Bunkyo-ku, Tokyo, 113 Japan

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1 Journal of Physiology (1991), 443, pp With 7 figures Printed in Great Britain IMPULSE CONDUCTION IN CAl APICAL DENDRITES OF RABBIT HIPPOCAMPUS: ITS POSSIBLE IMPLICATION IN NORMAL AND ABNORMAL ACTIVITIES BY YASUICHIRO FUJITA From the Department of Physiology, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo, 113 Japan (Received 3 January 1991) SUMMARY 1. Impulse conduction in CAl apical dendrites was studied by stimulating afferent fibres to the distal portion of the dendrites (Schaffer stimulation) and recording intracellularly from the pyramidal cell body in the hippocampus of the rabbit anaesthetized with sodium pentobarbitone and immobilized with d-tubocurarine. 2. The stimulation, when strong enough, produced a full spike in all the pyramidal cells (n = 48) which were capable of producing spikes of mv in amplitude. A single-shock Schaffer stimulation produced a single spike in forty-six cells and a twospike burst in two cells. All the single spikes and the first spike of the two-spike burst arose directly from the baseline. 3. By reducing the stimulus strength, three categories of small spikes (cx,, and y) could be distinguished in twenty-two pyramidal cells. cx was of the lowest threshold with an amplitude of less than 7 5 mv and the time from the foot of the spike to its peak (peak time) was more than 040 ms., was of the next lowest threshold with an amplitude of mv and had a peak time of ms (n = 3242). y was of the highest threshold with an amplitude of mv and had a peak time of ms (n = 1783). The duration of a was 1P5-40 ms, whereas that of /8 and y was ms. 4. Within a given pyramidal cell, the waveform of /3 and y was remarkably constant, being independent of stimulus strength. They were therefore regarded as units and referred to as unitary D-spikes. The unitary D-spikes tended to summate forming a larger, longer-lasting potential which was referred to as the D-spike. ac was probably a D-spike produced at a greater distance away from the recording microelectrode, as compared with /3 and y. 5. Within a given pyramidal cell unitary D-spikes, and y could be further subdivided into two subclasses, respectively, according to the differences in amplitude. Furthermore ac contained at least one unitary D-spike. Thus, at least five different unitary D-spikes could be distinguished in the same cell. They were thought to be dendritic in origin, because only the dendrites could possibly give rise to so many small spikes which could be seen with the intrasomatically placed microelectrode. 6. In most cases a full spike consisted of the A-. B- and D-spikes. As a rule, the NMS 9033

2 336 Y. FUJITA threshold of the A-spike was high and a depolarization of mv was needed to trigger the A-spike. In the case of apical dendritic activation, such a depolarization was provided by summation of unitary D-spikes. However, the threshold could become very low due to unknown causes in the same cell and the A-spike then arose directly from the baseline in response to apical dendritic activation. 7. A double-shock Schaffer stimulation produced more D-spikes with shorter latencies, as compared with a single-shock stimulation of the same strength (frequency potentiation). There was evidence indicating that the potentiation was related to the genesis of seizure discharges. These results indicated that D-spikes (unitary D-spikes) took part in abnormal activities. 8. A possibility of the unitary D-spike subserving normal functions was discussed. INTRODUCTION Ramon y Cajal (1893, 1894, 1972) observed a remarkable phylogenetic development of the pyramidal cell in mammals. He therefore thought that it was the pyramidal cell that subserved higher nervous functions such as memory. For this reason he referred to the cell as cellule psychique. In particular, he noted that the dendrites of the cell attained the highest development in man. Accordingly, he ascribed the development of higher nervous functions to formation of more axodendritic connections. If this is so the study of the pyramidal cell dendrites electrophysiologically becomes of utmost importance for understanding the function of the brain. The hippocampus has been considered to serve this purpose favourably because of its well-defined, laminated structures (Ramon y Cajal, 1893, 1972; Lorente de No, 1934). In fact, many a study, employing both intracellular and extracellular microelectrodes, has been carried out in order to disclose the electrophysiological properties of the pyramidal cell dendrites. It is now known that the dendrites can generate intrinsic slow depolarizations (e.g. Fujita & Iwasa, 1977; Wong, Prince & Basbaum, 1979; compare Kandel & Spencer, 1961). On the other hand, the evidence for impulse conduction in the apical dendrite was first reported by Cragg & Hamlyn (1955) employing extracellular recording. This was subsequently confirmed by a number of similar studies (Andersen, 1960; Fujita & Sakata, 1962; Andersen & L0mo, 1966; Miyakawa & Kato, 1986). In respect of the impulse conduction, however, intracellular recording has been unable to provide detailed information although fast spike activities of the dendrite such as the fast prepotential (FPP) (Spencer & Kandel, 1961) and D-spike (Andersen & L0mo, 1966; Schwartzkroin, 1975) have been described. The present experiments were designed to disclose detailed aspects of impulse conduction in the apical dendrite. The in situ hippocampus was employed because in the hippocampal slice no overt evidence was obtained for orthodromic impulse conduction in the apical dendrite, whereas antidromic conduction was clearly demonstrated to occur (Miyakawa & Kato, 1986). It will be shown below that a small spike, which was much shorter in peak time (the time from its foot to its peak) than FPP (referred to as the unitary D-spike), could be consistently produced by apical dendritic activation and a full spike was triggered usually through summation of unitary D-spikes.

