INCREASE IN THE EFFICACY OF SYNAPSES BETWEEN PARALLEL FIBERS AND PURKINJE CELLS AFTER JOINT STIMULATION OF CLIMBING AND PARALLEL FIBERS IN THE FROG V. L. Dunin-Barkovskii, N. M. Zhukovskaya,

N. P. Larionova, L. M. Chailakhyan, and L. I. Chudakov

UDC 612.827:611.817.1

A study was made of the susceptibility of Purkinje cells to long-term plasticity changes produced by joint stimulation of two inputs: the parallel and the climbing fibers. Experiments were conducted on a preparation of isolated frog cerebellum, joined to the medulla by one peduncle. A total of 18 neurons were investigated which showed a monosynaptic response to stimulation of the parallel fibers and maintained stable background activity over a 2 h period. Curves were plotted throughout this time for the likelihood of a reaction occurring in Purkinje cells in response to stimulation of the parallel fibers. Level of current required to stimulate a Purkinje cell firing index of 0.5 (I0.5) was calculated. Neurons in which compound response to the "climber" type had been produced by stimulating the medulla showed a 10.s of 0.7 (less than one unit) at the start and finish of experiments, which would suggest an increase in the efficacy of the synapses of parallel fibers in Purkinje cells when parallel and climbing fibers are stimulated simultaneously.

INTRODUCTION It has already been established [ii] that the cerebellar cortex has two fundamentally different afferent inputs, namely the mossy and climbing fibers (MF and CF respectively). The former produce an excitatory effect on Purkinje (PC) and other cells of the cerebellum via granular cells and their axons (parallel fibers - PF), while the latter exert a powerful excitatory action on the PC direct. Marr put forward a hypothesis in 1969 regarding plasticity changes occurring in PF--PC synapses. He postulated that joint activity of the CF and PF at work on a single PC produces an increase in the efficacy of the synaptic action of active PF and PC. Recent research has indicated that changes take place in the efficacy of PF--PC synapses after simultaneous activation of different afferent inputs into the cerebellum [8, 9]. This article describes research into the plasticity properties of PF synapses on PC in the frog cerebellum in vitro. Earlier articles on the same subject have already been published [2, 5]. METHODS Experiments were conducted on isolated preparations of the cerebellum of Rana temporaria, joined to the medulla by one peduncle. The preparation was placed in an experimental chamber containing Ringer's solution, composed as follows (mM): NaCI: 114, KCI: 1.8, CaCI: 2.0, and NaHCO3; 4; dextrose: 2 g/liter, sa£uratedwithcarbogen (O2+4.5%CO2); pHT.3. Temperature was 15-17QC. The preparation could remain alive for up to 24 h in these conditions. Intracellular recordings were made of the background and evoked activity of PC. Glass micropipets filled with a 2 M NaCl solution and measuring i-2 ~m at the tip, with a resistance of 20-30 M~ were used as recording electrodes (R) and 20-40 Dm nichrome wires in glass insulation as stimulating electrodes (Sl and $2). Stimulation was carried out using squarewave current pulses (I0-~-i0 -5 A, 100 ~sec). Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 2, pp. 156-164, March-April, 1987. Original article submitted March 14, 1986.

