP induction (assuming that the afferent 1-2 or in area 5a. There were no significant differences inputs to the recorded cells were synchronous), the TTP of between pyramidal and nonpyramidal neurons in memEPSPs recorded from the 39 analyzed cells were plotted brane potential, EPSP latency, amplitude, TTP, or the against the increase in EPSP amplitudes (Fig. 6 ) . There ability to produce LTP (Mann-Whitney U test, P > 0.5). was no correlation between the TTP and the magnitude of Furthermore, there were no morphological features that LTP (P > 0.3; Spearman rank correlation). This finding differentiated neurons that produced LTP, PTP, or the indicates that the potency of synapses to produce LTP is not neurons that were not potentiated. related to their spatial distribution on the postsynaptic cell. Cell bodies belonging to pyramidal neurons ranged from 8 to 15 +m in diameter for neurons in layer I1 and in upper Intracell~labelingwithbiocytin layer I11 (six cells), and 12 to 22 Frn for neurons in deep Biocytin was injected intracellularly into 13 of the 39 analyzed neurons, after they were examined for LTP induction. In no instance was there more than one neuron labeled following a single biocytin injection, indicating that Fig. 7. A biocytin-stained pyramidal neuron in deep layer 111 of the the results were not complicated by extracellular leakage of motor cortex (cell #16, Table 2 ) .LTP was induced in this cell by tetanic biocytin and uptake into neighboring cells, or by transneu- stimulation in area 2. A montage of micrographs of portions of this cell is shown on the right. Note labeling of dendritic spines and of boutons ronal transport (‘‘dye coupling”). along the axon collaterals (arrows). On the left is a drawing of this cell, There were no differences between neurons recorded reconstructed from serial sections. Note the arborizations of axon with biocytin-filled electrodes or with 3 M KCl electrodes in collaterals in the vicinity of the parent soma and their horizontal terms of membrane potentials, TTP, control EPSP ampli- projection within layer 111. Cortical laminae are indicated by roman tudes, or ability to induce LTP. In several instances, numerals; boundaries between cortical layers occur approximately intracellular recordings were obtained before and after halfway between these numerals. Scale bar = 0.1 mm. Inset depicts intracellular recording (ten averaged sweeps) of an EPSP recorded intracellular injections of biocytin, to determine the effects before (A), and after (B) 5 minutes of biocytin injection. The record of biocytin on these recordings. In Fig. 7 (inset), intracellu- below B was produced by subtraction of B from A and by increasing the lar recordings of EPSPs before, and after 5 minutes of gain, to demonstrate that the biocytin injection has little or no effect on biocytin injection, are compared. Biocytin injections did not the intracellular recording. Scale bars = 1mV and 2 ms.

>

54

A. KELLER Er AL.

Fig. 8. Electron micrograph of a thin section through the soma and proximal portion of the apical dendrite of the cell depicted in Fig. 7. The ultrastructural preservation of cytoarchitectonic details was main-

tained by using a low concentration of detergent (0.06%Triton X).Scale bar = 2 pm.

layer I11 (three cells).Apical dendrites belonging to pyramidal neurons in deep layer 111 projected towards the pial surface and branched mainly in upper layer I1 and in layer I (Fig. 7). Pyramidal neurons having somata in layer I1 and in upper layer I11 emitted branches from the proximal portions of their apical dendrites, and had a wider dendritic tree than their counterparts in deep layer I11 (cf. Figs. 7 and 9). The basal dendritic trees of all the pyramidal neurons were characterized by relatively thin and short dendrites that branched in the immediate vicinity of the cell body. Dendritic spines were located throughout the dendritic tree, but were more densely distributed along the more distal segments of the apical dendrites and their branches. In eight of the nine pyramidal neurons the axon could be traced from the soma to the subcortical white matter, at which point the labeling terminated. Axon collaterals branched extensively in the immediate vicinity of the cell

bodies, where they intermingled with the dendritic branches. In addition, axon collaterals projected for relatively long distances (up to 2 mm) horizontal to the pial surface, within the same layer in which their parent soma was located (Figs. 7 and 9). Only a single, long horizontal axon collateral projected from each pyramidal neuron, most commonly in the anterioposterior plane. Less frequently, axon collaterals projected vertically towards the pial surface, immediately above their parent soma. Punctate structures, presumably

Fig. 9. Drawing of a biocytin-labeled pyramidal neuron in layer XI of the motor cortex (cell #6, Table 2). Dendritic branches are depicted by thicker lines bearing spines; thinner lines depict arborization of axon collaterals. Inset: the time course of LTP induced in this cell by stimulation in area 5a.

