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Long-term potentiation-induced synaptic changes in hippocampal dentate gyrus of rats with an inborn low or high learning capacity
Brain Research, 537 (1990) 293-297 Elsevier
BRES 24434
293
Short Communications
Long-term potentiation-induced synaptic changes in hippocampal dentate gyrus of rats with an inborn low or high learning capacity Ratil A. G6mez 1, Lucas D. Pozzo Miller 2'*, Augustin Aoki 2 and Oscar A. Ramirez 1 1Departamento de Farmacolog[a, Facultad de Ciencias Qu[micas, Universidad Nacional de C6rdoba, C6rdoba (Argentina) and ZCentro de Microscop[a Electr6nica, Facultad de Ciencias Mddicas, Universidad Nacional de C6rdoba, C6rdoba (Argentina)
(Accepted 4 September 1990) Key words: Learning capacity; Dentate gyrus; Synaptic change; Hippocampal slice; Long-term potentiation
Structural changes induced by high frequency (I-IF) stimulation on granule cells of the hippocampal dentate gyrns of rats with inborn high (HP) and low (LP) learning capacity was examined in this study. Two types of axodendritic synapses were processed morphometrically; the axonal bouton on dendritic shafts and those making contact through dendritic spines. The occurrence of granule cell spine synapses on LP rats before tetanus is less than the one in control animals. However, after long-term potentiation (LTP) produced by I-IF stimulation the number of spine synapses decreased, as happened in the control rats. Moreover, the number of granule cells shaft synapses counted on LP rats, before tetanus is less than the one on control rats, and after LTP this number increased greatly. There was no change in the number of granule cell shaft synapses in HP rats before and after LTP. LP rats which did not show LTP in spite of I-IF stimulation did not show changes in the number of spine and shaft synapses. From these results we can conclude that: (1) the great hippocampal synaptic plasticity observed in HP rats compared to LP rats can be attributed, at least in part, to the increased number of spine and shaft synapses observed in HP rats; (2) the minor number of spine and shaft synapses in LP rats may account for the impossibility to obtain LTP after I-IF stimulation in these animals. Long-term potentiation (LTP) is characterized by a long-lasting (hours-days) increase in synaptic efficacy produced by high-frequency afferent stimulation to either the hippocampal granule or a pyramidal cell 6'7. This p h e n o m e n o n has been proposed as a model for studies of the SNC plasticity 1'19'21'28 and it is also a useful model to test learning and m e m o r y 4. A number of studies suggested that the primary site of LTP is postsynaptic 5"2°, and several postsynaptic ultrastructural correlates of LTP were described s,9,1s. There is also some evidence of a presynaptic contribution to hippocampal LTP 2,5,H. Different lines of evidence suggested a relationship between hipp0campal synaptic plasticity and learning 2225,32. Previous results from this laboratory showed a good correlation between the hippocampal synaptic plasticity and the performance in a shuttle box avoidance paradigm 29. Moreover, rats with an inborn low performance in a shock-induced escape behavior revealed a greater threshold to generate LTP in comparison to rats with an inborn high performance response in a shuttle box avoidance paradigm 3°. In the present investigation, a morphometric synapse analysis on the hippocampal granule cell was carried out at an ultrastructural level in both H P and LP rats under basal conditions, and the
structural changes observed before and after LTP on granule cell synapses were related with the different hippocampal synaptic plasticity exhibited in H P and LP rats 30. Male albino rats, 6 - 9 weeks old, were used. They were descendants of H P or LP rats. These strains were inbred for many generations at the time of this study a°. High performance rats were defined as those that made 70% or more conditioned responses (CRs) while LP rats were those with a 15% or less CRs in the shuttle box avoidance paradigm 12. Electrophysiological experiments were conducted in a hippocampal slice preparation in vitro, as essentially described by Y a m a m o t o et al. 33. To prevent circadian variations and non-specific stress 1° the animals were killed with a blow to the neck between 11.00 and 12.00 h. Transverse hippocampal slices of approximately 400/~m thick were placed in a recording chamber perfused with a standard salt solution 33 saturated with 95% 0 2 and 5% C O 2. The perfusion rate was 2 - 3 ml/min; the temperature of the bathing solution was 30 °C. U n d e r visual control, a stimulating electrode made of two twisted wires was placed in the perforant path (PP). This electrode was insulated except for the cut end (diameter
* Present address: Departments of Neurology and Neuroseiences, School of Medicine, E-604, Case Western Reserve University, 2119 Abington Road, Cleveland, 44106 OH, U.S.A. Correspondence: O.A. Ramfrez, Departamento de Farmacologia, Facultad de Cieneias Qufmicas, Universidad Nacional de C6rdoba, Sueursal 16, C.C. 61, 5016 C6rdoba, Argentina. 0006-8993/90/$03.50 ~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
