67
Brain Research, 111 (1976) 67-78 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
RETICULAR SUPPRESSION CORTEX NEURONS
OF
FLASH-EVOKED IPSPs
IN
VISUAL
V. G. SKREBITSKY and I. N. S H A R O N O V A
The Brain Institute, U.S.S.R. Academy of Medical Sciences, Moscow (U.S.S.R..) (Accepted December 2nd, 1975)
SUMMARY
In a majority of visual cortex (VC) neurons recorded intracellularly in chronically implanted rabbits, light flashes evoked responses consisting of protracted IPSPs (duration 160-200 msec) followed in some cases by a rebound. Inhibition of spontaneous activity through high frequency reticular stimulation (MRF) or through presentations of non-visual arousing stimuli, was not associated with strong hyperpolarization of the cell membrane. Reticular stimulation evoking both activation and inhibition of spontaneous neuronal activity elicited attenuation or complete elimination (disinhibition) of flash-evoked protracted IPSPs and of the postinhibitory rebounds. Rhythmic neuronal discharges elicited by stimulation of the visual pathway was also reduced during reticular activation, due to attenuation of the IPSP-rebound sequence.
INTRODUCTION
It is well known now that reticular activation leads to modulation of spontaneous and evoked cellular activity in different sensory systems. It has been shown in the visual system that transmission of information through the lateral geniculate body (LGB) is greatly enhanced during and after high frequency midbrain reticular stimulation6,7,21,2a,~7. Various non-visual arousing stimuli have similar effectsa,la, 14,1s. Changes in neuronal activity of the visual cortex (VC) during reticular and sensory arousal have also been described. They consist both of facilitation and inhibition of light evoked responses15,19,a0. Recently it has been shown that a reticular inhibition of internuncial cells in the LGB plays an important role in the modulation of visual transmission11. One may also point out that reticular stimulation induces the suppression of hyperpolarizing potentials in the LGB relay cells evoked by light stimuli and by electric stimulation
68 of the optic chiasm 27,z8. These data are in agreement with our previous observations showing the elimination of flash-evoked hyperpolarization along with diminution of' the slow negative wave at the VC surface of waking rabbits during arousing acoustic stimulation 29. This wave is associated with inhibition in the majority of flash-reacting neurons 17,a',35. In the present study we examined the changes of synaptic activity in visual cortex neurons during reticular activation. Some preliminary data have been published earlier ~1. METHODS
Experiments were carried out on 16 chronically implanted adult rabbits weighing about 3 kg. Several days before the experiments the scalp and the periosteum were removed under local anesthesia and the stimulating electrodes (bipolar pairs of glass-insulated stainless steel wires, tips 100/~m, separated by 0.5 mm) were stereotaxically 2G inserted into the midbrain reticular formation (MRF) and in some instances into the LGB. On the day of the experiment the animal was loosely fixed in a stand and the head placed in a special non-traumatic head-holder. A cranial opening (diameter about 1 mm) was made over the striate cortex 25 unilaterally and the exposed portion of the dura was removed. The trephine hole was made small enough to minimize cerebral pulsations. To reduce pulsations still more the hole was covered with paraffin after insertion of the microelectrode. As a rule the animal lay quietly and, judging from its spontaneous electrocortical activity (ECoG), was in a state of relaxed wakefulness (stages III and IV according to the classification of Mimura et al.22). Different arousing stimuli evoked an ECoG arousal. To drive the recording microelectrodes (glass micropipettes filled with 2 M potassium citrate) a head mounted hydraulic micromanipulator was used 4. The microelectrode resistances were usually in the range of from 50 to 100 Mr2. The surface ECoG was recorded in some experiments. Bright flashes (duration about 75/~sec) of a stroboscopic unit placed 50 cm in front of the rabbit's eyes served as visual stimuli. The animal's pupils were dilated with atropine. M R F stimulation (pulse duration 0.1-0.5 msec, frequency 100/sec) was of threshold intensity for pupillary reactions measured before atropinization and subthreshold for motor reactions. Various arousing stimuli (tones, clicks, whistles) were also used. During the experiment the animal was in dim light. After each experiment the rabbit was returned to its cage. One to four experiments were carried out on each rabbit. Histological control of the stimulating electrodes position was made after the experimental session. RESULTS
Flash-evoked I P S P s The activities of 76 flash-reacting neurons were analyzed. Twenty of the records were intracellular, with resting and spike potentials o f 30-60 mV; cell firing with an
69
A
B
1 50
C
I)
Fig. 1. Responses of 4 cells to light flashes (at arrows). In A and B the responses consist of plimary IPSPs followed by a prolonged inhibition without prominent hyperpolarization of cell membrane. In C and D, the protracted IPSPs reach their maximum at the end of the inhibitory periods. Upper trace in D: extracellular recording of the same cell before impalement. Notice correspondence b~tween time course of protracted IPSP and extracellular slow positive wave. Here and in other figures spikes were retouched. DC potentials in A, B, C and D: about 60, 45, 45 and 40 mV, respectively.