LTP-PRODUCING NEURONS IN CAT MOTOR CORTEX

55

rn

rn rn rn rn

rn rn rn rn rn

:

A. KELLER ET AL.

56

rn rn

U

LTP-PRODUCING NEURONS IN CAT MOTOR CORTEX

57

TABLE 3. Motor CorticalNeurons in Which LTP Induction Was Attempted by Tetanic Stimulationin Area 2 or Area 5a Nonpyramidal neurons

Pyramidal neurons

Unidentified neurons

Total (n = 39)

Potentiated

Not Potentiated

Area 5a

3

21

2

4

0

1 2

0

Area 2

1

13

Total

7

2

3

1

15

11

Potentiated

Not Potentiated

Potentiated

Not potentiated

Not

Potentiated

Potentiated

1

6

10

19

3' 11

25

14

'These neurons produced short-termpmttetmic potentiation (PTF').

synaptic boutons, were commonly found along the axon collateral branches (Figs. 7 and 9). The four identified nonpyramidal neurons had somata in upper layer 111, and their cell bodies ranged from 10 to 25 km in diameter. All the cells emitted dendrites from multiple points on the soma, and the dendrites had a beaded appearance and were devoid of spines (Fig. 10). In three of the four nonpyramidal neurons the axon was labeled; the axons projected from the soma or from a proximal dendrite and ramified in the immediate vicinity of the cell bodies, or slightly above them. The form and distribution of the dendritic and axonal plexuses of these cells resemble that of local plexus neurons that have been described in sensory areas of the cortex (Peters and Saint Marie, '84).