294 50 Mm). A recording microelectrode made with a micropipette (tip 10-20 Mm in diameter) was inserted in the dentate granule cell body layer. Evoked-field potentials were conventionally amplified and displayed in an oscilloscope for monitoring and recording. In response to the stimuli, 10 averaged field potentials, were sampled at 0.2 Hz on line using a PC microcomputer and the data stored on diskette for further analysis. Tetani, consisting of a
PRE TETANUS
train of pulses (0.5 ms) of 1 s duration, were delivered to the slices at 200 Hz. The intensity of stimuli was kept constant at approximately 50% above threshold. LTP was produced when the amplitude of the evoked population spike (PS, Fig. 1) recorded 20 min after the tetanus had increased at least 30%. Slices of the hippocampus from each electrophysiological treatment were picked up from the medium with a
POSTTETANUS
Fig. L Upper panel: photographs showing responses on granule cell layer of the hippocampus dentate gyrus evoked by stimulation to the perforant path. Left panel: responses recorded with stimuli delivered at 0.2 Hz. Right panel: the response from the same animals after a train of high frequency stimulation (PS, population spike). Calibration: 360/~V, 4 mS. Lower panel: micrographs of synapses from control animal (left) and from LTP animals (right) showing an overall depletion and shift of remaining yfsiclfs toward the,active zone during LTP; 36,000x.
295 Pasteur pipette and fixed in 1.5% glutaraldehyde, 1.5% formaldehyde and 0.1 M phosphate buffer (pH 7.2) 15. The tissue was diced around 2 m m from the recording electrode insertion position in the dentate gyms. This block was rinsed twice in a buffer and then treated with 2% osmium tetroxide for 2 h, stained in block with 2% uranylacetate, washed, dehydrated in a series of cold acetones and embedded in Araldite. The blocks were cut serially in 1/~m thick sections and stained with 1% Toluidine blue in 1% sodium borate for localization at light microscopic level of the molecular layer of the dentate gyrus; the tissue adjacent to the recording electrode position was excluded. For electronmicroscopy thin sections of the outer and middle level of the molecular layer about 80 nm in thickness were obtained in a Jeol JUM-7 ultramicrotome. To avoid duplicate counting of the same structure only one large section per 200 mesh copper grid was mounted, stained with lead citate and uranyl acetate and recorded photographically at 4000x magnification in a Siemens Elmiskop 101 electron microscope operating at 80 kV. Each electron micrograph covered an area of 225/~m 2 avoiding capillaries, glial and granular cell somas. Ten electron micrographs were randomly taken from each grid covering an area of 2250/./m 2 from each hippocampal slice. Two types of axodendritic synapses were processed morphometrically: the axonal boutons on dendritic shafts and those making contact through dendritic spines s. The mean values of 3 rats for each electrophysiological treatment were taken as independent observations. Fig. 1 shows a typical example of field potential response recorded in the granule cell body layer of the dentate gyrus evoked by stimuli (0.2 Hz) given to the P E The upper right panel shows the increased amplitude of the population spike after an effective tetanus (1 s train of 200 Hz). After LTP treatment, PS amplitude was increased by 200% on average (range: 106 + 30%, n = 3) when measured 20 min after the stimulus train. Electron micrographs of synapses in slices from control rats which showed LTP exhibited a consistent migration of synaptic vesicles toward the synaptic active zone during LTP; this finding is in accordance with earlier results 3. (Fig. 1, lower right panel.) Blind morphometric analysis revealed that the numbers of spine synapses in normal and H P rats were higher than that of LP rats (Fig. 2), The occurrence of shaft synapses per 225 ~ m 2 counted on granule cell layer of hippocampal dentate gyms of LP rats are less compared to control rats (Table I). Normal rats in which was obtained LTP after H F stimulation exhibited a significant reduction in the number of spine synapses of dentate gyrus (Fig. 2). O n the other hand, the increment in the number of shaft
Im50
[
] PRE- TETANUS POST-TETANUS
1.35'
~.I.20 ~" 1.05 UJ
~ 90 Z >u) I,I Z
75
u_ 0
ne 45 uJ m
g
Y////A
30
z
15 CONTROL
HP
LP
RATS
Fig. 2. Change in number of spine synapses per 225 # m 2 counted on granule cell layer of the hippocampal gyrus dentatus from control, HP and LP rats before and after LTP. All data are expressed as means + S.E.M. (n = 3 animals). Significance level was revealed by two-way analysis of variance (ANOVA) F1,17= 9.831 (P < 0.01) and F2,17 = 16.400 (P < 0.01). Post-hoe comparisons were performed using the Fisher test (P < 0.05). * Significantly different from control pretetanus (P < 0.05). ** Significantly different from control and HP rats pretetanus (P < 0.05). *** Significantly different from LP rats pretetanus (P < 0.05).