3 IAIPUTLSE CONDUCTION IN APICAL DENDRITE337 METHODS Fifty-four rabbits ( kg) were used. They were initially anaesthetized with an intravenous injection of mg/kg of sodium pentobarbitone (Nembutal) and subsequently given mg/kg of the same drug intramuscularly for maintenance of the anaesthesia. Additional Nembutal was given when required. The depth of the anaesthesia was so controlled that hippocampal electroencephalograms (EEGs) consisted mainly of irregular slow waves of various amplitudes (compare Fujita & Sato, 1964). All the animals were immobilized with d-tubocurarine (Amerizol: 1-2 mg/kg, i.v. and i.m.). The capillary microelectrode for recording was filled with 4 M-potassium acetate solution. The resistance of the microelectrode originally ranged from 50 to 120 MQ2, but it was reduced to MQ by moving the microelectrode repeatedly into and out of 2-4 % Ringer-agar, because the artificial reduction in the resistance yielded better results in intracellular recording (Fujita, 1979). The potentials recorded with the microelectrode were displayed on a cathode-ray oscillograph (Nihonkoden VC11, Japan). They were also monitored with a pen-writing oscillograph (Nihonkoden RM80, Japan). The baseline of the pen-writer usually exhibited incessant changes corresponding to EEG activities. Occasionally, the baseline could become quiescent for about 0 5 s or more. The steady membrane potential was then measured. Intracellular potentials were recorded from the right hippocampus, whereas EEGs were monitored from the left hippocampus with a conventional concentric electrode by means of the pen-writer. Another concentric electrode was placed in the fornix for stimulation. A capillary microelectrode, filled with equal parts of 1 M-sodium acetate and 4% Pontamine Sky Blue solutions (resistance: less than 0 5 MQ) was employed to record field potentials or stimulate intrahippocampal structures. The location of the tip was identified by passing a DC current of 8,uA for 6 min through the microelectrode which produced a spot of am in diameter (Fig. 1 J). Identification of pyramidal cells The hippocampus was not exposed because otherwise it rapidly lost EEG activities (Fujita & Sato, 1964). CAI pyramidal cells were studied. Their identifications were made primarily by referring to the waveforms of Schaffer stimulation-evoked potentials (see below) which exhibited waveforms peculiar to the depths from the hippocampal surface (Fujita & Sakata, 1962; Fujita & Sato, 1964, Fig. 1). Of 134 cells impaled, forty-eight cells could exhibit spikes of (S.D.) mv (range: mv). Their steady membrane potentials (see above) were mv ( mv) (compare Kandel, Spencer & Brinley, 1961). Input resistance was measured in five out of twenty-two cells which exhibited unitary D-spikes (see below). The resistance ranged from 12 to 16 MQ. In all the forty-eight cells the rate of spontaneous discharges never exceeded 15/s except during the intrinsic spike burst (Kanel & Spencer, 1961). All of them exhibited a hyperpolarization of 8-19 mv lasting ms following Schaffer (Fujita & Sato, 1964) or fornix (Fujita, 1979) stimulation. They were all located in the layer of pyramidal cell bodies, as judged from the waveform of the Schaffer stimulation-evoked potential, and thirty-four cells exhibited the intrinsic spike burst (inactivation response) spontaneously and/or in response to a depolarizing current injection which was characteristic of the pyramidal cell (Kandel & Spencer, 1961; Fujita, 1975; Schwartzkroin, 1975). In fourteen cells, however, there occurred antidromic spikes in response to supramaximal fornix stimulation. This was considered to be due to current spread to the hippocampal commissure which was quite close to the fornix. Finally, the microelectrode which yielded good results was further advanced considerably so as to make its tract clearer and it was ascertained that the tract traversed across the CAI stratum pyramidale. Placement of the capillary microelectrode for intrahippocampal stimulation The capillary microelectrode containing Pontamine Sky Blue (see above) was introduced into the distal portion of CAI apical dendrites by referring to the waveform of the fornix-evoked potential, which was essentially the same as Schaffer collaterals-evoked potentials (Fujita & Nakamura, 1961). As shown in Fig. 1 G, in the layer of pyramidal cell bodies (stratum pyramidale), the first population spike arose almost directly from the slow positivity with a small negative pre-potential (arrow). As the microelectrode was advanced into the stratum radiatum, the latency and the amplitude of the pre-potential became considerably shorter and larger, respectively (Fig. 1 H, arrow). These changes occurred within /am below the stratum pyramidale, as ascertained

4 338 Y. FUt.JITA, by making a spot (compare Fujita & Sato. 1964, Fig 1). The microelectrode was then advanced further by about pm. In this region, the fornix-evoked potential consisted mainly of a slow negative wave (Fig. 11). The microelectrode was fixed there and used as the monopolar stimulating electrode. In twenty-one experiments, the location of the tip of the stimulating microelectrode was examined histologically by making a spot (see above). It was found that the spots were all located in the region of the distal arborizations of the apical dendrite (Fig. 1 J). The most conspicuous of the afferent fibres in this region are the Schaffer collaterals. Consequentlv. the stimulation with the microelectrode was referred to as Schaffer stimulation for descriptive purposes. Subtraction of field potentials from intracellularly recorded potentials Inasmuch as field potentials in the hippocampus attain a considerable magnitude (Fig. 1 G-I. Fujita & Sakata. 1962), it is necessarv to subtract field potentials from intracellularly recorded potentials in order to know the accurate time course of intracellular potentials. The field potentials for subtraction were recorded with the same microelectrodes as used for recording intracellular potentials after the steady membrane potential fell to -IO to 0 mv with no observable intracellular transients. To use a different microelectrode for recording the field potentials was considered infeasible because of the following reasons. (a) It was not easy to place the tips of two microelectrodes even at a distance of 500,am from each other. (b) The difference in the locations of the tips could cause a difference in latency or waveform of the field potentials. (c) The difference in electrode resistance could have a similar effect. The onlyt possible drawback in using the same microelectrode at different times was that the state of the hippocampus might change, resulting in a change in the waveform of the field potentials. This posed. however. no observable problems as far as the present experiments were concerned. The subtraction was made graphically by projecting the potentials on a large screen. The distance between the stimulating and recording microelectrodes was less than 1 mm in terms of septo-temporal (longitudinal) axis and less than 3 mm in terms of subiculo-dentate (transverse) axis. The latencies of' fornix- and Schaffer stimulation-induced population spikes were in a similar order, being ms. They depended largely upon the relative positions of recording and stimulating electrodes, as well as the strength of stimulation. No attempts were made to investigate this problem. RESULTS Small spikes of ms in peak time (unitary D-spikes) In Fig. 1A, Schaffer stimulation (see Methods) at 2 6 T produced two kinds of potentials. (1 T was defined as the threshold for Schaffer stimulation-evoked potentials.) One was potential dl and the other a full spike having three notches on its rising phase (arrows a. d, d'). In Fig. 1B-D, the stimulus strength was reduced to 2 0 T and potential dl was still produced as before, but the full spike was no longer elicited. In lieu of the full spike, there appeared a small potential with a complex waveform (Fig. 1B-D, d2). The waveforms of these two small potentials, i.e. dl and d2, could not be changed by grading the stimulus strength. This suggested that both potentials were all-or-none in nature, and they will be referred to as D-spikes for descriptive purposes. Although D-spike d2 occurred in all-or-none fashion in response to stimulation, its waveform underwent spontaneous changes despite the stimulus strength having been maintained at 2-0 T. That is to say, in Fig. lb it consists of three components (arrow-heads), but in Fig. 1 C and D, it has two components. It is to be noted that the time relationship between these two components also changed spontaneously (Fig. 1 C and D, d2). Comparison of Fig. la-d with Fig. 1 E gives the impression that all the slow potentials recorded with the intracellular microelectrode were field potentials and as a result all the spikes arose directly from the baseline. In full agreement with a previous work (Andersen & L0mo, 1966), the impression was ascertained by subtracting the field potentials from the intracellularly recorded