neurons analyzed in the present study. LTP could be induced only in neurons that produced short latency, presumably monosynaptic EPSPs in response to area 1-2 or area 5a stimulation. These cells included both pyramidal and nonpyramidal neurons having somata in layers I1 or 111 of the motor cortex. Although neurons in layers VNI of the motor cortex have been demonstrated to produce monosynaptic EPSPs in response to stimulation in the superficial layers of area 2 (Asanuma et al., '85; Herman et al., '85; Zarzecki, '891, none of these cells exhibited LTP, even when they produced short-latency EPSPs (Table 2). The superficial layers of the motor cortex contain mainly corticocortical and intracortical neurons, whereas pyramidal tract neurons are located in layers V and VI (Groos et al., '78). This suggests that tetanic stimulation of corticocortical pathways does not produce LTP in pyramidal tract DISCUSSION neurons, even though these cells receive monosynaptic The data obtained in this study support and expand our corticocortical input (Zarzecki, '89). Thus, the function of previous observations on the induction of LTP in the motor plasticity in synaptic transmission in the motor cortex is cortex (Iriki et al., '89; Sakamoto et al., '87). Our findings likely to be related to intracortical information processing. indicate that LTP of synaptic transmission in motor corti- Findings that LTP can be induced in both pyramidal cal neurons can be induced by tetanic stimulation of (presumably excitatory) and nonpyramidal (presumably different cortical areas that provide direct input to the inhibitory) neurons indicate that this intracortical processmotor cortex (areas 1-2 and 5a). Furthermore, the com- ing involves both excitatory and inhibitory interactions. bined intracellular recording and labeling techniques emThe specificity of LTP t o a particular subset of neurons ployed in the present study enabled the morphological implies that different classes of neurons in the motor cortex identification of the motor cortical neurons in which LTP exhibit inherently different biophysical properties. For was induced. example, different classes of cells may utilize different postsynaptic receptors and second messenger systems. PerLTP in the motor cortex tinent to this assumption are findings that NMDA-sensitive In the present study, LTP was induced by a single train of glutamate receptors, which have been shown to be involved high-frequency stimulation (200 Hz, 20 seconds) in the in LTP induction (Artola and Singer, '87; Collingridge et somatosensory cortex. Although some somatosensory corti- al., '83; Kimura et al., '89), are distributed preferentially in cal neurons can sustain even higher rates of spike frequen- the superficial layers of the cerebral cortex (Monaghan and cies (e.g., Mountcastle et al., '69),it is not likely that these Cotman, '85). The heterogeneity of biophysical properties cells normally fire at these frequencies for more than of cortical neurons is supported also by findings showing several hundred milliseconds. Since the present study was that neurons having similar morphological features may aimed mainly at elucidating neural elements involved in exhibit distinctly different electrophysiological properties LTP induction, we employed stimulation parameters that (Dykes et al., '88; McCormick et al., '85). In the present were previously established as being optimal for LTP study, only subthreshold synaptic potentials were recorded induction (Sakamoto et al., '87). Future studies are neces- from both pyramidal and nonpyramidal neurons; therefore sary to determine whether LTP can be induced in corticocor- differences in the firing patterns of these cell types were not detected. tical pathways using more "physiological" conditions. Most of the analyses in the present study were of neurons The results of the present study indicate that LTP can be induced in a specific group of neurons in the motor cortex. that produced short-latency, monosynaptic EPSPs in reTable 3 summarizes the effects of tetanic stimulation on the sponse to microstimulation in areas 1-2 or 5a. These EPSPs could have been induced by activation of corticocortical axons whose parent somata were in the stimulating sites. Alternatively, the microstimulation could have antidromiFig. 10. Drawing of a biocytin-labeled nonpyramidal (local circuit) cally activated axons of corticocortical cells having somata neuron in upper layer I11 of the motor cortex (cell #lo, Table 2). Note in the motor cortex, which produced EPSPs in other motor the beaded appearance of the dendritic branches (thicker lines). The cortical neurons via their local axon collaterals. Attempts to axon is emitted from a proximal dendrite (arrowhead), and arborizes substantiate one of these possibilities by selective neuroimmediately above the parent soma (thinner lines). Inset: the time toxin-induced lesions of projection neurons in the stimulatcourse of LTP induced in this cell by stimulation in area 2.