synapses on these animals was not statistically different (Table I). In H P rats after LTP there were no changes in the n u m b e r of both spine (Fig. 2) and shaft (Table I) synapses. A significant increase in the n u m b e r of shaft synapses was observed in LP rats with LTP after H F stimulation (Table I); concomitantly, a reduction in the number of spine synapses was observed under similar conditions (Fig. 2). However, in LP rats in which LTP was not detected no changes in the numbers of both spine (67 + 1.41 vs 69 + 2.82) and shaft (29.33 _+ 4.2 vs 34 + 2.44) synapses were found. Several lines of evidence strongly support the relevant
TABLE I Effects of LTP on number of shaft synapses per 225 ion 2 counted on granule cell layer of the hippocampal gyrus dentatus from control, HP and L P rats before and after HFstimulation
All data are expressed as means _+S.E.M. (n = 3 animals).
Control HP LP
Pretetanus
Post-tetanus
37.66 + 4 35.60 + 1.7 28.00 + 1.8+
41.00 + 6.2 33.60 + 4.2 40.33 + 5.71"
* Significantly different from LP rats (pretetanus); P < 0.05 Student's t-test. + Significantly different from control; P < 0.05 one-way ANOVA F2,s = 5.131 posthoc comparisons Fisher test P < 0.05.
296 not increase their basal number of dendritic shafts although the H F stimulation was delivered to PP. This observation may explain the impossibility to generate LTP on some LP rats. The fact that H P rats showed no increase in the number of synapses on dendritic shaft on the hippocampal dentate gyrus after LTP is a paradoxal effect, since it has been described as one of the structural changes subserving LTP 17'1s. Other pre- and postsynaptic mechanisms, such as increase in glutamate release 11, changes in the number or sensitivity of N receptor 2° membrane rearrangement leading to formation of new synapses 9 increase of Ca levels 16, and increments in the concentration of spectrin break-down product 23 may account for this effect. Moreover, unpublished observations (Ramirez et al., unpublished results) from this laboratory demonstrated that H P rats are less sensitive to inhibitory effect of 2-amino-5-phosphonovalerate, compared to LP rats, on blockade of LTP generation. The ultrastructural changes observed in H P or L P rats in the hippocampal dentate gyrus synapses, pre- or posttetanus can be correlated with the different hippocampal synaptic plasticity described on these rats 3°. The very high frequency of stimulation necessary to induce LTP and the lack of response in some LP rats may be due to the low number of spine and shaft synapses observed on the hippocampal dentate gyms of LP rats. We cannot rule out that other pre- and postsynaptic mechanisms subserved the different hippocampal synaptic plasticity observed on H P and LP rats.