0090-2977/87/1902-0119512.50

~ 1987 Plenum Publishing Corporation

119

Electrode S l which served to stimulate the PF, was placed on the surface of the molecular layer to the contralateral side of the cerebellum (in relation to the test cell - see Fig. la and b) and electrode S 2 on the medulla, at sites traversed by the CF, according to Hacket [6]. This electrode was capable of excitating the CF, the MF, and PC axons. Stimulation of the PF (by electrode S I) consisted of a train of stimuli at an intensity fluctuating between threshold and twice or thrice threshold level to produce monosynaptic response in the PC. This range could be divided into 5-11 equal steps. The cell was stimulated by a current at each intensity, starting with the lowest, repeated 3-10 times at a frequency of 0.3-0.5 Hz. A 20-sec interval separated each current application from its successor of raised intensity. We named this technique for stimulating PF via electrode $I procedure A. Procedure C~ consisted of stimulating PC via electrode S 2 using a current 20100 times above threshold intensity at the rate of 0.1-0.5 Hz, while simultaneous stimulation performed via both these electrodes (at stimulation parameters similar to those just mentioned) was known as procedure C 2. Conditioning could be produced with both procedures C I and C 2, namely simultaneous excitation of the PF and the CF; the PP populations activated by these two procedures were different. The trial consisted of a series of successive procedures: A-~]I-A-C2-A. Duration of procedure A measured 3-6 min and the interval between separate procedures lasted 10-15 min. Data were processed from experiments of no less than 2 h duration. During procedure A, neuronal background activity was consistently recorded at the intervals occurring between electrical stimuli of different intensities. Activity evoked in PC by electrical stimulation at each of the intensities used during A-procedure was identified by firing index (v), i.e., the ratio between the number of stimuli evoking monosynaptic response in PC (see below) and total number of stimuli. Each procedure A thus enabled us to ascertain the relationship between PC firing index and intensity of stimulation. An approximation curve was plotted for these experimentally obtained relationships (a broken line consisting of three portions: horizontal sections corresponding to values of v = 0 at less intense and v = 1.0 at more intense currents, together with a sloping line joining these horizontal segments). Stimulus intensity corresponding to the point where this broken line transected the straight line v = 0.5 was designated as I0. 5. In order to evaluate the changes occurring in the efficacy of PC activation via the PF, graphs were plotted during the course of the experimental procedures showing the probability of PC response occurring while these procedures were carried out. The value I0. 5 at the end in relation to the same parameter at the start of the experiment (i.e., I0. 5 beg and I0. s end) was also calculated. The plasticity properties of one PC only were investigated in each preparation. RESULTS When the PF were stimulated via electrode Sl, an excitatory wave spread laterally from this electrode (along the PF fibers) and could be recorded throughout the entire extent of the cerebellum in the form of parallel fiber field potentials (PFFP). Amplitude of the latter depended on stimulus intensity (see Fig. ic). The cerebellar strip from which PFPP were recorded with $I in a fixed position measured 50-100 ~m in width. Readings were taken of background and evoked activity in individual cells and action potentials (AP) distinguishable from background noise. Neurons were selected showing monosynaptic excitation in response to PF stimulation at a certain starting current intensity, i.eo, those with AP arising "on the tail" of PFPP with short and barely fluctuating latencies (see Fig. ic). Only PC activity displays these features and gives steady recordings over a prolonged period in the isolated frog cerebellum. Once the stimulating current was increased, PC response was elicited by all the stimuli presented (Fig. ic, series 2 and 3). We took the lowest stimulation intensity at which the neuron produced a monosynaptic response to all stimuli presentations as the saturation threshold. When stimuli were applied at electrode S 2, three types of response were observed in different cells: standard PC complex discharge ("climber" type discharge) - a set of 2-6 impulses, occurring with fixed latency and conforming to the "all or none" principle, the first and subsequent impulses being of standard and (generally) reduced types respectively, the latter usually taking a different form from the former, with an interspike interval which did not vary between responses of one unit or within one response of a single unit (see Fig. Ig,

120

b a

b

C

:,--.-.~,~ -4

C

¢,,.~.,,~__._.:..,.,.~.,_,--.__ d

d

Fig. i

Fig. 2

Fig. i. Diagram showing preparation and sample patterns of response in Purkinje cells to stimulation of afferent inputs; a) location of recording (R) and stimulating (S I and S2) electrons in the preparation; C: cerebellum; M; medulla; b) sketch of afferent inputs to Purkinje cells (PC) and stimulation of the PC; PCA: Purkinje cell axons; GC: granular cells; MF, CF, and PF; mossy, climbing, and parallel fibers respectively; c) monosynaptic response in PC to stimulation of the PF using three graded current intensities (I, 1.25, and 1.5 times threshold level for 1-3 respectively). Progressive growth is observed in the amplitude of PF field potentials (arrowed). Calibration: 3 mV, I0 msec; d) PC response to bulbar stimulation; series I: "climbing," 2: "Climber-type" complex discharges, and 3: response to MF stimulation. Calibration: 1 mV, 20 msec. Fig. 2. Oscillograms of Pirkinje cell (PC) response, generating clear-cut "climbing" discharges ("+" neurons) to bulbar and parallel fiber (PF) stimulation and (a-c); graphs of latency plotted against current intensity (d) and changing PC firing index on the course of experimentation (e); a) PC response to bulbar stimulation at abovethreshold intensity; b) PC response to PF stimulation by currents of graded intensities (0.25, 0.75, and 1.25 times threshold level for series 1-3 respectively at the start of trials; c) as for b, but at end of trials, after completing conditioning procedure. Calibration: i mV, I0 msec; d) abscissa; times min; ordinate: likelihood of PC response to PF stimulation. Curves with unfilled circles indicate changes in the firing index (v) at different intensities (stimulus intensity shown for each curve, in relative units); minimum stimulation strength required for monosynaptic PC response to occur after PF stimulation at the start of trials taken as unity. Curve with filled circles represents changing mean firing index (v) at all stimulus intensities. Arrows mark point of carrying out conditioning procedures C I and C 2.