overshoot of several millivolts could sometimes be recorded. Fifty six of the records were 'quasi-intracellular '2° ('partially-intraceilular'a2). These records were characterized by a D C potential of 10-30 mV and by spike potentials of 10-25 mV. Recording time varied between several minutes and one hour. Many features of synaptic activity in chronic rabbits were the same as those previously described in anesthetized or immobilized rabbits 12. Light flashes elicited protracted hyperpolarizing potentials in 80 9/00of the neurons. It was shown earlier 87 that long-lasting inhibitions in visual cortex neurons evoked by stimulation of afferent pathways are true IPSPs. Therefore, we shall further designate these hyperpolarizing potentials as IPSPs although direct evidence, e.g., with measurements of the membrane resistance, are lacking in this study. In a few instances they were preceded by excitatory postsynaptic potentials (EPSPs) with latencies of 15-30 msec. Two types of protreacted IPSPs with distinct configurations were the most common. The first type was characterized by the primary component with the sharp slope of the initial falling phase. The latency of this component was 25-30 msec, the duration 20-30 msec and the amplitude 3-5 mV. It was followed by a long-lasting inhibition
70 (160-200 msec), during which, as a rule, the polarization of the cell membrane decreased. Figs. IA, B and 4 show typical examples of this type of IPSP. Several single EPSPs were often superimposed on the ascending phase of the primary component and sometimes led to cell discharge (Fig. 1A). The second type of protracted IPSP had a 'slow' initial phase and a gradual increase in polarization, reaching a maximum at the end of the inhibitory period. Two examples are provided by records C and D of Fig. 1. Unit C shows how the gradual increase in the amplitude of the flash-evoked IPSP is accompanied by the superposition of single IPSPs which strongly summated in the last period of inhibition. Protracted IPSPs exhibiting other time courses than those described above were also recorded in several units. For example, a relatively 'sharp' initial IPSP may be followed by a gradual increase in membrane polarization (Fig. 3B1). One may see in Fig. 3At,a and in Fig. 4 that in one cell the amplitudes and the durations of the protracted IPSPs are rather inconstant for several successive stimuli. These synaptic potential changes depend on the level of wakefulness and of alertness which constantly fluctuates in a waking animal. At the same time the membrane potential level and spike features were unchanged during the period analyzed, showing the stability of the recording conditions. The latency and duration of some of the protracted IPSPs were approximately the same as those of photically evoked, extracellularly recorded slow cortical wave (50-100 msec, and 120-200 msec, respectively), negative at the surface and positive in deep cortical layersal (Figs. I D and 5). After cessation of the protracted inhibition a rebound was often observed (Figs. 1C, D, 3 and 4). ' Non-specific inhibition'
It was shown earlier 3° that about 40 ~ of rabbit visual cortex neurons react to different non-visual arousing stimuli and to reticular stimulation. In approximately 7 5 ~ of these neurons spontaneous activity is inhibited ('non-specific inhibition'). There are some difficulties in recording the synaptic events during this kind of inhibition because it easily disappears when the frequency of neuronal firing increases in the course of cell impalement. In this respect 'non-specific inhibition' differs greatly from the flash-evoked inhibition. The latter is detectable in damaged cells even during high frequency discharge of deterioration. A few quasi-intracellular records of 'non-specific inhibition' (10 neurons) that we were able to obtain in the present study showed the absence of strong hyperpolarization and of protracted IPSPs. Fig. 2 demonstrates this 'non-specific inhibition' evoked by MRF stimulation in 4 cells. One may see that activity of unit A (Fig. 2A1,2) was characterized at the beginning of the record by the groups of spikes separated by the inhibitory periods (phasic discharge); during this time the animal was in a drowsy state (judging from its ECoG) and reticular stimulation led to replacement of the phasic discharge with high amplitude EPSPs and IPSPs by low amplitude synaptic activity not reaching the firing level.
71
u
ct
!t ~
-,-..,.,./,,u ~
,I.,' ~
t
tk ~ -.-.--- x - - , . . i ~ ~ _ . _
~ , v k . , . , . v . , . _
I
~
A
HU}!kjcu,,m !t!IH L1 :
'
tt i
h
!
'!
Ill ~
tl tll/iili
tlil l o,,v
~
120"'V' 250~sec
500~c 1
1
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ll0
Fig. 2. Inhibition of spontaneous activity in 4 ceils (A, B, C, and D) during stimulation of midbrain reticular formation (MRF) (interrupted line). Notice absence of strong hyperpolarization and of large protracted IPSPs during the inhibitory period. DC potentials in A, B, C and D: about 30, 20, 25, and 15 mV, respectively.
Inhibition of spontaneous activity without strong hyperpolarization of the cell membrane is also demonstrated in records B and D of Fig. 2. However, some repolarization did take place and manifested itself by restoration of the mechanism of spike generation when the latter was partially destroyed by cell deterioration (Fig. 2C).