participation of hippocampus in learning and memory processes 4'24'32. LP rats revealed different kinds of alterations on the hippocampal physiology 13'14'3°. More recently it has been reported that the inborn learning ability of H P and LP rats can be closely related with the plasticity of hippocampal synaptic transmission, as evidenced by the difference in threshold for LTP induction 3°. Lee et al. 17'18 using in vitro and in vivo methods demonstrated an increase in the number of synapses on dendritic shafts and a reduction in dendritic spines, the length of post-synaptic thickenings on dendritic spines, and the width of spine stalks, after LTP in hippocampal subfield CA1. These authors suggested that these structural changes could subserve LTP. In the present investigation LP rats displayed a lower n u m b e r of spine and shaft synapses with respect to H P and normal rats. LP rats also showed decreased numbers of hippocampus dentate gyms spine synapses in comparison to normal or H P rats after H F stimulation (200 Hz) to P E The decreased number of spine synapses found in hippocampal dentate gyrus of LP rats may account for the high threshold necessary to induce LTP and the impossibility to obtain it, in spite of the supramaximal stimulation frequency applied, on some LP rats 3°. No differences occurred in the number of synapses on dendritic shafts after LTP on hippocampal dentate gyrus of H P rats compared to control (Table I). O n the other hand L P rats exhibited a decreased number of synapses on dendritic shaft on the hippocampus dentate gyrus before tetanus (Table I). The LP rats that showed LTP after H F stimulation (200 Hz) were able to increase the n u m b e r of synapses on dendritic shaft on dentate gyrus. These structural changes can be interpreted as necessary to induce LTP on the hippocampal dentate gyrus of LP rats. LP rats which did not show LTP after tetanus could
This study was supported by grants from the C43nsejoNacional de Investigaciones Cientfficas y T6cnicas (CONICET) of Argentina to O.A.R. and from the Rockefeller Foundation, New York to A.A. The authors wish to thank Ms. Mercedes Guevara for technical assistance. Emma M. G~lvez for secretarial assistance.
1 Alger, B.E. and Teyler, T.J., Long-term and short-term plasticity in the CA1, CA3 and dentate region of the rat hippocampal slice, Brain Research, 110 (1976) 463-480. 2 Andersen, E, Avoli, M. and Hvalby, O., Evidence for both preand postsynaptic mechanisms during long-term potentiation in hippocampal slices, Exp. Brain Res., (Suppl.) 9 (1984) 315-324. 3 Applegate, M.D., Kerr, D.S. and Landfield, P.W., Redistribution of synaptic vesicles during long-term potentiation in the hippocampus, Brain Research, 401 (1987) 401-406. 4 Berger, T.W., Long-term potentiation of hippocampai synaptic transmission affects rate of behavioral learning, Science, 224 (1984) 627-630. 5 Bliss, T.V.P. and Dolphin, A.C., Where is locus of long-term potentiation? In G. Lynch, J.L. McGaugh and M.N. Weinberg (Eds.), Neurobiology of Learning and Memory, New York, 1984, pp. 451-458. 6 Bliss, T.V.P. and Garner-Medwin, A.R., Long-lasting potentiation of synaptic transmission in the dentate area of the unanaesthetized rabbit following stimulation of the perforant path, J. Physiol., 232 (1973) 357-374. 7 Bliss, T.V.E and Lomo, T., Longqasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit
following stimulation of perforant path, J. Physiol., 232 (1973) 331-356. Chang, EL.E and Greenough, W.T., Transient and enduring morphological correlates of synaptic activity and efficacy change in the rat hippocampal slice, Brain Research, 309 0984) 35-46. Desmond, N.L and Levy, W.B., Synaptic correlates of associative potentiation-depression: an ultrastructurai study in the hippocampus, Brain Research, 265 (1983) 21-30. Di Scenna, P., Method and myth in maintaining brain slices. In A. Schurr, T.J. Teyler and M.T. Tscng (Eds.), Brain Slices: Fundamentals, Applications and Implications, Karger, Basel, 1987, pp. 10-21. Dolphin, A.C., Errington, M.D. and Bliss, T.V.P., Long-term potentiation of perforant path in vivo associated with increased glutamate release, Nature, 297 (1982) 496--498. Evagelista, A.M. and Izquierdo, I., The effect of pre- and post-trial amphetamine injections on avoidance responses of rats, Psychopharmacolo$ia, 23 (1972) 42-47. Izqulerdo, I. and Orsingher, O.A., A physiological difference in the hippocampus of rats with a low inborn learning ability, Psychopharmacologia, 23 (1972) 386-396. Izqulerdo, I., Orsingher, O.A. and Ogura, O.A., Hippocampal