121

a

b

I ,t~,----. 2vv-

d

_./

msec

I? ~

0,5

I,o

J,5

2o

2,5

~o

Fig. 3. As for Fig. 2, but applies to Purkinje cells (PC) generating "climber-type" complexes in response to bulbar stimulating (i.e., "+" neuron). First peak on oscillograms under (a): antidromic PC response to bulbar stimulation; b and c) wave of potentials produced by summated excitation of PC arrowed. Calibration: 1 mV, 10 msec.

series i). In the case of complex responses, interspike intervals, amplitude, and latencies changed following an arbitrary pattern (see Fig. id, series 2). Finally, a few (1-4) AP were also observed, which followed the same pattern as single impulses - apparently representing the pattern of PC response to stimulating the MF (Fig. id, series 3). This study presents experimental findings obtained from 18 PC. All these neurons were divided into three groups depending on the pattern of PC response to stimulation via electrode S 2, The first group comprised "+" neurons (Fig. id, series 1 and Fig. 2a) - eight units in all; the second was composed of "!" cells (Fig. id, series 2 and 3a) - 7 units, and the third consisted of "-" neurons (Fig. id, series 3 and 4a) three units. Figures 2-4 show findings from trials on three groups of neurons. The following changes occurred in the activity of PC during trials on a sample "+" neuron (Fig. 2). A "climber" firing pattern could be clearly discerned in PC response to bulbar stimulation (see Fig. 2a). Comparison response pre- and post-conditioning revealed that saturation threshold was reached at lower current intensities following conditioning (see Fig. 2b, series 1-3 and c, 1-3), whereupon latency decreased in length from 6-7 to 3-4 msec (frame d). Once the conditioning procedure had been carried out, overall probability of monosynaptic PC response increased in procedure A (marked by a thick continuous line in Fig. 2e). Saturation threshold decreased substantially once conditioning had been completed and remained at the reduced level for 1.5 h (see Fig. 2e). Excitation threshold fell approximately threefold, as emerges very clearly from Fig. 2d. Only slight and unstable alterations (whether upwards or downwards) in the efficacy of PF at different stimulus intensities were observed during trials involving "!" and "-" neurons. Mean value of v remained virtually static (Fig. 3e and 4e). No changes were observed in PC latencies during the A procedure (see Fig. 3d and 4d). One curious fact emerges from comparing Fig. 3b and 3c; although the test unit showed no rise in the efficacy of stimulation after conditioning, this parameter did increase in many other PC in this preparation; the amplitude of field potentials produced by summated excitation of the PC (arrowed) did increase considerably after conditioning. Table 1 shows findings on effectiveness of PC activation via the PF at the start and finish of experiments in the 18 test neurons figuring in the table. It will be seen that the mean level of I0.send/I0 sbeg for "+" neurons equals 0.70, as against 0.94 for "+" neurons and 0.99 for "-" cells• It may be seen by comparing these measurements than in instances where the occurrence of a "climber" response pattern could not be doubted, concurrent stimu-

122

a

c

-1

_2 b

_3 C

tZJ3.'67./1,0"% 7

..R

"

Fig. 4

.~

,

.

Fig. 5

Fig. 4. As for Fig. 2, but applies to Purkinje cells responding to bulbar stimulation consisting of separate impulses (or "-" neuron). Fig. 5. Plasticity changes occurring in the background and evoked activity of Pirkinje cells (PC) in various preparations; a) spontaneous "climber', complex discharge before (tracing i) and after (2) conditioning; b) changes in evoked and "climber" complex discharges during the course of procedure C I (on left): i) Ist, 2) 20th, and 3) 40th response to bulbar stimulation at suprathreshold intensity and at the rate of 5 Hz, together with evoked "climber" complex discharges after completion of C i and C= procedures (to right). Arrows mark additional peaks (fully explained in text); c) response evoked in PC by stimulating the parallel fibers before carrying out conditioning procedures (series !), after procedure C I (2), and procedure C 2 (3). Constant stimulus intensity. Calibration: 1 mV, I0 msec.