Changes of IPSPs during reticular stimulation The general 'modulating' effect of reticular stimulation consisted of a diminution in the amplitude (and sometimes of the duration) of protracted IPSPs leading to their elimination (disinhibition). Modulation of flash-evoked IPSPs during M R F stimulation was observed in 43 of the 61 units exhibiting protracted inhibition in response to light flashes. Several examples are shown in Fig. 3. It is seen that during 100/sec reticular stimulation flash-evoked IPSPs are either attenuated (Fig. 3A3,4) or completely eliminated (Fig. 3AI,8, B). Duration of the M R F stimulation in these experiments was 1-5 sec. The changes of flash-evoked IPSPs were tested at different intervals after onset of the reticular stimulation. (Their maximum was inconstant and varied in different cases from several msec to 1 sec and more.) These changes of IPSPs represent one of the components of modulation of neuronal activity during arousal; its intensity depends on the intensity of arousal. Any sensory stimulus evoking E C o G arousal (sound, electrocutaneous stimulation)
72
A
2
40mY ¢
t
gOO ec
B 30mY
2
2oo.n .c, Fig. 3. Attenuation or suppression of flash-evoked protracted IPSPs during MRF stimulation in two cells (A and B). Light flashes at arrows. Left column: before MRF stimulation. Right column: during MRF stimulation. Reticular stimulation was switched on 1 sec before flash in A~; 1.3 sec in A4, and 1 sec in B2. DC potentials in A and B are about 40 mV. elicits at its first presentation the same changes of IPSPs as reticular stimulation (Fig. 45). But after several repetitions, when arousal has been extinguished, the sensory stimulus also fails to provoke modulation of synaptic activity. The suppression was not identical for the different inhibitory components of flash-evoked responses. A diminution in the amplitudes of short latency 'wellsynchronized' IPSPs was never observed. On the other hand, long-lasting IPSPs with various latencies were easily suppressed. As a rule, their suppression was correlated with diminution of the slow negative wave registered on the surface of the visual cortex 81. Fig. 5 shows the correspondence between the flash-evoked IPSPs and the slow negative waves. One may see that the flash-evoked negative-positive complex is followed by a slow afterdischarge having a negative component which is also correlated with inhibition of neuronal discharges. Arousing reticular stimulation led to the elimination of the protracted IPSPs and to an attenuation of both primary and secondary flash-evoked slow negative waves. After cessation of reticular stimulation both ]PSPs and the slow waves are restored. It is of interest that along with the diminution of flash-evoked IPSPs, the postinhibitory rebound was either diminished or even completely eliminated (Figs. 3, 4 and 5).
73
t
!
3
4
t
!
t
200msec i
,200m~
t
Fig. 4. Sound-induced disinhibition of the inhibitory responses to light flashes in one cell. l~,, 5 (left): flash-evoked protracted IPSPs; at 5 (right) sound was switched on 160 msec after flash. Light flashes at arrows, sound during horizontal line. Notice that sound completely suppresses both the inhibitory pause and postinhibitory rebound. DC potentials about 35 mV.
No dependence was found between the changes in spontaneous neuronal activity during reticular stimulation and the attenuation of flash-evoked IPSPs: the latter effect was observed when spontaneous discharges were inhibited, facilitated or remained unchanged. In some instances, light flashes or single shocks applied to LGB elicited phasic neuronal discharges consisting of IPSPs - - a rebound sequence. MRF stimulation resulted in 'disturbance' of this sequence owing to disinhibition of inhibitory periods and to elimination of postinhibitory rebound. Fig. 5 demonstrates that each flash (Fig. 61,3) and each shock (Fig. 68) evoked several protracted IPSPs followed by burst discharges. During MRF stimulation only the first IPSPs were distinguishable while the others were abolished. DISCUSSION
It has been shown in the present intracellular study that flash-evoked protracted IPSPs in the rabbit visual cortex are suppressed during high frequency reticular stimulation or during presentation of sensory arousing stimuli. The cells whose activities were recorded were not identified. One may suggest that they are the largest elements of the visual cortex, i.e., the pyramidal cells, although a proof for this statement is lacking.
74
o ta~
Fig. 5. Correspondence between neuronal and ECoG activities before and during MRF stimulation. Top: ECoG activity. Bottom: intracellular record of the same cell in 1 and 2. Records 2 were taken 10 sec after cessation of reticular stimulation. Light flashes at arrows, MRF stimulation at interrupted line. DC potential about 40 mV. The results r e p o r t e d a b o v e show t h a t in some cells the I P S P s consist o f two c o m p o n e n t s differentiated by their degree o f m e m b r a n e p o l a r i z a t i o n . T h e first c o m p o n e n t (latency 25-30 msec, d u r a t i o n 20-30 msec) is a c c o m p a n i e d by s t r o n g e r p o l a r i z a t i o n t h a n the second one (latency 40-80 msec, d u r a t i o n 160-200 msec). T h e
75
=-
I
-.i
I
?---
__-
~iJ
~
N
--.
Fig. 6. Modification of rhythmic neuronal discharges during MRF stimulation. Light flashes (1 and 2: at arrows) and single shocks applied to LGB (3: triangles) elicit several IPSPs followed by dis-
charges. During MRF stimulation (interrupted lines) only the first IPSPs are distinguishable. DC potential about 25 mV.
former may result from a greater synchronization of single IPSPs and possibly from a more efficient position of the involved inhibitory synapses on the somatodendritic membrane. Only the long latency component is suppressed during reticular stimulation whereas the short latency one remains unchanged.