8 9 10
11 12 13 14
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15 16 17
18 19 20 21 22
23 24
facifitation and RNA build-up in response to stimulation in rats with a low inborn learning ability, Behav. Biol., 7 (1972) 699-707. Karnovsky, M.J., A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microsopy, J. Cell Biol., 27 (1965) 137 (Abstr.). Kuhnt, U., Mihaly, A. and Joo, E, Increased binding of calcium in the hippocampal slice during long-term potentiation, Neurosci. Lett., 53 (1985) 149-154. Lee, K.S., Oliver, M., Schottler, E and Lynch, G., Electron microscopic studies of brain slices: the effects of high frequency stimulation on dendritic ultrastructure. In: G. Kerkut and H.V. Wheal (Eds.), Electrical Activity in Isolated Mammalian C.N.S. Preparations, Academic Press, New York, 1981, pp. 189-212. Lee, K.S., Shottler, E, Oliver, M. and Lynch, G., Brief bursts of high-frequency stimulation produce two types of structural change in rat hippocampus, J. Neurophysiol., 44 (1980) 247-258. Levy, W.B. and Steward, O., Synapses as associative memory elements in the hippoeampal formation, Brain Research, 175 (1979) 233-245. Lynch, G. and Bandry, M., The biochemistry of memory: a new and specific hypothesis, Science, 224 (1984) 1057-1063. Lynch, G., Dunwiddie, T. and Gribkoff, V., Heterosynaptic depression: a post-synaptic correlate of long-term potentiation, Nature, 266 (1977) 736-737. Lynch, G., Gall, C. and Dunwiddie, T.V., Neuroplasticity in the hippocampal formation. In M.A. Coner (Ed.), Maturation of Nervous System: Progress in Brain Research, Vol. 48, Elsevier, Amsterdam, 1978, pp. 113-128. Lynch, G., Muller, D., Seubert, P. and Larson, J., Long-term potentiation: persisting problems and recent results, Brain Res. Bull., 21 (3) (1988) 363-372. Mamounas, L.A., Tbompsom, R.E, Lynch, G. and Baudry, M., Classical conditioning of the rabbit eyelid response increases
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26
27 28 29
30 31 32 33
glutamate receptor binding in hippocampai synaptic membranes, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 2548-2552. Morris, R.G.M., Anderson, E., Lynch, (3. and Bandry, M., Selective impairment of learning and blockade of long-term potentiation by an N-methyl-v-aspartate receptor antagonist AP5, Nature, 319 (1986) 774-776. Ott, T., Ruthrich, H., Reymann, K., Lindenau, L. and Matthies, H., Direct evidence for the participation of changes in synaptic efficacy in the development of behavioral plasticity. In C. Ajmone Marsan and H. Matthies (Eds.), Neuronal Plasticity and Memory Formation, Raven Press, New York, 1982, pp. 441-452. Pozzo Miller, L.D. and Aoki, A., Stereological analysis of the hypothaiamic ventromedial nucleus. I: Distribution of synapses, Com. Biol., 6 (4) (1988) 377-382. Racine, R.3., Milgram, N.W. and Harrier, S., Long-term potentiation phenomena in the rat in limbic forebrain, Brain Research, 260 (1983) 217-231. Ramfrez, O.A. and Carrer, H.E, Correlation between threshold to induce long-term potentiation in the hippocampus and performance in shuttle box avoidance response in rats, Neurosci. Lett., 104 (1,2) (1989) 152-156. Ramfrez, O.A., Orsingher, O.A. and Carter, H.E, Differential threshold for LTP in rats with high or low learning capacity, Neurosci. Lett., 92 (1988) 275-279. Tyler, J. and Di Scena, P., Long-term potentiation, Annu. Rev. Neurosci., 10 (1987) 131-161. Weisz, D.J., Clark, G.A. and Thompson, R., Increased responsivity of dentate granule cells during nictitating membrane response conditioning, Behav. Brain Res., 12 (1984) 145-154. Yamamoto, C., Matsumoto, K. and Takagi, M., Potentiation of excitatory postsynaptic potentials during and after repetitive stimulation in thin hippocampal sections, Exp. Brain Res., 38 (1980) 469-477.