A. KELLER ET AL.

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motor learning and memory of corticocortical pathways linking the somatosensory and the motor cortex (Asanuma, '89; Asanuma and Mackel, '89). Ablation of the somatosensory cortex, which does not produce marked motor deficits, dramatically impedes the process of acquiring novel motor skills (Sakamoto et al,, '89). Furthermore, axons from the somatosensory to the motor cortex have been shown to sprout and to form functionally active synapses following lesions of the deep cerebellar nuclei (Keller et al., '90). Thus, these corticocortical afferents can form new synapses without deafferentiation and the creation of synaptic vacancies. Whether the formation of new synapses requires the induction of LTP is still unknown. This possibility is supported by findings that the induction of LTP is correcorticalLTPvs.hip~pdLTP lated with structural alternations in both pre- and postsynLong-term potentiation of synaptic transmission in neo- aptic elements of the activated synapses (reviewed by cortical areas has many features in common with hippocam- Desmond and Levy, '88; Markus and Petit, '89). pal LTP. In both the neocortex (Komatsu et al., '88; These findings and the data in the present study indicate Stripling et al., '88) and the hippocampus (Douglas and that corticocortical pathways to the motor cortex are Goddard, '75) LTP has been shown to persist for hours and capable of dramatic synaptic plasticity. The long-term even days. In the present study, the longest LTP observed enhancement of synaptic efficacy reported here may be was for 90 minutes. Were it not for the technical limitations related to the initial stages of motor learning, which are inherent in in vivo intracellular recordings, it is likely that dependent on somatosensory feedback. The repetitive somaLTP in the motor cortex could have been demonstrated to tosensory input evoked during the practice of a particular persist for longer periods. Cortical and hippocampal LTP motor task may induce LTP in a subset of motor cortical are similar also in the methods by which they can be neurons. The potentiated cells may then affect the firing induced. In both areas, LTP can be induced by tetanic patterns of additional motor cortical neurons that are stimulation of a single afferent pathway (cf. Bliss and Ldmo, involved in the execution of that particular motor task. '73 and the present study). LTP can also be induced in both Thus, LTP may serve to augment and refine the circulation areas by combined stimulation of two separate pathways, in of nerve impulses between the motor cortex and the a process termed associativity. Thus, if a cell receives periphery (Favorov et al., '88), thereby controlling the converging inputs from two pathways, one that does and neuronal network responsible for the execution of skilled the other that does not induce LTP, the latter input can be movements. potentiated if both pathways are coactivated (hippocampus: Levy and Steward, '79; motor cortex: Iriki et al., '89). Furthermore, in both the motor cortex (Baranyi and Szente, ACKNOWLEDGMENTS '87) and the hippocampus (Gustafsson and Wigstrom, '86), The expert technical assistance of Mrs. K. Arissian and E. long-lasting potentiation can be induced by activating an Ruiz is deeply appreciated. This work was supported by afferent input while depolarizing the postsynaptic cell. In both the neocortex and the hippocampus, similar NIH NS-08626 to A.K., and by NIH NS-10705 to H.A. biophysical processes may be involved in the induction of LTP. Afferent pathways in the hippocampus (StormLJJERAmcFTED Mathisen and Ottersen, '88) and corticocortical pathways in the cerebral cortex (Conti et al., '88; Giuffrida and Artola, A,, and W. Singer (1987) Long-term potentiation and NMDA receptors in rat visual cortex. Nature 3303349-652. Rustioni, '89) are believed to utilize the excitatory neurotransmitter glutamate. Activation of NMDA-sensitive gluta- Asanuma, H. (1989) The Motor Cortex. New York: Raven Press. mate receptors has been demonstrated to be a prerequisite Asanuma, H., and R. Mackel (1989) Direct and indirect sensory input pathways to the motor cortex; its structure and function in relation to for LTP induction in both the hippocampus (Collingridge et learning motor skills. Jpn. J. Physiol. 39:l-19. al., '83) and in the visual cortex (Artola and Singer, '87; Asanuma, H., A. Arnold, and P . Zarzecki (1976) Further study on the Kimura et al., '89). excitation of pyramidal tract cells by intracortical microstimulation. Exp. However, neocortical and hippocampal LTP differ in Brain Res. 26:443461. other features. For example, in the hippocampus, LTP can Asanuma, H., E. Kosar, N. Tsukahara, and H. Robinson (1985) Modification be triggered by a brief and low-frequency tetanic train of of the projection from the sensory cortex to the motor cortex following the elimination of thalamic projections to the motor cortex in cats. Brain stimulation (Bliss and Lbno, '73). In comparison, signifiRes. 345:79-86. cantly longer trains of stimulation, or higher frequency A., and M.B. Szente (1987) Long-lasting potentiation of synaptic stimulation are needed for the induction of LTP in the Baranyi, transmission requires postsynaptic modifications in the neocortex. Brain cerebral cortex (Iriki et al., '89; Sakamoto et al., '87; Res. 423t378-384. Stripling et al., '88). Whether these differences in LTP Berger, T.W., and R.J. Sclabassi (1988) Long-term potentiation and its characteristics are related to differences in experimental relation to hippocampal pyramidal cell activity and behavioral learning during classical conditioning. In P.W. Landfield and S.A. Deadwyler paradigms, or whether they reflect differences in the mech(eds): Long-Term Potentiation: From Biophysics to Behavior. New York: anisms of LTP induction. remains to be clarified. Alan R. Liss, pp. 467-497.

ing sites, leaving the fibers from the somatosensory to the motor cortex intact, have thus far been unsuccessful (Asanuma, Iriki, and Keller, unpublished observations). Both the afferents from area 2 (Ichikawa et al., '87; Porter and Sakamoto, '88) and the local axon collaterals of motor cortical neurons that project to area 2 (A. Keller, in preparation) synapse with a variety of neuronal elements in layers 11-VI of the motor cortex. Thus, at present it is not possible to determine whether the LTP recorded in this study was produced by activation of corticocortical afferents to the motor cortex, by activation of axon collaterals of motor cortical neurons, or by both pathways.