lation of the CF PC via the PFo

and the PF produced an evident rise in the effectiveness of activating the

In addition to the described plastic change resulting from conditioning, there was a change in the form of the PC response to stimulation of the CF and PF. Figure 5a illustrates spontaneous firing occurring in the same PC before (i) and after (2) carrying out procedures, C I and C 2. In the case of the latter, extra peaks could be discerned within the discharge. The same was true of evoked potentials (see Fig. 5b). Changes in response pattern were already occurring concurrently with procedure CI, with an increase in the number of spikes per complex discharge (see Fig. 5b, left, series 1-3), as well as the occurrence of peaks, clearly distinguishable once procedures Cl and C 2 had been carried out (b, series 2, arrowed). F~gure 5c gives oscil!ograms for the response of "+" neurons before conditioning and after procedures C I and C 2. The relationship between the current intensities producing a firing index of 0.5 in this neuron at the start and finish of trials equaled one. The response elicited in PC by presentation of a stimulus of the sane (initial) intensity consisted of 2 instead of 1 spike following procedure C2, however. This change could indicate that the efficacy of PF-PC synapses after conditioning by procedure C 2 continues to rise.

123

TABLE i. Changes in the Efficacy of Purkinje Cell Stimulation due to Conditioning |

l

Cell group INo. of cells Durationof trial, rain ¢-+-, e::h* ¢--~

8 7

3

140--305 120--240

140--160

I IO-5end/Io-5beg !

0,38-- 1,00 (0,70) 0,58-- l,l 4 (0,94)

0,96--1,04 (0,99)

Note: Mean Io.send/I0.sbeg for each group given in brackets. Student's t-test between pairs of groups: for "+" and "-": p < 0.i; for "+" and "±": p < 0.05; for "+" and the combined "+-" group: p < 0.02, and for "~" and "-": p > 0.5. DISCUSSION It has thus been shown that eight out of 18 test PC (namely, "+" cells - see Table l) showed a clear-cut and consistent "climber" peak in response to bulbar stimulation. The level of PF stimulating current required to produce monosynaptic response fell noticeably after joint PF and CF stimulation in seven of these neurons, while the mean value of the ratio I0.send/I0.bbeg for "+" neurons equaled 0.7. This finding could be attributed to one of the following reasons: either changes were occurring in the effectiveness of PF-PC synapses, or else excitation was rising in the PF, or alternatively the PC threshold was decreasing. The two last-mentioned reasons appear unlikely since, as stated earlier, the rate of PC background activity and amplitude of PFFP remained steady throughout the trial at a fixed stimulus intensity. Irregular, complex responses occurred in the seven "~" cells amongst the 18 neurons tested following stimulation of bulbar sites traversed by CF. Mean value of the ratio I0.bend/I0.bbeg approached unity in these neurons, at 0.94. Latency of response also remained unchanged in length in these neurons. It might be presumed that these neurons undergo no significant plasticity changes once conditioning has been carried out. Lack of effect on "~" neurons can probably be explained by the discrepancy between "climber" and irregular, complex PC activity. Irregular, complex PC response appears to result from extracellular recording of dendritic slow waves, of which intracellular recordings had been made during studies on guinea pig cerebellar slices [12] and on PC of isolated frog cerebellum [i]. We ourselves, like Hackett and Cochran [6], and only succeeded in obtaining clear"climber" recordings from preparations containing the medulla. A reorganization could be occurring in PC activity, bound up with the absence of some trophic CF factor which determines "normal" PC activity, in the preparations excluding the medulla (and thus excluding the cells where CF originate) [9]. As shown by our findings, a distinction must be made between true "climbing" and "climber type" PC discharges in interpreting experimental data. Llinas's hypothesis [ii] that CF are present in the vestibular nerve was put forward as a result of indistinct differentiation between short response latencies with complex patterns produced by stimulating the vestibular nerve. This hypothesis afterwards failed to find support [14]. In the case of "-" neurons (see Table i), the value of the ratio I0 send/10.sbeR equals 0.99. No plasticity changes are apparent and this tallies with the absence of "climber" response in these cells. It may therefore be deduced that the efficacy of PF-PC synapses may increase following joint stimulation of the PF and the CF. This finding provides a concrete confirmation of Marr's hypothesis in its original form [13]. A number of studies have appeared in recent years [8, 9] presenting findings thought to confirm that Albus's version [4] of Marr's basic hypothesis holds true of the cerebellar cortex, namely, that the efficacy of PF-PC synapses diminishes following simultaneous CF and PF activity. The possibility remains that the discrepancy between the latter findings and our own could be due to a possible increase or reduction in the efficacy of PF-PC synapses depending on the functional state of the organism as a whole, or on the animal species or, alternatively, peculiarities of individual PC. This view