BRES 24434
293
Short Communications
Long-term potentiation-induced synaptic changes in hippocampal dentate gyrus of rats with an inborn low or high learning capacity Ratil A. G6mez 1, Lucas D. Pozzo Miller 2'*, Augustin Aoki 2 and Oscar A. Ramirez 1 1Departamento de Farmacolog[a, Facultad de Ciencias Qu[micas, Universidad Nacional de C6rdoba, C6rdoba (Argentina) and ZCentro de Microscop[a Electr6nica, Facultad de Ciencias Mddicas, Universidad Nacional de C6rdoba, C6rdoba (Argentina)
(Accepted 4 September 1990) Key words: Learning capacity; Dentate gyrus; Synaptic change; Hippocampal slice; Long-term potentiation
Structural changes induced by high frequency (I-IF) stimulation on granule cells of the hippocampal dentate gyrns of rats with inborn high (HP) and low (LP) learning capacity was examined in this study. Two types of axodendritic synapses were processed morphometrically; the axonal bouton on dendritic shafts and those making contact through dendritic spines. The occurrence of granule cell spine synapses on LP rats before tetanus is less than the one in control animals. However, after long-term potentiation (LTP) produced by I-IF stimulation the number of spine synapses decreased, as happened in the control rats. Moreover, the number of granule cells shaft synapses counted on LP rats, before tetanus is less than the one on control rats, and after LTP this number increased greatly. There was no change in the number of granule cell shaft synapses in HP rats before and after LTP. LP rats which did not show LTP in spite of I-IF stimulation did not show changes in the number of spine and shaft synapses. From these results we can conclude that: (1) the great hippocampal synaptic plasticity observed in HP rats compared to LP rats can be attributed, at least in part, to the increased number of spine and shaft synapses observed in HP rats; (2) the minor number of spine and shaft synapses in LP rats may account for the impossibility to obtain LTP after I-IF stimulation in these animals. Long-term potentiation (LTP) is characterized by a long-lasting (hours-days) increase in synaptic efficacy produced by high-frequency afferent stimulation to either the hippocampal granule or a pyramidal cell 6'7. This p h e n o m e n o n has been proposed as a model for studies of the SNC plasticity 1'19'21'28 and it is also a useful model to test learning and m e m o r y 4. A number of studies suggested that the primary site of LTP is postsynaptic 5"2°, and several postsynaptic ultrastructural correlates of LTP were described s,9,1s. There is also some evidence of a presynaptic contribution to hippocampal LTP 2,5,H. Different lines of evidence suggested a relationship between hipp0campal synaptic plasticity and learning 2225,32. Previous results from this laboratory showed a good correlation between the hippocampal synaptic plasticity and the performance in a shuttle box avoidance paradigm 29. Moreover, rats with an inborn low performance in a shock-induced escape behavior revealed a greater threshold to generate LTP in comparison to rats with an inborn high performance response in a shuttle box avoidance paradigm 3°. In the present investigation, a morphometric synapse analysis on the hippocampal granule cell was carried out at an ultrastructural level in both H P and LP rats under basal conditions, and the
structural changes observed before and after LTP on granule cell synapses were related with the different hippocampal synaptic plasticity exhibited in H P and LP rats 30. Male albino rats, 6 - 9 weeks old, were used. They were descendants of H P or LP rats. These strains were inbred for many generations at the time of this study a°. High performance rats were defined as those that made 70% or more conditioned responses (CRs) while LP rats were those with a 15% or less CRs in the shuttle box avoidance paradigm 12. Electrophysiological experiments were conducted in a hippocampal slice preparation in vitro, as essentially described by Y a m a m o t o et al. 33. To prevent circadian variations and non-specific stress 1° the animals were killed with a blow to the neck between 11.00 and 12.00 h. Transverse hippocampal slices of approximately 400/~m thick were placed in a recording chamber perfused with a standard salt solution 33 saturated with 95% 0 2 and 5% C O 2. The perfusion rate was 2 - 3 ml/min; the temperature of the bathing solution was 30 °C. U n d e r visual control, a stimulating electrode made of two twisted wires was placed in the perforant path (PP). This electrode was insulated except for the cut end (diameter
* Present address: Departments of Neurology and Neuroseiences, School of Medicine, E-604, Case Western Reserve University, 2119 Abington Road, Cleveland, 44106 OH, U.S.A. Correspondence: O.A. Ramfrez, Departamento de Farmacologia, Facultad de Cieneias Qufmicas, Universidad Nacional de C6rdoba, Sueursal 16, C.C. 61, 5016 C6rdoba, Argentina. 0006-8993/90/$03.50 ~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
294 50 Mm). A recording microelectrode made with a micropipette (tip 10-20 Mm in diameter) was inserted in the dentate granule cell body layer. Evoked-field potentials were conventionally amplified and displayed in an oscilloscope for monitoring and recording. In response to the stimuli, 10 averaged field potentials, were sampled at 0.2 Hz on line using a PC microcomputer and the data stored on diskette for further analysis. Tetani, consisting of a
PRE TETANUS
train of pulses (0.5 ms) of 1 s duration, were delivered to the slices at 200 Hz. The intensity of stimuli was kept constant at approximately 50% above threshold. LTP was produced when the amplitude of the evoked population spike (PS, Fig. 1) recorded 20 min after the tetanus had increased at least 30%. Slices of the hippocampus from each electrophysiological treatment were picked up from the medium with a
POSTTETANUS
Fig. L Upper panel: photographs showing responses on granule cell layer of the hippocampus dentate gyrus evoked by stimulation to the perforant path. Left panel: responses recorded with stimuli delivered at 0.2 Hz. Right panel: the response from the same animals after a train of high frequency stimulation (PS, population spike). Calibration: 360/~V, 4 mS. Lower panel: micrographs of synapses from control animal (left) and from LTP animals (right) showing an overall depletion and shift of remaining yfsiclfs toward the,active zone during LTP; 36,000x.