76 Spontaneous activity of the neurons whose flash-evoked IPSPs are suppressed may be facilitated, inhibited ('non-specific inhibition') or unaltered by reticular stimulation or by various non-visual arousing stimuli. 'Non-specific inhibition' is not associated with strong hyperpolarization of the cell membrane. It disappears when the frequency of neuronal firing increases due to current injection or to damage of the cell by the microelectrode. The dependence of the responsiveness of the cortical neurons on the rate of spontaneous activity was noted earlier 5. Inhibition of neuronal firing without strong membrane hyperpolarization after reticular stimulation was also described in the cat motor cortex 16. Thus, one may assume that 'non-specific inhibition' is remote dendritic, or that it is due to disfacilitation of the cortical neurons during reticular activation. A suppression of optic radiation-evoked inhibition during 'non-specific inhibition' in VC neurons was described in cats 10. However, the opposite effect - - a facilitation of evoked inhibition during reticular-induced excitation of spontaneous activity noted in this paper - - was never observed in our experiments, possibly owing to the different conditions of the visual stimulation or to the species used. Suppression of protracted flash-evoked IPSPs could result from reticular inhibition of cortical inhibitory neurons. Besides, the events displayed at the cortical level could, to some extent, be determined at the thalamic level. In fact, it was demonstrated in rats and cats that reticular stimulation induces the suppression of inhibition in LGB relay c~lls 11,27. Similar effects were also manifested in other systems (for example, attenuation or elimination of IPSPs elicited in the ventrolateral thalamic neurons by brachium conjunctivum stimulation during brain stem reticular activation 24 and depression of the motor cortex inhibition during waking34). Slow thalamic and cortical waves suppressed during arousal are often considered as manifestation of feedback inhibition 1,9,36. Hence, it may be concluded that inhibition of the feedback inhibitory system is one of the mechanisms underlying arousal. It leads to the facilitation of sensory transmission which manifests itself at the thalamic level of the visual system in the increase of the visual receptive fields and in the improvement of the reactions in the directionally selective cells 21. Diminution of slow cortical waves along with attenuation of protracted IPSPs during the action of arousing stimuli was previously described and considered in terms of the animal's alertness 31. There is also the suggestion that the modulation of the LGB activity by mesencephalic stimulation is linked to the process of 'saccadic sampling '7. It is also of importance that suppression of flash-evoked 1PSPs is accompanied by attenuation or cessation of postinhibitory discharge. This finding confirms previous data of the abolition of postinhibitory rebound in the cat's ventrolateral thalamic nucleus during arousal a3. The role of rebound excitation in the phasing of the neuronal discharges in thalamic nuclei has been discussed previously 1,8. According to the proposed hypotheses, the burst response appearing in the hyperexcitable phase of postanodal excitation, would again excite the inhibitory neurons via the axon collaterals, and so terminate itself by the IPSPs so generated, and so on. From Eccles TMpoint of view the rhythmic cortical responses are secondary to the thalamic
77
discharges. However, there is some evidence that rhythmic discharges in the somatosensory and visual cortical areas may have an intracortical origin~,aL The pronounced IPSPs which are followed by spike discharges obtained in the present study also allow the possibility of participation of VC neurons in the generation of the IPSP-rebound sequences. In conclusion, the generation and suppression of this sequence may be mediated by processes taking place both at thalamic and cortical levels.
REFERENCES 1 Andersen, P., Brooks, C.McC., Eccles, J. C. and Sears, T. A., The ventro-basal nucleus of thalamus: potential fields, synaptic transmission and excitability of both pre ynaptic and postsynaptic components, J. Physiol. (Lond.), 174 (1965) 348-369. 2 Andersson, S. A., Intracellular postsynaptic potentials in the somatosensory cortex of cat, Nature (Lond.), 205 (1965) 297-298. 3 Arden, Y. and S/Sderberg, V., The transfer of optic information through the lateral geniculate body of the rabbit. In N. A. Rosenblith (Ed.), Sensory Communication, M.I.T. Press, New York, 1961, pp. 521-544. 4 Blaivas, A. S., Bomstein, O.Z. and Voronin, L. L., Oil micromanipulator for the microelectrode investigation of the neuronal activity, Sechenov physiol. J., U.S.S.R., 53 (1967) 1240-1242. 5 Buser, P. and Imbert, M., Sensory projections to the motor cortex in cats; microelectrode study. In W. A. Rosenblith and J. Willy (Eds.), Sensory Communication, M.I.T. Press, New York, 1961, pp. 607-626. 6 Coenen, A. M. L. and Vendrik, A. I. H., Determination of the transfer ratio of cat's geniculate neurons through quasi-intracellular recordings and the relation with the level of alertness, Exp. Brain Res., 14 (1972) 227-242. 7 Doty, R. W., Wilson P. D., Bartlett J. R. and Pecci-Saavedra, J., Mesencephalic control of lateral geniculate nucleus in primates. I. Electrophysiology, Exp. Brain Res., 18 (1973) 189-203. 8 Eccles, J. C., Inhibition in thalamic and cortical neurons and its role in phasing neuronal discharges, Epilepsia (Amst.), 6 (1965) 89-115. 9 Eccles, J. C., The Inhibitory Pathways of the Central Nervous System, The Sherrrington Lectures IX, Liverpool University Press, Liverpool, 1969. 10 Feeney, D. M. and Orem, J. M., Modulation of visual cortex inhibition during reticular evoked arousal, Physiol. Behav., 9 (1972) 805-808. 