124

seems all the more likely, in that type of cerebellar plasticity (Marr's or Albus's variant) could well be associated with the relative efficacy of inhibitory connections in the cerebella, cortex [3, 7]. The frog cerebellum differs from that of mammalian species by the virtual absence of inhibitory interneurons [9], which could be responsible for divergence in the plasticity profile of cerebellar neurons in the frog (see our own findings), in the rabbit [9], and in the monkey [8]. One other possible reason for the disparate changes in the efficacy of PF synapses d~ring combined PF and CF fiber activity should be mentioned. The character of this effect can apparently be determined by the integral action exerted on PC by CF activation. Findings are available on how, despite the powerful excitatory action exerted by the CF on the PC, the excitatory effect is confined to a 2-5 msec time spell in mammals, followed by an inhibitory period lasting approximately 50-100 msec [9]. In the frog, however, according to our own findings (Figs. 2a and 5a and b) and those contained in the literature [6], an excitatory effect can predominate in CF action on PC. It should be noted that increased efficacy of PF influence was not noted immediately after conditioning processes during our experiments. It will be seen from Fig. 2e that the rise in the effectiveness of stimulation took place after a considerable delay (of about 40 min). None of the findings described to date enable us to make deductions on the mechanisms underlying the plasticity changes observed. We can only postulate that the changed efficacy of PF synapses onto the PC is mediated by alterations in the postsynaptic dendrites [9]. This would be supported by the pattern of change in complex PC activity observed during conditioning namely, the appearance of additional dendritic peaks (Fig. 5b, series 2) and occurrence of a second spike in PC response to stimulation of the PF (bb, series 1 and c, series 3).

LITERATURE CITED i. 2.

3.

4. 5.

6. 7. 8. 9. 10.

Ii. 12. 13. 14.

V. L. Dunin-Barkovskii and A. L. Byzov, "Intracellular recordings of potentials in cells of the isolated frog cerebellum," Neirofiziologiya, 15, No. 4, 434-436 (1983). V. L. Dunin-Barkovskii, N. M. Zhukovskaya, N. P. Larionova, et el., "Efficacy of synapses of parallel fibers onto Purkinje cells following joint stimulation of climbing and parallel fibers," in: Proceedings of Seventh All-Union Conference on the Electrophysiology of the CNS, Izd. AN Arm. SSR (1980), pp. 281-282. V. L. Dunin-Barkovskii, N. P. Larionova, L. M. Chailakhyan, and L. Chudakov, "Simulated model of the process for noting information in Marr's cell in the cerebellar cortex," in: Current Views of Cerebellar Function [in Russian], Izd. AN Arm-SSR (1984), pp. 407-417. J. C. Albus, "'A theory of cerebellar function," Math. Biosci., i__O0,No. i, 25-61 (1971). L. M. Chajlkhian, L. I. Chudakov, W. L. Dunin-Barkowski, et al., "An analysis of the synaptic plasticity in the frog cerebellum in vitro," in: Synaptic Constituents in Health and Disease, M. Brzin, D. Sket, and H. Bachelard, eds., Mladinska knjiga, Pergamon Press, Ljublana, Oxford (1980). S. L. Cochran and J. T. Hackett, "The climbing fiber afferent system of the frog," Brain Res., 321, No. 2, 362-367 (1977). W. L. Dunin-Barkowski and N. P. Larionowa, "Computer simulation of a cerebellar cortex compartment," Biol. Cybern., 51, No. 5, 399-415 (1985). P. F. C. Gilbert and W. T. Thach, "Purkinje cell activity during motor learning," Brain Res., 128, No. 2, 309-328 (1977). M. Ito, The Cerebellum and Neural Control, Raven Press, New York (1984). M. Kano and M. Kato, "Specific glutamate sensitivity involved in the long-term depression of PF-PS transmission in rabbit cerebellar cortex," Neurosci. Lett., 2_~2, Suppl., p. 26 (1985). R. Llinas, J. R. Bloedel, and D. E. Hillman, "Functional characterization of neuronal circuitry of frog cerebellar cortex," J. Neurophysiol., 32, No. 5, 817-870 (1969). R. Llinas and M. Sugimori, "Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices," J. Physiol., 305, 197-214 (1980). D. Mar,, "A theory of cerebella, cortex," J. Physiol., 202, No. 2, 437-4~0 (1969)o C. Matesz, "Central projection of the VIII cranial nerves in the frog," Neuroscience, i, No. 12, 2661-2671 (1979).

125