5 IMPUTLSE CONDUCTION IN APICAL DENDRITE 339 A R d2 C d2 dl 0 dl 0 dl /,W%- 0 D d2 v d\i 10mV E F d2 0 1 ms 0ll dl G H l J i. CAl S Gr 5 ms. t CA4 5~~~ ~ Ms Fig. In this figure and Figs 2, 4-6, each sweep was triggered by the stimulus artifact. All the data in this figure and Figs 2-4 were obtained from the same pyramidal cell which remained in a good state for about an hour. The responses in A-F were produced by a single shock stimulation (pulse duration: ms, given every 3 s) of the afferent fibres to the distal portion of the CAl apical dendrites (Schaffer stimulation, see Methods). In A stimulus strength was 2-6 T and B-F 2 0 T. A-D, and F, intracellular records. E, field potentials recorded with the same intracellular microelectrode after the death of the cell. 0: population spikes. Note in A that the full spike had three notches on its rising phase (arrows, a, d, d'). Potentials dl and d2 were all-or-none in nature as a whole and they were referred to as D-spikes. Note that D-spike d2 consists of two to three components (B-D, arrow-heads). All the slow potentials in A-D are the field potentials recorded intracellularly. For example, in F the field potentials were subtracted from the intracellularly recorded potentials; note that D-spikes dl and d2 arise directly from the baseline without any depolarizing pre-potential. The responses in G-I were recorded with the 'stimulating microelectrode' (see Methods) during the process of its introduction into the distal portion of the apical dendrite layer. The fornix was supramaximally stimulated with a ms pulse. Downward deflections denote negativity. G, recorded from the stratum pyramidale (str. pyr.); H, from 100,um below str. pyr.; I, from 600,um below str. pyr. The microelectrode was fixed at this point and used as a stimulating electrode. In J, arrow indicates the lesion where the microelectrode was located (see Methods); CAl and CA4 indicate pyramidal cells; Gr, granule cells. potentials (e.g. Fig. IF). This has been confirmed in all other records except in a few cases (see below). The D-spike in the same cell was further studied by double-shock Schaffer stimulation. Five D-spikes, dl-d5, were distinguished according to the differences in

6 340 Y. FUJITA latency (Figs 2 and 3). Namely, the interval between the two shocks was 8 ms and the second shock happened to be delivered during the population spike produced by the first shock (compare Fig. 2I with Fig. 1 E). Not only D-spike dl, but also D-spike d2 was produced by the first shock, as judged by their latencies (Figs 1, 2 and 3). On A d2 d3 B d2 d3 d5 C dl dl *dl D E F S ds l g - d1l * dl ims fms G ~~~~~~ d2 d4 d5 H d2s~~~~~~1m d4 dl dl dl Fig. 2. Responses produced by double-shock Schaffer stimulation at 1-6 T (A), 2-0 T (B and C), 2-2 T (D and E) and 1-9 T (F-I). The interval between the two shocks was 8 ms. A-H, intracellular records. I, field potentials for subtraction. dl-d5, D-spikes. D-spikes dt and d2 were produced by the first shock, whereas D-spikes d3, d4 and d5 by the second shock. * indicates population spikes produced by the second shock. s, subtracted drawing in which field potentials were subtracted from intracellular records. s in F is the subtracted drawing of the full spike (the spike is truncated); s in G, the subtracted drawings of D-spikes d4 and d5; s in H, the subtracted drawing of D-spike d2. In these subtracted drawings, note that all of the spikes arose directly from the baseline. the other hand, D-spikes d3, d4 and d5 were produced by the second shock (Figs 2 and 3). It was thought that D-spikes dl and d2 corresponded to D-spikes d3 and d4, respectively, because both D-spikes dl and d3 appeared shortly before, or during the early phase of, the population spike (Figs 1A-D and 2B and C); both D-spikes d2 and d4 appeared during the later phase of the population spike (Figs 1B-D and 2G and H). D-spike d5 appeared some time after the population spike. In no case was a D- spike corresponding to D-spike d5 produced by the first shock. D-spikes d2 and d4 were of comparable waveform, both consisting of one to three components of similar magnitudes (Figs 1B-D, and 2G, H, and H,s). In contrast, D-spikes dl and d3 were entirely different in waveform from each other. The latter was, for example, much larger in amplitude than the former (e.g. Fig. 2B). As a result, D-spike d3 rather resembled D-spikes d2 and d4 in waveform (e.g. Fig. 2B and H), although the latency of D-spike d3, as measured from the second shock, was the same as that of D-spike