295 Pasteur pipette and fixed in 1.5% glutaraldehyde, 1.5% formaldehyde and 0.1 M phosphate buffer (pH 7.2) 15. The tissue was diced around 2 m m from the recording electrode insertion position in the dentate gyms. This block was rinsed twice in a buffer and then treated with 2% osmium tetroxide for 2 h, stained in block with 2% uranylacetate, washed, dehydrated in a series of cold acetones and embedded in Araldite. The blocks were cut serially in 1/~m thick sections and stained with 1% Toluidine blue in 1% sodium borate for localization at light microscopic level of the molecular layer of the dentate gyrus; the tissue adjacent to the recording electrode position was excluded. For electronmicroscopy thin sections of the outer and middle level of the molecular layer about 80 nm in thickness were obtained in a Jeol JUM-7 ultramicrotome. To avoid duplicate counting of the same structure only one large section per 200 mesh copper grid was mounted, stained with lead citate and uranyl acetate and recorded photographically at 4000x magnification in a Siemens Elmiskop 101 electron microscope operating at 80 kV. Each electron micrograph covered an area of 225/~m 2 avoiding capillaries, glial and granular cell somas. Ten electron micrographs were randomly taken from each grid covering an area of 2250/./m 2 from each hippocampal slice. Two types of axodendritic synapses were processed morphometrically: the axonal boutons on dendritic shafts and those making contact through dendritic spines s. The mean values of 3 rats for each electrophysiological treatment were taken as independent observations. Fig. 1 shows a typical example of field potential response recorded in the granule cell body layer of the dentate gyrus evoked by stimuli (0.2 Hz) given to the P E The upper right panel shows the increased amplitude of the population spike after an effective tetanus (1 s train of 200 Hz). After LTP treatment, PS amplitude was increased by 200% on average (range: 106 + 30%, n = 3) when measured 20 min after the stimulus train. Electron micrographs of synapses in slices from control rats which showed LTP exhibited a consistent migration of synaptic vesicles toward the synaptic active zone during LTP; this finding is in accordance with earlier results 3. (Fig. 1, lower right panel.) Blind morphometric analysis revealed that the numbers of spine synapses in normal and H P rats were higher than that of LP rats (Fig. 2), The occurrence of shaft synapses per 225 ~ m 2 counted on granule cell layer of hippocampal dentate gyms of LP rats are less compared to control rats (Table I). Normal rats in which was obtained LTP after H F stimulation exhibited a significant reduction in the number of spine synapses of dentate gyrus (Fig. 2). O n the other hand, the increment in the number of shaft
Im50
[
] PRE- TETANUS POST-TETANUS
1.35'
~.I.20 ~" 1.05 UJ
~ 90 Z >u) I,I Z
75
u_ 0
ne 45 uJ m
g
Y////A
30
z
15 CONTROL
HP
LP
RATS
Fig. 2. Change in number of spine synapses per 225 # m 2 counted on granule cell layer of the hippocampal gyrus dentatus from control, HP and LP rats before and after LTP. All data are expressed as means + S.E.M. (n = 3 animals). Significance level was revealed by two-way analysis of variance (ANOVA) F1,17= 9.831 (P < 0.01) and F2,17 = 16.400 (P < 0.01). Post-hoe comparisons were performed using the Fisher test (P < 0.05). * Significantly different from control pretetanus (P < 0.05). ** Significantly different from control and HP rats pretetanus (P < 0.05). *** Significantly different from LP rats pretetanus (P < 0.05).
synapses on these animals was not statistically different (Table I). In H P rats after LTP there were no changes in the n u m b e r of both spine (Fig. 2) and shaft (Table I) synapses. A significant increase in the n u m b e r of shaft synapses was observed in LP rats with LTP after H F stimulation (Table I); concomitantly, a reduction in the number of spine synapses was observed under similar conditions (Fig. 2). However, in LP rats in which LTP was not detected no changes in the numbers of both spine (67 + 1.41 vs 69 + 2.82) and shaft (29.33 _+ 4.2 vs 34 + 2.44) synapses were found. Several lines of evidence strongly support the relevant
TABLE I Effects of LTP on number of shaft synapses per 225 ion 2 counted on granule cell layer of the hippocampal gyrus dentatus from control, HP and L P rats before and after HFstimulation
All data are expressed as means _+S.E.M. (n = 3 animals).