11 Fukuda, Y. an5 Iwama, K., Reticular inhibition of internuncial cells in the rat lateral geniculate body, Brain Research, 35 (1971) 107-118. 12 Fuster, J. M,, Creutzfeldt, O. D. and Straschill, M., IntraceUular recording of neuronal activity in the visual system, Z. vergl. Physiol., 49 (1965) 605-622. 13 Godfraind, J. M. et Meulders, M., Effets de la stimulation sensorielle somatique sur les champs visuels des neurones de la r6gion genouill6e chez le Chat anesth6si6 au chloralose, Exp. Brain Res., 9 (1969) 183-200. 14 Hotta, T. and Kameda, K., Interactions between somatic and visual or auditory responses in the thalamus of the cat, Exp. Neurol., 8 (1963) 1-13. 15 Jung, R., Kornhuber, H. H. and Da Fonseca J. S., Multisensoryconvergence on cortical neurons. In G. Moruzzi, A. Fessard and H. Jasper (Eds.), Brain Mechanisms, Progr. Brain Res., 1Iol. 1, Elsevier, Amsterdam, 1963, pp. 207-234. 16 Klee, M. R., Different effects on the membrane potential of motor cortex units after thalamic and reticular stimulation. In D. P. Purpura and M. D. Yahr (Eds.), The Thalamus, Columbia University Press, New York, 1966, pp. 287-322. 17 Kondratjeva, I. N. and Polyansky, V. B., Inhibition in the neuronal systems of the visual cortex, Activ. herr. sup. (Praha), 10 (1968) 1-11. 18 Kuman, E. A., and Skrebitsky, V. G., Interaction of visual and auditory afferents in the lateral geniculate body in rabbits, Parlor J. higher herr. Activ., 3 (1968) 507-512. 19 L/Smo,T. and Mollica, A., Activity of single units in the primary optic cortex in the unanesthetized
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20 21
22
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25 26 27 28
29 30 3l 32 33 34
35 36 37
rabbit during visual, acoustic, olfactory and painful stimulation, Arch. ital. Biol., 100 (1962) 86-120. Mcltvain, J. T., and Creutzfeldt, O. D., Microelectrode study of synaptic excitation and inhibition in the lateral geniculate nucleus of the cat, J. Neurophysiol., 30 (1967) 1 -21. Meulders, M. et Godfraind, J. M., Influence du r6veil d'origine r6ticulaire sur l'6tendue des champs visuels des neurones de la r6gion genouill6e chez le Chat avec cerveau intact ou avec cerveau isol6, Exp. Brain Res., 9 (1969) 201-220. Mimura, K., Sato, K., Kitajima, H., Ochi, N. and lshino, T., Photically evoked potentials in the visual cortex of rabbits in relation to various electroencephalographic stages, Brain Research, 5 (1967) 306-318. Ogawa, T., Midbrain reticular influences upon single neurons in lateral geniculate nucleus, Science, 139 (1963) 343-344. Purpura, D. P., McMurtry, J. G. and Maekawa, K., Synaptic events in ventrolateral thalamic neurons during suppression of recruiting responses by brain stem reticular stimulation, Brain Research, 1 (1966)63-76. Rose, M., Cytoarchitektonischer Atlas der Grosshirnrinde des Kaninchens, J. Psyehol. Neurol. (Lpz.), 43 (1931) 353-440. Sawyer, C. H., Everett, J. W. and Green, T. D., The rabbit diencephalon in stereotaxic coordinates, J. comp. Neural., 101 (1954) 801-824. Singer, W., The effect of mesencephalic reticular stimulation on intracellular potentials of cat lateral geniculate neurons, Brain Research, 61 (1973) 35-54. Singer, W., and Dr/iger, U., Postsynaptic potentials in relay neurons of cat lateral geniculate nucleus after stimulation of the mesencephalic reticular formation, Brain Research, 41 (1972) 214-220. Skrebitsky, V. G., Auditory suppression of the postsynaptic inhibition in photically evoked responses, Parlor J. higher herr. Aetiv., 17 (1967) 158-160. Skrebitsky, V. G. Nonspecific influences on neuronal firing in the central visual pathway, Exp. Brain Res., 9 (1969) 269-283. Skrebitsky, V. G., Postsynaptic inhibition in photically evoked responses and its change during distraction of the animal, Brain Research, 14 (1969) 510-512. Skrebitsky, V. G. and Voronin, L. L., Intracellular records of the single unit activity of the visual cortex in nonanaesthetized rabbits, Parlor J. higher herr. Activ., 16 (1966) 864-873. Steriade, M., Apostol, V. and Oakson, G., Control of unitary activities in cerebellothalamic pathway during wakefulness and synchronized sleep, J. Neurophysiol., 34 (1971) 389-413. Steriade, M. and Deschenes, M., Inhibitory processes and interneuronal apparatus in motor cortex during sleep and waking II. Recurrent and afferent inhibition of pyramidal tract neurons, J. Neurophysiol., 37 (1974) 1093-1113. Supin, A. Ya., Some mechanisms of rhythmical activity in the cerebral cortex, Parlor J. higher nerv. Activ., 17 (1957) 287-294. Supin, A. Ya., On the mechanisms of reticulo-cortical activation, Sechenovphysiol. J. U.S.S.R., 54 (1968) 893-898. Watanabe. S., Konichi, M. and Creutzfeldt, O., Postsynaptic p~tentials in the cat's visual cortex following electrical stimulation of afferent pathways, Exp. BrJin Res., 1 (1966) 272-283.
Brain Research, 111 (1976) 67-78 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
RETICULAR SUPPRESSION CORTEX NEURONS
OF
FLASH-EVOKED IPSPs
IN
VISUAL
V. G. SKREBITSKY and I. N. S H A R O N O V A
The Brain Institute, U.S.S.R. Academy of Medical Sciences, Moscow (U.S.S.R..) (Accepted December 2nd, 1975)
SUMMARY
In a majority of visual cortex (VC) neurons recorded intracellularly in chronically implanted rabbits, light flashes evoked responses consisting of protracted IPSPs (duration 160-200 msec) followed in some cases by a rebound. Inhibition of spontaneous activity through high frequency reticular stimulation (MRF) or through presentations of non-visual arousing stimuli, was not associated with strong hyperpolarization of the cell membrane. Reticular stimulation evoking both activation and inhibition of spontaneous neuronal activity elicited attenuation or complete elimination (disinhibition) of flash-evoked protracted IPSPs and of the postinhibitory rebounds. Rhythmic neuronal discharges elicited by stimulation of the visual pathway was also reduced during reticular activation, due to attenuation of the IPSP-rebound sequence.