7 IMPULSE CONDUCTION IN APICAL DENDRITE dl, both being 6-5 ms (Fig. 3A). Since the waveform of D-spike dl could not be changed by grading the stimulus strength (compare D-spike dl produced with 1P6 T in Fig. 2A with that produced with 2-2 T in Fig. 2E), the larger amplitude of D-spike d3 could be accounted for if it is assumed that more components were added to D-spike dl to become D-spike d dl d2 d3 d5-10 A o 0 : o O 0 ~ co E 10 E z 0 ~~ ~~0 5 1 dl d2 d4 B 0 0 o 0 0 O 0 d ~~ O. o5 l mime 0 0 ~~~~ ~ie(ins) Fig. 3. At zero time and arrow, a single-shock Schaffer stimulation was given. dl-d5, D- spikes. 0, the latencies of the D-spikes (dl and d2) produced by the first shock. *, the latencies of D-spikes (d3-d5) produced by the second shock. In A, the latencies of the D- spikes produced at 2 0 T, such as shown in Fig. 2B and C, were measured, whereas inb those produced at 1 9 T, such as shown in Fig. 2F-H, were measured. Note in A that the latency of D-spike dl is approximately the same (6-5 ms) as that of D-spike d3, as measured from the second shock (arrow). Also note that D-spikes d3 and d4 do not occur in succession. One of the purposes of the double-shock stimulation was to measure accurately the duration of the refractoriness of the D-spike. However, due to the fact that a D-spike consisted of several components and there were two D-spikes produced by a single shock, it was not possible to know the accurate duration of the refractoriness of a D- spike. Nevertheless, the following facts revealed the existence of the refractoriness. First, D-spikes d3 and d4 did not occur in succession in the experiments illustrated in Fig. 2. The appearance of the former always resulted in the disappearance of the latter in all-or-none fashion (Fig. 2B and C). Second, when a full spike was produced in place of D-spike d2, D-spike d3 disappeared in all-or-none fashion and, instead, D- spike d4 made its appearance (Fig. 2D). Thirdly, an increase in the latency of D-spike d2 (compare the latency of D-spike d2 in Fig. 2F-H with that in Fig. 2B and C) resulted in the disappearance of D-spike d3 in all-or-none fashion and D-spike d4 appeared. Since the interval between D-spikes d2 and d3 was about 4 ms (Figs 2B, C, and 3A), it may be assumed that the refractoriness of these D-spikes was approximately 4 ms. D-spike d5 was not influenced by the preceding D-spikes (Fig. 3). As a rule, a full spike consisted of three components: the A-spike, B-spike and a

8 342 Y. FUJITA potential that triggered the A-spike (which, in turn, triggered the B-spike) (Kandel, Spencer & Brinley, 1961). In Fig. 1A and Fig. 2D-F, a denotes the peak of the A- spike. In these figures, it should be noted that the A-spike was of much higher threshold than the initial segment (IS)-spike of the spinal motoneurone (Eccles, /3 a dl d d2 dd3 GHI K tl d4 d4 d4 d5 d5 Fig. 4. The main components forming a D-spike were referred to as unitary D-spikes, because they had a constant waveform. A-J illustrate D-spikes or unitary D-spikes produced by a double-shock Schaffer stimulation at 1-8 T. The interval between the two shocks was 8 ms. dl-d5, D-spikes (or unitary D-spikes in isolation). As shown in Fig. 2, D-spikes dl and d2 were produced by the first shock, while D-spikes d3, d4 and d5 by the second shock. The vertical line in each record was drawn so that the differences in peak time (time from the foot to the peak of a spike) might be easily recognized. Two categories of unitary D-spikes, /6 and y, were distinguished according to the difference in peak time. The peak time of /1 was ms (n = 281) and that of y was ms (n = 154). Peak times of,: 0 30 (B), 0-32 (C), 0-28 (D), 0-26 (F), 0-29 (I) and 0-32 (J) ms. Peak times of y: 0-20 (E), 0-22 (F) and 0-24 (I) ms. a was thought of as a D-spike produced at various distances from the recording microelectrode because of its longer peak time (more than 0 40 ms) and large variations in amplitude, as compared with, and y. Note that, whereas /6 could occur in isolation (J), y was produced only when triggered by a (E) or by,3 (C, F, I). s in I is the subtracted drawing of y. The drawing was made by assuming that, from which y arose, had a falling phase similar in time course to that of, in J. In K, the stimulus strength was increased to 2-3 T and a larger spike having a complex waveform was produced. In L, the stimulus strength was further increased to 2-5 T and a full spike was produced. Note that the full spike had three notches (arrows a, d, d'). Upward arrows in A-C, K and L indicate the second shock artifacts. * indicates population spikes produced by the second shock. An upward deviation of the baseline in each record is due to field potentials. 1957). It was a rule, except in a few cases (see below), that a depolarization of mv was needed to trigger the A-spike. In Fig. 4 the components forming a D-spike were studied in 412 D-spikes recorded from the same pyramidal cell and classified into three categories according to the peak time (the time from the foot to the peak of a potential). Namely: a, more than 040 ms; /3, ms (range, ms; n = 281) and y, ms (range: ms, n = 154). Thus y had shorter peak time than /,. y was also