Control HP LP
Pretetanus
Post-tetanus
37.66 + 4 35.60 + 1.7 28.00 + 1.8+
41.00 + 6.2 33.60 + 4.2 40.33 + 5.71"
* Significantly different from LP rats (pretetanus); P < 0.05 Student's t-test. + Significantly different from control; P < 0.05 one-way ANOVA F2,s = 5.131 posthoc comparisons Fisher test P < 0.05.
296 not increase their basal number of dendritic shafts although the H F stimulation was delivered to PP. This observation may explain the impossibility to generate LTP on some LP rats. The fact that H P rats showed no increase in the number of synapses on dendritic shaft on the hippocampal dentate gyrus after LTP is a paradoxal effect, since it has been described as one of the structural changes subserving LTP 17'1s. Other pre- and postsynaptic mechanisms, such as increase in glutamate release 11, changes in the number or sensitivity of N receptor 2° membrane rearrangement leading to formation of new synapses 9 increase of Ca levels 16, and increments in the concentration of spectrin break-down product 23 may account for this effect. Moreover, unpublished observations (Ramirez et al., unpublished results) from this laboratory demonstrated that H P rats are less sensitive to inhibitory effect of 2-amino-5-phosphonovalerate, compared to LP rats, on blockade of LTP generation. The ultrastructural changes observed in H P or L P rats in the hippocampal dentate gyrus synapses, pre- or posttetanus can be correlated with the different hippocampal synaptic plasticity described on these rats 3°. The very high frequency of stimulation necessary to induce LTP and the lack of response in some LP rats may be due to the low number of spine and shaft synapses observed on the hippocampal dentate gyms of LP rats. We cannot rule out that other pre- and postsynaptic mechanisms subserved the different hippocampal synaptic plasticity observed on H P and LP rats.
participation of hippocampus in learning and memory processes 4'24'32. LP rats revealed different kinds of alterations on the hippocampal physiology 13'14'3°. More recently it has been reported that the inborn learning ability of H P and LP rats can be closely related with the plasticity of hippocampal synaptic transmission, as evidenced by the difference in threshold for LTP induction 3°. Lee et al. 17'18 using in vitro and in vivo methods demonstrated an increase in the number of synapses on dendritic shafts and a reduction in dendritic spines, the length of post-synaptic thickenings on dendritic spines, and the width of spine stalks, after LTP in hippocampal subfield CA1. These authors suggested that these structural changes could subserve LTP. In the present investigation LP rats displayed a lower n u m b e r of spine and shaft synapses with respect to H P and normal rats. LP rats also showed decreased numbers of hippocampus dentate gyms spine synapses in comparison to normal or H P rats after H F stimulation (200 Hz) to P E The decreased number of spine synapses found in hippocampal dentate gyrus of LP rats may account for the high threshold necessary to induce LTP and the impossibility to obtain it, in spite of the supramaximal stimulation frequency applied, on some LP rats 3°. No differences occurred in the number of synapses on dendritic shafts after LTP on hippocampal dentate gyrus of H P rats compared to control (Table I). O n the other hand L P rats exhibited a decreased number of synapses on dendritic shaft on the hippocampus dentate gyrus before tetanus (Table I). The LP rats that showed LTP after H F stimulation (200 Hz) were able to increase the n u m b e r of synapses on dendritic shaft on dentate gyrus. These structural changes can be interpreted as necessary to induce LTP on the hippocampal dentate gyrus of LP rats. LP rats which did not show LTP after tetanus could
This study was supported by grants from the C43nsejoNacional de Investigaciones Cientfficas y T6cnicas (CONICET) of Argentina to O.A.R. and from the Rockefeller Foundation, New York to A.A. The authors wish to thank Ms. Mercedes Guevara for technical assistance. Emma M. G~lvez for secretarial assistance.
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