Role ofLTP in motor learning The results of the present study provide further evidence for the growing body of data demonstrating the role in

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LTP-PRODUCING NEURONS IN CAT MOTOR CORTEX Collingridge, G.L., S.J. Kehl, and H. McLennan (1983) Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J. Physiol. 334r33-46. Conti, F., M. Fabri, and T. Manzoni (1988)Glutamate-positive corticocortical neurons in the somatic sensory areas I and I1 of cats. J. Neurosci. 82948-2960. Corkin, S.(1965) Tactually guided maze learning in man: Effect of unilateral cortical excision in bilateral hippocampal lesions. Neuropsychologia 3t339-35 1. Davies, S.N., R.A.J. Lester, K.G. Reymann, and G.L. Collingridge (1989) Temporally distinct pre- and post-synaptic mechanisms mediate longterm potentiation. Nature 338r500-503. Desmond, N.L., and W.B. Levy (1988) Anatomy of associative long-term synaptic modification. In P.W. Landfield and S.A. Deadwyler (eds): Long-Term Potentiation: From Biophysics to Behavior. New York: Alan R. Liss, pp. 265-305. Douglas, R.M., and G.V. Goddard (1975) Long-term potentiation of the perforant path-granule cell synapses in the rat hippocampus. Brain Res. 86:205-2 15. Dykes, R.W., Y. Lamour, P. Diadori, P. Landry, and P. Dutar (1988) Somatosensory cortical neurons with an identifiable electrophysiological signature. Brain Res. 441r4EL58. Favorov, O., T. Sakamoto, and H. Asanuma (1988) Functional role of corticoperipheral loop circuits during voluntary movements in the monkey: A preferential bias theory. J. Neurosci. 8:3266-3277. Giuffrida, R., and A. Rustioni (1989)Glutamate and aspartate immunoreactivity in corticocortical neurons of the sensorimotor cortex of rats. Exp. Brain Res. 74t41-46. Groos, W.P., L.K. Ewing, C.M. Carter, and J.D. Coulter (1978) Organization of corticospinal neurons in the cat. Brain Res. 143:393-419. Gustafsson, G., and H. Wigstrom (1986) Hippocampal long lasting potentiation produced by pairing single volley and brief tetani evoked in separate afferents. J. Neurosci. 6t1575-1582. Hassler, R., and K. Muhs-Clement (1964) Architektonischer aufbau des sensomotorischen und parietalen Cortex der Katze. J. Hirnforsch. 6:377-420. Herman, D., R. Kang, M. MacGillis, and P. Zarzecki (1985) Responses of cat motor cortex neurons to cortico-cortical somatosensory inputs. Exp. Brain Res. 57:598-604. Horikawa, K., and W.E. Armstrong (1988) A versatile means of intracellular labeling: Injection of biocytin and its detection with avidin conjugates. J. Neurosci. Methods 25t1-11. Hsu, S.M., L. Raine, and H. Fanger (1981) Use of avidin-biotin-peroxidase complex (ABC)in immunoperoxidase techniques. A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29:577-580. Ichikawa, M., K. Arissian, and H. Asanuma (1987) Reorganization of the projection from the sensory cortex to the motor cortex following elimination of the thalamic projection to the motor cortex in cats: Golgi, electron microscope and degeneration study. Brain Res. 437:131-141. Iriki, A., C. Pavlides, A. Keller, and H. Asanuma (1989) Long term potentiation in the motor cortex. Science 245t1385-1387. Kauer, J.A., R.C. Malenka, and R.A. Nicoll (1988)A persistent postsynaptic modification mediates long-term potentiation in the hippocampus. Neuron 1:911-917. Keller, A,, and E.L. White (1987) Synaptic organization of GABAergic neurons in the mouse SmI cortex. J. Comp. Neurol. 2621-12. Keller, A,, K. Arissian, and H. Asanuma (1990) Formation of new synapses in the cat motor cortex following lesions of the deep cerebellar nuclei. Exp. Brain Res. 8023-33. Kimura, F., A. Nishigori, T. Shirokawa, and T. Tsumoto (1989) Long-term potentiation and N-methyl-D-aspartate receptors in the visual cortex of young rats. J. Physiol. (Lond.) 414t125-144. Komatsu, Y., K. Fujii, J. Maeda, H. Sakaguchi, and K. Toyama (1988) Long-term potentiation of synaptic transmission in kitten visual cortex. J. Neurophysiol. 59:124-141. Kosar, E., R.S. Waters, N. Tsukahara, and H. Asanuma (1985) Anatomical and physiological properties of the projection from the sensory cortex to the motor cortex in normal cats: The difference between corticocortical and thalamocortical projections. Brain Res. 34568-78. Levy, W.B., and 0.Steward (1979) Synapses as associative memory elements in the hippocampal formation. Brain Res. 175233-245. Markus, E.J., and T.L. Petit (1989) Synaptic structural plasticity: Role of synaptic shape. Synapse 3:l-11.