9 IMPULSE CONDUCTION IN APICAL DENDRITE distinguished from, by the fact that the former was always of a higher threshold than the latter. /3 and y were further divided into two subclasses, respectively, according to differences in amplitude. For example, /8 in D-spike d2 (Fig. 4B and C) belonged to subclass 'large /3' ( mv; range, mv; n = 101), whereas that in D-spikes d4 and d5 (Fig. 4D, F, I, J) belonged to subclass 'small /3' ( mv; range, mv; n = 180). Similarly, y in D-spike d2 (Fig. 4C) was a large y ( mv; range, mv; n = 58), whereas that in D-spikes d4 and d5 (Fig. 4F, I) was a small y ( mv; range, mv; n = 96). The waveform of /3 or y in each subclass was thus fairly constant. Consequently, they may be regarded as units (unitary D-spikes). The three components forming a D-spike always appeared in the order of a, / and y. Thus a D-spike was a sequential discharge of o, /l and y. (Fig. 2 C, d3); or that of a and / (Fig. 4D); or that of o and y (Fig. 4E); or that of, and y (Fig. 4C, F, I). a (Fig. 4A) and /3 (Fig. 4B,J) could appear in isolation. In contrast, y was produced only when triggered by ac or /. a was smaller in amplitude, as compared with / and y. Its amplitude was usually less than 3 0 mv, although it could be as large as 7-5 mv. Because of its smaller amplitude in most cases, it was thought that it was a D-spike produced at a greater distance away from the recording site (presumably the cell body) than /3 and y. However, some of them could be a unitary D-spike occurring in isolation at a distance. There was a spike whose peak time fell in the category of a, but its amplitude was considerably larger than that of a. Such a spike had a notch on its rising phase and could be explained as being formed by two unitary D-spikes (Fig. 4G and H). In Fig. 4H, a appeared in isolation. Since it appeared during the early phase of the population spike, it corresponded to D-spike d3. However D-spike d4 occurred after it (Fig. 4H), whereas in Fig. 2 D-spikes d3 and d4 never occurred in succession. The difference could be explained if it is assumed that in this case D-spikes d3 and d4 were formed by the same unitary D-spikes because their waveforms were quite similar, whereas in that case they were formed by different unitary D-spikes as suggested by a large difference in amplitude. The above results on the three main components of a D-spike, as well as those on the two main types of unitary D-spikes, were confirmed also on 3948 D-spikes recorded from twenty-one other pyramidal cells. In each cell, at least two unitary D- spikes could be distinguished: /3 was ms in peak time and mv in amplitude (n = 2961) and y was ms and mv (n = 1629). Even when the membrane potential was decreased due to injury toward the end of the recording from each cell, unitary D-spikes could still be observed, provided the cell could fire mv spikes. Their peak time however could be as long as 0 70 ms and their amplitude as low as 2-5 mv. The most peculiar feature of the unitary D-spike was that in no case could a single unitary D-spike trigger a full spike except the one arising in the portion presumably nearest to the soma (see Discussion). As a rule production of a full spike entailed a summation of unitary D-spikes. For example, in Fig. 4A-J, no D-spikes or unitary D-spikes could trigger a full spike. In Fig. 4K, the stimulus strength was increased from 1-8 T in Fig. 4A-J to 2-3 T and a larger spike with a complex waveform (complex spike) was produced, suggesting a recruitment of unitary D-spikes. The stimulus was further increased to 2-5 T, and a full spike was produced (Fig. 4L). In 343

10 344 Y. FUJITA Fig. 4L, arrow d indicates a notch which corresponds in potential level to the peak of the complex spike in Fig. 4K. There is another notch (Fig. 4L, arrow d') which is obviously at a higher potential level than the peak of the complex spike shown in Fig. 4K). This suggests that recruitment of more unitary D-spikes was needed to trigger A =~~~~~~~~~~~~~~~~~ 1d 0 d 1 O B c dl1d2ja D 1000 Fig. 5. Potentials produced by a double-shock Schaffer stimulation. Sweeps 1 and 2 were triggered by the first and second shocks, respectively. The interval between the two shocks was 30 ms. The first shock produced a unitary D-spike (A, d). The second shock produced either a full spike (A, B) or D-spikes (C, dl, d2). D is the field potentials for subtraction. 0 and * indicate population spikes produced by the first and second shocks, respectively. Note that in A the full spike had four notches (arrows a, d, d', d"), but in B it had only one notch (arrow a). s indicates subtracted drawings. Note that the full spike (B) and D-spikes (C) arise directly from the baseline. Also note that potentials produced by the second shock had shorter latencies than those produced by the first shock. a full spike. This agrees with the above results that the A-spike was of high threshold, and in Fig. 4L a depolarization of about 30 mv was necessary to trigger it. That the A-spike was of high threshold was ascertained in seventeen other pyramidal cells. Potentiation of D-spikes by double-shock Schaffer stimulation (frequency potentiation) Figure 5 illustrates the effects of double-shock Schaffer stimulation upon a pyramidal cell. The interval of the two shocks was 30 ms. The stimulus strength (2f4 T) was just threshold for this cell. Accordingly, the first shock produced either a unitary D-spike of 0-26 ms in peak time (Fig. 5A, sweep 1) or nothing (Fig. 5B and C, sweep 1). The second shock produced either a full spike (Fig. 5A and B, sweep 2) or D- spikes of 0 4 ms in peak time (Fig. 5C, sweep 2). The full spike in Fig. 5A had four notches on its rising phase. The first notch (Fig. 5A, arrow d) is approximately at the same potential level as the peak of the unitary D-spike (Fig. 5A, d). It was therefore thought that the notch indicated the unitary D-spike. However, the unitary D-spike

11 IMPULTSE CONDL YCTION IN AIPICAL DENDRITE4345 could not trigger a full spike by itself. A summation of more potentials (probably unitary D-spikes) was needed to trigger it (Fig. 5A, arrows d' and d"). Thus the A- spike's being of high threshold was demonstrated also by double-shock stimulation. The full spike in Fig. 5B had only one notch, and it arose directly from the baseline (Fig. 5B, s). This means that the A-spike arose directly from the baseline, inasmuch as the notch denotes the peak of the A-spike (Fig. 5B, arrow a). Thus, here was an A-spike which was of very low threshold, and comparable to the IS-spike of the motoneurone (Eccles, 1957). The time from the foot of the full spike to the notch, i.e. the peak time of the A-spike, was 0 34 ms. Both Fig. 5A and B were recorded from the same pyramidal cell with an interval of about 10 min. These results show that the threshold of the A-spike of this cell varied considerably. In contrast, the threshold of the B-spike was fairly constant (e.g. arrow a in Fig. 5A and B). The considerable variation in the threshold of the A-spike was observed in four out of eighteen pyramidal cells in which the notches on the rising phase of the full spike were studied in detail. In the remaining fourteen cells, the A-spike was invariably of high threshold, such as illustrated in Figs 1A and 2D-F. In Fig. 5A. it can be seen that there was a decrease in the latency of the full spike produced by the second shock, as compared with the latency of the unitary D-spike produced by the first shock. Thus the effects of double-shock stimulation were twofold: one was generation of more potentials and the other a decrease in latency. The dual effects can be clearly appreciated also in the field potentials. Namely, while the first shock produced a single population spike (Fig. 5A-D, 0), the second shock produced two population spikes (Fig. 5D, *), and the first population spike produced by the second shock was of considerably shorter latency than the population spike produced by the first shock (Fig. 5D). These phenomena are exactly the same as those observed by previous investigators on the pyramidal (Andersen & L0mo, 1966) and dentate granule (Bliss & L0mo, 1973) cells following a repetitive stimulation of the perforant path. They referred to these phenomena as frequency potentiation. The term will be employed in the present paper. Figure 6 illustrates another example of frequency potentiation. In Fig. 6A, a single-shock Schaffer stimulation was given at 1f4 T and a full spike occurred. The stimulus strength was just threshold for the spike. Neither gradation in the stimulus strength nor a hyperpolarizing current injection could disclose component potentials forming the full spike. The spike arose directly from the baseline (Fig. 6A, s). In Fig. 6B, a double-shock Schaffer stimulation was given with an interval of 80 ms and the latency of the spike was decreased. In Fig. 6C, the stimulus strength was increased to 1-6 T and a two-spike burst was produced. The two-spike burst in response to a single-shock stimulation occurred only in two out of forty-eight pyramidal cells studied in the present experiments. The first spike arose directly from the baseline (Fig. 6C, s). All of these spikes had after-hyperpolarizations (Fig. 6A-C). Yet the cell under study was a pyramidal cell, not only because it exhibited some electrical activities characteristic of the pyramidal cell (see Methods), but also because fornix stimulation produced in this cell an antidromic spike, presumably due to current spread to the hippocampal commissure. That the pyramidal cell could exhibit an after-hyperpolarization has already been reported (Fujita, 1975). It is noted that the after-hyperpolarization of the first spike of the two-spike burst was considerably