INTRODUCTION
It is well known now that reticular activation leads to modulation of spontaneous and evoked cellular activity in different sensory systems. It has been shown in the visual system that transmission of information through the lateral geniculate body (LGB) is greatly enhanced during and after high frequency midbrain reticular stimulation6,7,21,2a,~7. Various non-visual arousing stimuli have similar effectsa,la, 14,1s. Changes in neuronal activity of the visual cortex (VC) during reticular and sensory arousal have also been described. They consist both of facilitation and inhibition of light evoked responses15,19,a0. Recently it has been shown that a reticular inhibition of internuncial cells in the LGB plays an important role in the modulation of visual transmission11. One may also point out that reticular stimulation induces the suppression of hyperpolarizing potentials in the LGB relay cells evoked by light stimuli and by electric stimulation
68 of the optic chiasm 27,z8. These data are in agreement with our previous observations showing the elimination of flash-evoked hyperpolarization along with diminution of' the slow negative wave at the VC surface of waking rabbits during arousing acoustic stimulation 29. This wave is associated with inhibition in the majority of flash-reacting neurons 17,a',35. In the present study we examined the changes of synaptic activity in visual cortex neurons during reticular activation. Some preliminary data have been published earlier ~1. METHODS
Experiments were carried out on 16 chronically implanted adult rabbits weighing about 3 kg. Several days before the experiments the scalp and the periosteum were removed under local anesthesia and the stimulating electrodes (bipolar pairs of glass-insulated stainless steel wires, tips 100/~m, separated by 0.5 mm) were stereotaxically 2G inserted into the midbrain reticular formation (MRF) and in some instances into the LGB. On the day of the experiment the animal was loosely fixed in a stand and the head placed in a special non-traumatic head-holder. A cranial opening (diameter about 1 mm) was made over the striate cortex 25 unilaterally and the exposed portion of the dura was removed. The trephine hole was made small enough to minimize cerebral pulsations. To reduce pulsations still more the hole was covered with paraffin after insertion of the microelectrode. As a rule the animal lay quietly and, judging from its spontaneous electrocortical activity (ECoG), was in a state of relaxed wakefulness (stages III and IV according to the classification of Mimura et al.22). Different arousing stimuli evoked an ECoG arousal. To drive the recording microelectrodes (glass micropipettes filled with 2 M potassium citrate) a head mounted hydraulic micromanipulator was used 4. The microelectrode resistances were usually in the range of from 50 to 100 Mr2. The surface ECoG was recorded in some experiments. Bright flashes (duration about 75/~sec) of a stroboscopic unit placed 50 cm in front of the rabbit's eyes served as visual stimuli. The animal's pupils were dilated with atropine. M R F stimulation (pulse duration 0.1-0.5 msec, frequency 100/sec) was of threshold intensity for pupillary reactions measured before atropinization and subthreshold for motor reactions. Various arousing stimuli (tones, clicks, whistles) were also used. During the experiment the animal was in dim light. After each experiment the rabbit was returned to its cage. One to four experiments were carried out on each rabbit. Histological control of the stimulating electrodes position was made after the experimental session. RESULTS
Flash-evoked I P S P s The activities of 76 flash-reacting neurons were analyzed. Twenty of the records were intracellular, with resting and spike potentials o f 30-60 mV; cell firing with an
69
A
B
1 50
C
I)
Fig. 1. Responses of 4 cells to light flashes (at arrows). In A and B the responses consist of plimary IPSPs followed by a prolonged inhibition without prominent hyperpolarization of cell membrane. In C and D, the protracted IPSPs reach their maximum at the end of the inhibitory periods. Upper trace in D: extracellular recording of the same cell before impalement. Notice correspondence b~tween time course of protracted IPSP and extracellular slow positive wave. Here and in other figures spikes were retouched. DC potentials in A, B, C and D: about 60, 45, 45 and 40 mV, respectively.
overshoot of several millivolts could sometimes be recorded. Fifty six of the records were 'quasi-intracellular '2° ('partially-intraceilular'a2). These records were characterized by a D C potential of 10-30 mV and by spike potentials of 10-25 mV. Recording time varied between several minutes and one hour. Many features of synaptic activity in chronic rabbits were the same as those previously described in anesthetized or immobilized rabbits 12. Light flashes elicited protracted hyperpolarizing potentials in 80 9/00of the neurons. It was shown earlier 87 that long-lasting inhibitions in visual cortex neurons evoked by stimulation of afferent pathways are true IPSPs. Therefore, we shall further designate these hyperpolarizing potentials as IPSPs although direct evidence, e.g., with measurements of the membrane resistance, are lacking in this study. In a few instances they were preceded by excitatory postsynaptic potentials (EPSPs) with latencies of 15-30 msec. Two types of protreacted IPSPs with distinct configurations were the most common. The first type was characterized by the primary component with the sharp slope of the initial falling phase. The latency of this component was 25-30 msec, the duration 20-30 msec and the amplitude 3-5 mV. It was followed by a long-lasting inhibition