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McCormick, D.A., B.W. Connors, J.W. Lighthall, and D.A. Prince (1985) Comparative electrophysiology of pyramidal and sparsely spiny stellate neurons of the neocortex. J. Neurophysiol. 54~782-805. Monaghan, D.T., and C.W. Cotman (1985) Distribution of NMDA-sensitive LPH-glutamate binding sites in rat brain as determined by quantitative autoradiography. J. Neurosci. 52909-2919. Mountcastle, V.B., W.H. Talbot, H. Sakata, and J. Hyvarinen (1969)Cortical neuronal mechanisms in flutter-vibration studied in unanesthetized monkeys. Neuronal periodicity and frequency discrimination. J. Neurophysiol. 32452-484. Peters, A., and E.G. Jones (1984) Classification of cortical neurons. In A. Peters and E.G. Jones (eds): Cerebral Cortex. Val. 1. Cellular Components of the Cerebral Cortex. New York: Plenum Press, pp. 107-121. Peters, A., and R.L. Saint Marie (1984) Smooth and sparsely spinous nonpyramidal cells forming local axonal plexuses. In A. Peters and E.G. Jones (eds): Cerebral Cortex. Vol. 1. Cellular Components of the Cerebral Cortex. New York: Plenum Press, pp. 419-445. Porter, L., and K. Sakamoto (1988)Organization and synaptic relationships of the projection from the primary sensory to the primary motor cortex in the cat. J. Comp. Neural. 271:387-396. Rall, W. (1967) Distinguishing theoretical synaptic potentials computed for different somadendritic distributions of synaptic input. J. Neurophysiol. 3O:1138-1168. Ramon y Cajal, S. (1911) Anatomicophysiological considerations on the cerebrum. In J. DeFelipe and E.G. Jones (eds): Cajal on the Cerebral Cortex. An Annotated Translation of the Complete Writings. New York: Oxford University Press, pp. 465-490. Sakamoto, T., L.L. Porter, and H. Asanuma (1987) Long lastingpotentiation of synaptic potentials in the motor cortex produced hy stimulation of the sensory cortex in the cat: A basis of motor learning. Brain Res. 413:360-364. Sakamoto, T., K. Arissian, and H. Asanuma (1989) Functional role of the sensory cortex in learning motor skills in cats. Brain Res. 503:258-264. Squire, L.R. (1982) Human memory: Neurophysiological and anatomical aspects. Annu. Rev. Neurosci. 5241-273. Storm-Mathisen,J . , and O.P. Ottersen (1988) Anatomy of putative glutaminergic neurons. In M. Avoli, T.A. Reader, R.W. Dykes, and P. Gloor (eds): Neurotransmitters and Cortical Function. New York: Plenum Publishing Co., pp. 39-70. Stripling, J.S., D.K. Patneau, and C.A. Gramlich (1988)Selective long-term potentiation in the pyriform cortex. Brain Res. 441:281-291. Tanji, D.G., S.P. Wise, R.W. Dykes, and E.G. Jones (1978) Cytoarchitecture and thalamic connectivity of third somatosensory area of cat cerebral cortex. J. Neurophysiol. 41t268-284. Thompson, W.D., S.D. Stoney, and H. Asanuma (1970) Characteristics of projections from primary sensory cortex to motorsensory cortex in cats. Brain Res. 2215-27. Waters, R.S., 0. Favorov, and H. Asanuma (1982) Pattern of projection and physiological properties of cortico-cortical connections from the posterior bank of the ansate sulcus to the motor cortex, area 4y, in the cat. Exp. Brain Res. 48:335-344. Woody, C.D. (1986) Understanding the cellular basis of memory and learning. Annu. Rev. Psychol. 37:433-493. Zarzecki, P. (1989) Influence of somatosensory cortex on different classes of cat motor cortex output neuron. J. Neurophysiol. 622487494.