12 346 Y. FUJITA curtailed in time and magnitude, as compared with that of a single spike (compare Fig. 6C with Fig. 6A and B). This means that the second spike arose from a depolarizing pre-potential (Fig. 6C, s). The pre-potential could be the excitatory postsynaptic potentials (EPSPs) observed by Andersen & L0mo (1966) in frequency potentiation. A B cj D d ~50mV 22 4 ms E F G H 10 ms Fig. 6. A and(c illustrate potentials produced by a single-shock Schaffer stimulation at 1 4 and 16 T, respectively. B. produced by a double-shock Schaffer stimulation at 1-4 T. D, produced by a supramaximal double-shock Schaffer stimulation. Sweeps 1 and 2 were triggered by the first and second shocks, respectively. The interval between the two shocks was 80 ms. s shows subtracted drawings. Note that the spike in A and the 1st spike in C arise directly from the baseline. Downward arrows in D indicate full spikes produced by the second shock. Arrow d in D indicates a D-spike. Note that in B and D the latencies of the full spikes on the second sweep are shorter than those on the first sweep and in D a third potential which happens to be a D-spike is produced (frequency potentiation). 0 and 0 show population spikes produced by the first and second shocks, respectively. E and F show field potentials for subtraction for A and C, respectively. G and H, field potentials produced by a supramaximal Schaffer stimulation. G and H were obtained before and after supramaximal 10 Hz Schaffer stimulation was delivered for 5 s, respectively. Note that after repetitive stimulation, frequency potentiation occurred. Voltage calibration in D applies to all records. Time calibration in D is for A-F, whereas that in H applies also to G. In H, six traces were superimposed. In Fig. 6D, two shocks were delivered at an interval of 80 ms. The second shock produced not only two full spikes with shorter latencies (Fig. 6D, downward arrows), but also a D-spike (Fig. 6D, arrow d). As is already known, an antidromic or orthodromic stimulation of the pyramidal cell could produce in the cell inhibitory postsynaptic potentials (IPSPs) (Kandel et al. 1961; Andersen, Eccles & Loyning, 1964a, b; Fujita, 1979; Alger & Nicoll, 1982). Accordingly, with a double-shock stimulation at an interval of 80 ms, the second shock inevitably landed on the IPSPs (Fig. 6B and D, sweep 2). In Fig. 6D, note that the first spike on sweep 2 arose from a more hyperpolarized level than the level from which the first spike on sweep 1 took off. Thus the IPSPs could not inhibit the spike, as earlier noted by Schwartzkroin (1975). Figure 6G shows field potentials produced by a supramaximal single-shock Schaffer stimulation. Note that two spikes were produced and the second spike was

13 IMPULSE CONDUCTION IN APICAL DENDRITE less than one-fourth of the first spike in amplitude. Figure 6H illustrates field potentials produced by the same stimulation after 10 Hz Schaffer stimulation with the same strength was given for 5 s. The field potentials were considerably enhanced: four spikes could occur with shorter latencies. In eleven experiments such a frequency potentiation was observed and in eight cases it ended up in a seizure discharge. A single-shock Schaffer stimulation never produced three or more population spikes, unless a repetitive stimulation was given prior to the single-shock stimulation. In this respect, it was an abnormal sign that a third spike which was a D-spike was produced by the double-shock stimulation (Fig. 6D). Small spikes in deteriorating pyramidal cells The unitary D-spike was observed, only in twenty-two out of forty-eight pyramidal cells. Whereas the cells showing unitary D-spikes were kept in a good state (spike height, mv; steady membrane potential, mv) for min, all the other cells (n = 26) remained in a good state ( mv; mv) only for 5-10 min. As a consequence, it was not possible in these cells to test in detail if they could exhibit the unitary D-spike. However, as in the case of the cells showing the unitary D-spike, in all of them it was similarly ascertained that full spikes produced by Schaffer stimulation arose directly from the baseline. During the process of deterioration, in nineteen out of the twenty-six cells Schaffer stimulation could induce a small spike ( mv) which sometimes triggered a large spike ( mv) (not illustrated). The small spike was ms in duration with one to three peaks. A shorter duration indicated less peaks. A small spike with only one peak, i.e. a monophasic spike, for example, had a duration of about 2 ms. The time to the first peak ranged from 0 45 to 0-78 ms. These peaks could be interpreted as the peaks of unitary D-spikes whose peak time was prolonged due to low membrane potential caused by injury. In fact, a prolongation of the peak time of the unitary D-spike was observed as the cell deteriorated (see above). Thus, it could be assumed that the small spike was a D-spike which was originally produced when the cell was in a good state, but became easily identifiable as the cell began to deteriorate because the large spike (presumably the soma spike) then tended to be inactivated. If this assumption is accepted, it follows that the unitary D-spike occurred at least in forty-one out of forty-eight cells. 347 DISCUSSION Dendritic origin of the unitary D-spike and its possible relationship to FPP (fast prepotential) The present investigation revealed that small spikes, referred to as unitary D- spikes (peak time, ms; amplitude, mv), could be produced in the pyramidal cell by apical dendritic activation. They were categorized into four classes according to the differences in peak time and amplitude. In addition there was at least one more unitary D-spike which was produced presumably at a greater distance away from the soma than the four unitary D-spikes. Only the dendrites could generate such multiple spikes of small amplitude which could be observed with the intrasomatically placed microelectrode.