70 (160-200 msec), during which, as a rule, the polarization of the cell membrane decreased. Figs. IA, B and 4 show typical examples of this type of IPSP. Several single EPSPs were often superimposed on the ascending phase of the primary component and sometimes led to cell discharge (Fig. 1A). The second type of protracted IPSP had a 'slow' initial phase and a gradual increase in polarization, reaching a maximum at the end of the inhibitory period. Two examples are provided by records C and D of Fig. 1. Unit C shows how the gradual increase in the amplitude of the flash-evoked IPSP is accompanied by the superposition of single IPSPs which strongly summated in the last period of inhibition. Protracted IPSPs exhibiting other time courses than those described above were also recorded in several units. For example, a relatively 'sharp' initial IPSP may be followed by a gradual increase in membrane polarization (Fig. 3B1). One may see in Fig. 3At,a and in Fig. 4 that in one cell the amplitudes and the durations of the protracted IPSPs are rather inconstant for several successive stimuli. These synaptic potential changes depend on the level of wakefulness and of alertness which constantly fluctuates in a waking animal. At the same time the membrane potential level and spike features were unchanged during the period analyzed, showing the stability of the recording conditions. The latency and duration of some of the protracted IPSPs were approximately the same as those of photically evoked, extracellularly recorded slow cortical wave (50-100 msec, and 120-200 msec, respectively), negative at the surface and positive in deep cortical layersal (Figs. I D and 5). After cessation of the protracted inhibition a rebound was often observed (Figs. 1C, D, 3 and 4). ' Non-specific inhibition'
It was shown earlier 3° that about 40 ~ of rabbit visual cortex neurons react to different non-visual arousing stimuli and to reticular stimulation. In approximately 7 5 ~ of these neurons spontaneous activity is inhibited ('non-specific inhibition'). There are some difficulties in recording the synaptic events during this kind of inhibition because it easily disappears when the frequency of neuronal firing increases in the course of cell impalement. In this respect 'non-specific inhibition' differs greatly from the flash-evoked inhibition. The latter is detectable in damaged cells even during high frequency discharge of deterioration. A few quasi-intracellular records of 'non-specific inhibition' (10 neurons) that we were able to obtain in the present study showed the absence of strong hyperpolarization and of protracted IPSPs. Fig. 2 demonstrates this 'non-specific inhibition' evoked by MRF stimulation in 4 cells. One may see that activity of unit A (Fig. 2A1,2) was characterized at the beginning of the record by the groups of spikes separated by the inhibitory periods (phasic discharge); during this time the animal was in a drowsy state (judging from its ECoG) and reticular stimulation led to replacement of the phasic discharge with high amplitude EPSPs and IPSPs by low amplitude synaptic activity not reaching the firing level.
71
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ct
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Fig. 2. Inhibition of spontaneous activity in 4 ceils (A, B, C, and D) during stimulation of midbrain reticular formation (MRF) (interrupted line). Notice absence of strong hyperpolarization and of large protracted IPSPs during the inhibitory period. DC potentials in A, B, C and D: about 30, 20, 25, and 15 mV, respectively.
Inhibition of spontaneous activity without strong hyperpolarization of the cell membrane is also demonstrated in records B and D of Fig. 2. However, some repolarization did take place and manifested itself by restoration of the mechanism of spike generation when the latter was partially destroyed by cell deterioration (Fig. 2C).
Changes of IPSPs during reticular stimulation The general 'modulating' effect of reticular stimulation consisted of a diminution in the amplitude (and sometimes of the duration) of protracted IPSPs leading to their elimination (disinhibition). Modulation of flash-evoked IPSPs during M R F stimulation was observed in 43 of the 61 units exhibiting protracted inhibition in response to light flashes. Several examples are shown in Fig. 3. It is seen that during 100/sec reticular stimulation flash-evoked IPSPs are either attenuated (Fig. 3A3,4) or completely eliminated (Fig. 3AI,8, B). Duration of the M R F stimulation in these experiments was 1-5 sec. The changes of flash-evoked IPSPs were tested at different intervals after onset of the reticular stimulation. (Their maximum was inconstant and varied in different cases from several msec to 1 sec and more.) These changes of IPSPs represent one of the components of modulation of neuronal activity during arousal; its intensity depends on the intensity of arousal. Any sensory stimulus evoking E C o G arousal (sound, electrocutaneous stimulation)
72
A
2
40mY ¢
t
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B 30mY
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2oo.n .c, Fig. 3. Attenuation or suppression of flash-evoked protracted IPSPs during MRF stimulation in two cells (A and B). Light flashes at arrows. Left column: before MRF stimulation. Right column: during MRF stimulation. Reticular stimulation was switched on 1 sec before flash in A~; 1.3 sec in A4, and 1 sec in B2. DC potentials in A and B are about 40 mV. elicits at its first presentation the same changes of IPSPs as reticular stimulation (Fig. 45). But after several repetitions, when arousal has been extinguished, the sensory stimulus also fails to provoke modulation of synaptic activity. The suppression was not identical for the different inhibitory components of flash-evoked responses. A diminution in the amplitudes of short latency 'wellsynchronized' IPSPs was never observed. On the other hand, long-lasting IPSPs with various latencies were easily suppressed. As a rule, their suppression was correlated with diminution of the slow negative wave registered on the surface of the visual cortex 81. Fig. 5 shows the correspondence between the flash-evoked IPSPs and the slow negative waves. One may see that the flash-evoked negative-positive complex is followed by a slow afterdischarge having a negative component which is also correlated with inhibition of neuronal discharges. Arousing reticular stimulation led to the elimination of the protracted IPSPs and to an attenuation of both primary and secondary flash-evoked slow negative waves. After cessation of reticular stimulation both ]PSPs and the slow waves are restored. It is of interest that along with the diminution of flash-evoked IPSPs, the postinhibitory rebound was either diminished or even completely eliminated (Figs. 3, 4 and 5).
73
t
!
3
4
t
!
t
200msec i
,200m~
t
Fig. 4. Sound-induced disinhibition of the inhibitory responses to light flashes in one cell. l~,, 5 (left): flash-evoked protracted IPSPs; at 5 (right) sound was switched on 160 msec after flash. Light flashes at arrows, sound during horizontal line. Notice that sound completely suppresses both the inhibitory pause and postinhibitory rebound. DC potentials about 35 mV.