APPENDIX Biocytinasanidye Since intracellular biocytin injection is a relatively new approach, it seems worthwhile to discuss the merits and limitations of this technique, as well as the modifications to the technique introduced in the present study. The advantages and limitations of the biocytin technique, as compared to other intracellular dyes, were discussed in detail by Horikawa and Armstrong ('88), who introduced this method. The relatively small size and high solubility of the biocytin molecule results in rapid diffusion throughout the injected cells, and enables the use of electrodes having relatively low resistance. In the present study, biocytin was dissolved in a high concentration salt solution (2.5 M KC1 at 37"C), and electrodes filled with this solution were shown not to differ significantly in their impedance from elec-

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trodes filled with 3 M KCl only. Thus, fine-tipped biocytin electrodes could be used for stable and relatively long recordings from small cortical neurons. Furthermore, the quality of intracellular recordings was not effected by the intracellular injection of biocytin. Neurons were successfully labeled even after short ( < 1 minutes) injections of biocytin, using either constant hyperpolarizing current (3 nA) or hyperpolarizing current pulses (3 nA, 5 Hz; 100 ms). However, labeling of fine processes such as dendritic spines, thin distal dendritic segments, and axon collaterals was enhanced when longer injection times were used (5 to 10 minutes). The low toxicity of biocytin was beneficial also in allowing for relatively long postinjection survival times, which resulted in complete filling of even the finest and most distal neuronal processes. Survival times of up to 8 to 10 hours resulted in labeling of axon collaterals located 1.5 to 2 mm from the parent soma. There were no noticeable pathological changes in the morphologies of the injected neurons using survival periods of up to 15 hours. The quality of labeling was dependent also on methods of tissue preparation and on the histochemical reaction. The extent and intensity of labeling were significantly reduced when the fixative solution contained more than 0.1% glutaralehyde. There was no significant difference in the

A. KELLER ET AL. quality of staining of cells in sections cut with a freezing microtome, compared to vibratome sections. The main limitation of the biocytin technique pertains to its compatibility with electron microscopy, since the histochemical reaction requires the use of a detergent to allow for diffusion of reagents through the cell membrane. The use of a detergent results in disruption of the ultrastructural characteristics of neuronal cytoarchitectonics. This limitation was rectified in the present study by modifying the histochemical reaction, such that the concentrations of detergent could be reduced to levels that are compatible with electron microscopy. Although higher concentrations of detergent (Triton-X)resulted in a significantly enhanced labeling, particularly of thin neuronal processes, Triton-X concentrations as low as 0.06% proved adequate, and resulted in adequate ultrastructural preservation of t,he tissue, as determined by electron microscopical examination (Fig. 8). The reduction in detergent concentration was made possibly by incubating the sections with avidin, and then with biotinylated HRP (Table 1).The separate incubation steps allowed for easier penetration of these macromolecules. In comparison, the significantly larger avidin-biotinHRP complex employed in the one-step ABC technique (Hsu et al., '81) required the use of at least 0.2% Triton-X to label fine neuronal processes.