14 348 Y'. FU,JITA Small spikes of presumably dendritic origin were observed in the chromatolysing motoneurone (Kuno & Llina's, 1970) and the immature pyramidal cell (Purpura, Prelevic & Santini, 1968). Therefore, a question might arise as to the possibility that an unhealthy state of the cell such as that caused by microelectrode penetration E A B a-. a-. d - 13 Fig. 7. A schema illustrating functional organization of a pyramidal cell. A full spike in the pyramidal cell exhibits several notches (A, arrows a, d, d', d") or only one notch (B, arrow a). S. soma of the pyramidal cell. IS, initial segment (Eccles, 1957). D, the base of the apical dendrite, /1l, /32, yl, y2: sites giving rise to unitary D-spikes. T, dendritic trigger zone (Spencer & Kandel, 1961). c, sites producing spikes which presumably contribute to formation of a large, slow depolarization (inactivation response) (Fujita & Iwasa, 1977). It and 12 are feedback inhibitory interneurones for the soma and apical dendrite, respectively. 13, feedforward inhibitory interneurone for the dendrite. E, excitatory input to the dendrite. For convenience, the recurrent inhibition on the dendrite is shown only on one half of its branches, and excitatory and feedforward inhibitory inputs only on the other half. See text for explanation. might have played a role in producing the unitary D-spike. However, all the pyramidal cells showing the unitary D-spikes were thought to be healthy for the following reasons. First, they exhibited reasonably large spikes and high steady membrane potentials (see above). Second, the rate of spontaneous discharges never exceeded 15/s except during the intrinsic burst. Third, in peak time the unitary D- spike was the shortest of all the small spikes so far described as being dendritic in origin (e.g. FPP). Accordingly, it seems unlikely that the unitary D-spike was derived from an unhealthy cell membrane. Finally, the unitary D-spike occurred with a definite time relationship to the population spike which could be observed without cell penetration (e.g. Fig. 4; Fujita & Sakata, 1962). The peak time of FPP was 0-5-1V0 ms with one exception and its amplitude ranged from mv (Spencer & Kandel, 1961). This makes a conspicuous contrast with the unitary D-spike, because the latter exhibited much less variations

15 IMPULSE CONDUCTION IN APICAL DENDRITE 349 in these parameters among different cells. In the case of one of the two main types of unitary D-spikes, for example, the peak time was {27 ms and the range of amplitudes was mv in twenty-two pyramidal cells. The large variations in peak time and amplitude of FPP among different cells could be accounted for if FPP was formed by an assembly of unitary D-spikes produced at various distances away from the soma. In fact, there was evidence indicating that summation of unitary D- spikes could result in formation of a spike having a longer peak time and larger amplitude (Fig. 4 G and H). It is of interest that one FPP had a peak time of 0-3 ms and an amplitude of 81 mv (Spencer & Kandel, 1961). This falls in the category of the unitary D-spike. Impulse conduction in the apical dendrite with a low safety factor In agreement with a previous report (Andersen & L0mo, 1966), activation of the apical dendrite through afferent fibres to its distal portion did not produce EPSPs which could be observed with the intrasomatically placed microelectrode. As a result, a full spike arose directly from the baseline. This means that the full spike results only from a successive spike generation along the apical dendrite. In fact, a full spike had, as a rule, several notches on its rising phase (e.g. Fig. IA). On the other hand, according to the present experiments, a single unitary D-spike was unable to trigger a full spike except in a few cases (see below). The main reason for this was that the A-spike was of high threshold and a depolarization of mv was usually required to trigger it (Fig. 4L). Even the largest of unitary D-spikes had an amplitude of only 13 5 mv. As a result, summation of many unitary D-spikes was necessary to overcome the high threshold. This indicates that the apical dendrite is of low safety factor for impulse transmission. It is of interest that there were two unitary D-spikes which tended to make a pair (Fig. 4,,1 and y). One was lower in threshold than the other. The former was of higher amplitude and longer peak time than the latter. The difference in amplitude was, however, only about 20 %. Thus their amplitudes were comparable. This suggests that both unitary D-spikes were produced at comparable distances from the recording site. That the unitary D-spike with a shorter peak time (y) was of lower amplitude and triggered by the unitary D-spike of longer peak time and higher amplitude (,/) suggests that the difference in peak time was not due to the difference in the distances from the recording site, but was due to the difference in the electrophysiological properties of the cell membranes giving rise to these unitary D- spikes. If it is accepted that the larger unitary D-spike occurred at a site closer to the recording microelectrode than the smaller one, it could be assumed that /l was produced in a portion of the shaft of the apical dendrite and y in the branch nearest to it, as schematically shown in Fig. 7. Since at least two pairs of /-y of different amplitudes could occur in the same pyramidal cell (Fig. 4C and I), it may be suggested that a chain of such pairs is produced along the apical dendrite (Fig. 7, /31 and yl, /32 and y2). If so, this becomes the very mechanism for impulse conduction along the apical dendrite. One pair alone, however, could not trigger a full spike. In order to trigger it, more unitary D-spikes had to be recruited so as to overcome the high threshold for the A-spike. It may be suggested that these additional unitary D-spikes were generated further away in the branch or in other branches near the one