No dependence was found between the changes in spontaneous neuronal activity during reticular stimulation and the attenuation of flash-evoked IPSPs: the latter effect was observed when spontaneous discharges were inhibited, facilitated or remained unchanged. In some instances, light flashes or single shocks applied to LGB elicited phasic neuronal discharges consisting of IPSPs - - a rebound sequence. MRF stimulation resulted in 'disturbance' of this sequence owing to disinhibition of inhibitory periods and to elimination of postinhibitory rebound. Fig. 5 demonstrates that each flash (Fig. 61,3) and each shock (Fig. 68) evoked several protracted IPSPs followed by burst discharges. During MRF stimulation only the first IPSPs were distinguishable while the others were abolished. DISCUSSION
It has been shown in the present intracellular study that flash-evoked protracted IPSPs in the rabbit visual cortex are suppressed during high frequency reticular stimulation or during presentation of sensory arousing stimuli. The cells whose activities were recorded were not identified. One may suggest that they are the largest elements of the visual cortex, i.e., the pyramidal cells, although a proof for this statement is lacking.
74
o ta~
Fig. 5. Correspondence between neuronal and ECoG activities before and during MRF stimulation. Top: ECoG activity. Bottom: intracellular record of the same cell in 1 and 2. Records 2 were taken 10 sec after cessation of reticular stimulation. Light flashes at arrows, MRF stimulation at interrupted line. DC potential about 40 mV. The results r e p o r t e d a b o v e show t h a t in some cells the I P S P s consist o f two c o m p o n e n t s differentiated by their degree o f m e m b r a n e p o l a r i z a t i o n . T h e first c o m p o n e n t (latency 25-30 msec, d u r a t i o n 20-30 msec) is a c c o m p a n i e d by s t r o n g e r p o l a r i z a t i o n t h a n the second one (latency 40-80 msec, d u r a t i o n 160-200 msec). T h e
75
=-
I
-.i
I
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~iJ
~
N
--.
Fig. 6. Modification of rhythmic neuronal discharges during MRF stimulation. Light flashes (1 and 2: at arrows) and single shocks applied to LGB (3: triangles) elicit several IPSPs followed by dis-
charges. During MRF stimulation (interrupted lines) only the first IPSPs are distinguishable. DC potential about 25 mV.
former may result from a greater synchronization of single IPSPs and possibly from a more efficient position of the involved inhibitory synapses on the somatodendritic membrane. Only the long latency component is suppressed during reticular stimulation whereas the short latency one remains unchanged.
76 Spontaneous activity of the neurons whose flash-evoked IPSPs are suppressed may be facilitated, inhibited ('non-specific inhibition') or unaltered by reticular stimulation or by various non-visual arousing stimuli. 'Non-specific inhibition' is not associated with strong hyperpolarization of the cell membrane. It disappears when the frequency of neuronal firing increases due to current injection or to damage of the cell by the microelectrode. The dependence of the responsiveness of the cortical neurons on the rate of spontaneous activity was noted earlier 5. Inhibition of neuronal firing without strong membrane hyperpolarization after reticular stimulation was also described in the cat motor cortex 16. Thus, one may assume that 'non-specific inhibition' is remote dendritic, or that it is due to disfacilitation of the cortical neurons during reticular activation. A suppression of optic radiation-evoked inhibition during 'non-specific inhibition' in VC neurons was described in cats 10. However, the opposite effect - - a facilitation of evoked inhibition during reticular-induced excitation of spontaneous activity noted in this paper - - was never observed in our experiments, possibly owing to the different conditions of the visual stimulation or to the species used. Suppression of protracted flash-evoked IPSPs could result from reticular inhibition of cortical inhibitory neurons. Besides, the events displayed at the cortical level could, to some extent, be determined at the thalamic level. In fact, it was demonstrated in rats and cats that reticular stimulation induces the suppression of inhibition in LGB relay c~lls 11,27. Similar effects were also manifested in other systems (for example, attenuation or elimination of IPSPs elicited in the ventrolateral thalamic neurons by brachium conjunctivum stimulation during brain stem reticular activation 24 and depression of the motor cortex inhibition during waking34). Slow thalamic and cortical waves suppressed during arousal are often considered as manifestation of feedback inhibition 1,9,36. Hence, it may be concluded that inhibition of the feedback inhibitory system is one of the mechanisms underlying arousal. It leads to the facilitation of sensory transmission which manifests itself at the thalamic level of the visual system in the increase of the visual receptive fields and in the improvement of the reactions in the directionally selective cells 21. Diminution of slow cortical waves along with attenuation of protracted IPSPs during the action of arousing stimuli was previously described and considered in terms of the animal's alertness 31. There is also the suggestion that the modulation of the LGB activity by mesencephalic stimulation is linked to the process of 'saccadic sampling '7. It is also of importance that suppression of flash-evoked 1PSPs is accompanied by attenuation or cessation of postinhibitory discharge. This finding confirms previous data of the abolition of postinhibitory rebound in the cat's ventrolateral thalamic nucleus during arousal a3. The role of rebound excitation in the phasing of the neuronal discharges in thalamic nuclei has been discussed previously 1,8. According to the proposed hypotheses, the burst response appearing in the hyperexcitable phase of postanodal excitation, would again excite the inhibitory neurons via the axon collaterals, and so terminate itself by the IPSPs so generated, and so on. From Eccles TMpoint of view the rhythmic cortical responses are secondary to the thalamic
77
discharges. However, there is some evidence that rhythmic discharges in the somatosensory and visual cortical areas may have an intracortical origin~,aL The pronounced IPSPs which are followed by spike discharges obtained in the present study also allow the possibility of participation of VC neurons in the generation of the IPSP-rebound sequences. In conclusion, the generation and suppression of this sequence may be mediated by processes taking place both at thalamic and cortical levels.
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