16 350 Y. Fl rjita already firing. A chain of such events must have occurred all along the apical dendrite, inasmuch as no EPSPs were as a rule observed even when the stimulus strength was increased and the first impulse in response to Schaffer stimulation must have arisen somewhere in the distal portion of the apical dendrite. A corollary of these arguments is that the apical dendrite becomes functionally significant only if enough unitary D-spikes are recruited along it so as to ensure impulse transmission. It was first proposed in the cerebellar Purkinje cell that dendritic spikes summate to form a larger, longer-lasting potential so as to transmit impulses further forward (Fujita, 1968; compare Granit & Phillips, 1956). The same principle seems to be applicable to the apical dendrite, as discussed above. 7'he origin of the A-spike The A-spike of the pyramidal cell differed from the IS-spike of the spinal motoneurone (Eccles, 1957) in that the threshold could vary considerably in the same cell. Namely, as a rule, the A-spike was of high threshold and a depolarization of mv was needed to trigger it (e.g. Fig. 2D-F). Yet the threshold could become lower in the same cell due to unknown causes and the A-spike then arose directly from the baseline (Fig. 5B). The time from the foot of the spike to the notch denoting the peak of the A-spike was 0 34 ms. The peak time fell exactly in the category of the unitary D-spike. Accordingly, it is not possible to assume that transmission of impulses from the dendrite to the A-spike generating area was so rapid that no notches were discernible that distinguished the A-spike from the D-spike that triggered it. This means that it was the A-spike itself that arose directly from the baseline. In other words, the potential that triggered the A-spike was invisible with the intrasomatic microelectrode. This could be explained only if the A-spike originated at a site somewhere between the site of recording and that being stimulated. There seems to exist little doubt as to the former being the soma and the latter being the afferent fibres to the distal portion of the apical dendrite, because the locations of the tips of both recording and stimulating microelectrodes were checked electrophysiologically and histologically (see Methods). If these arguments are accepted, a possibility looms that the A-spike is also dendritic in origin. In this case, the most likely site of origin of the A-spike would be the base of the apical dendrite (Fig. 7D), for it was much higher in amplitude than the unitary D-spike. Further studies are needed to ascertain the origin of the A-spike. Does the unitary D-spike subserve normal functions? The unitary D-spike was produced by a synchronized stimulation which made many pyramidal cells fire in synchrony. Such a synchronized excitation of pyramidal cells could spontaneously occur in the kindled hippocampus, but not in the normal hippocampus, and the excitation was followed by a large hyperpolarization (Fujita & Sakuranaga, 1981; Fujita, Harada, Takeuchi, Sato & Minami, 1983; Fujita, Sato, Takeuchi & Minami, 1983). In this respect, it is of interest to note that even in the seizure-infested human hippocampus inhibitory interneurones are probably well preserved (Babb, Pretorius, Kupfer & Grandall, 1989). Thus the spontaneously occurring synchronized excitation in the kindled hippocampus was essentially the duplicate of the stimulus-induced synchronized excitation in the normal hip-

17 IMPUlL>SE CONDUCTION IL APICAL DENDRITE pocampus. Conversely, any responses, including the unitary D-spike, produced by a synchronized stimulation could be abnormal. In fact the D-spike (unitary D-spike) took part in frequency potentiation which was somehow related to the genesis of seizure discharges (Green & Adey, 1956; also see above). A close relationship between seizure discharges and dendritic spikes has also been reported (Green & Petsche, 1961; Purpura, McMurtry, Leonard & Milliani, 1966). It appears therefore that during seizure discharges the low safety factor of the apical dendrite is somehow compensated for and vigorous activation of pyramidal cells ensues through summation of unitary D-spikes. Yet there is a possibility that the unitary D-spike might subserve normal functions. Namely, FPP could occur spontaneously (Spencer & Kandel, 1961); two kinds of small fast spikes of possible origins in the dendrites were produced by high-frequency stimulation of the sciatic nerve (Fujita & Iwasa, 1977). These small spikes and FPP could well have been formed by unitary D-spikes. If the above arguments are accepted, it becomes of interest that the human hippocampus seems to serve two 'masters', so-to-speak, i.e. epilepsy (e.g. Babb et al. 1989) and memory (e.g. Zola-Morgan, Squire & Amaral, 1986). As shown in the present experiments, the safety factor for impulse conduction in the apical dendrite is low. How the safety factor is increased under normal conditions is unknown at present. A possibility may be that it is increased by 'learning', because there is a report that chronic stimulation of a dorsal root enhances dendritic activities of the motoneurone (Fujita, Harada, Kitamura, Minami & Sato, 1987). Through learning plastic changes might be induced in the apical dendritic membrane, akin to the potentiation which could occur postsynaptically and independently from presynaptic changes in the case of long-term potentiation (E-S potentiation) (Taube & Schwartzkroin, 1988 a, b). Conversely, the low safety factor could offer the very room for plastic changes to occur in the apical dendrite. I am greatly indebted to Mrs Masayo Saito for her co-operation in the present investigation including data processing and histological work. I am also grateful to MIrs Elizabeth Wilson for her advice on English usage. 351 REFERENCES ALGER, B. E. & NICOLL, R. A. (1982). Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studied in vitro. Journal of Physiology 328, ANDERSEN, P. (1960). Apical dendritic activation of CAI neurons. Acta Physiologica Scandinavica 48, ANDERSEN, P., ECCLES, J. C. & LoYNING, Y. (1964a). Location of postsynaptic inhibitory synapses on hippocampal pyramids. Journal of Neurophysiology 27, ANDERSEN, P., ECcLES, J. C. & LoYNING, Y. (1964b). Pathway of postsynaptic inhibition in the hippocampus. Journal ofn.europhysiology 27, ANDERSEN, P. & LoMo, T. (1966). Mode of activation of hippocampal pyramidal cells by excitatory synapses on dendrites. Experimental Brain Research 2, BABB, T. L., PRETORIUS, J. K., KUPFER, W. R. & GRANDALL, P. H. (1989). Glutamate decarboxylase-immuno-reactive neurons are preserved in human epileptic hippocampus. Journal of Neuroscience 9, BLISS, T. V. P. & LoMo, T. (1973). Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. Journal of Physiology 232,

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