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Differences in hippocampal synaptic plasticity in rats with inborn high or low learning ability may be related to different sensitivity of aspartate receptors
Brain Research Bulletin, Vol. 27, pp. 291-293. * Pergamon Press plc, 1991. Printed
in the UXA
0361-9230/91 $3.00 + .OTI
BRIEF COMMUNICATION
Differences in Hippocampal Synaptic Plasticity in Rats With Inborn High or Low Learning Ability May Be Related to Different Sensitivity of Aspartate Receptors OSCAR A. RAMIREZ,“’
RAUL A. GOMEZ” AND HUGO F. CARRERt
*Departamento de Farmacologia, Facultad de Ciencias Quimicas, Universidad National de Cordoba and i_Institutode Investigation Medica Mercedes y Martin Ferreyra, Cordoba, Argentina
Received 15 November 1990 RAMIREZ, 0. A., R. A. GOMEZ AND H. F. CARRBR. Differences in hippocampal synaptic plasticity in rats with inborn high or low learning ability may be related ro different sensitivity of aspartate receptors. BRAIN RBS BULL 27(2) 291-293, 1991, -
Rats with an inborn high (HP) or low (LP) learning capacity were used to study the sensitivity to the blocking effect of 2-aminophosphono~n~oic acid (AP5; 10 and 20 uM) on long-term ~ten&iation (LTP) produced in hip~ampal slices by a l-s tetanus at 200 Hz. The potential evoked by stimulation of the perfomnt path was recorded from the granule cell layer of the dentate gyms in 400 pm slices perfused with standard Krebs’ solution or the APS. Under perfusion with IO pM of AP5, in 100% of slices from HP rats, LTP generation was not blocked; when AP5 20 pM was used, in 85% of the cases LTP was not blocked. In 60% of slices from LP rats, AP5 10 +M and in 100% of the cases at 20 p,M AP5 blocked LTP generation. These results are coherent with the hypothesis that the different inborn learning ability of HP and LP rats is related to the different population or sensitivity of N-methyl-D-aspartate (NMDA) receptors. Long-term ~tentiation
Learning ability
Hip~~pus
Gyms dentatus
PREVIOUS work from this laboratory has shown that hippocampal function is different in rats with an inborn high (HP) or low (LP) learning capacity (8,9). Evidence has been obtained (12) indicating that the inborn learning ability of these animals is closely related with the plasticity of hippocampaf synaptic transmission. This phenomenon was demonstrated in HP rats by the lower threshold to induce long-lasting potentiation following a brief burst of high frequency stimulation (l), i.e., long-term potentiation (LTP). This phenomenon is considered a useful model of learning and memory (15). More recently, a good correlation between performance in a shuttle box avoidance response and the threshold to induce LTP has been demonstrated in normal rats (13). The mechanisms generating LTP are little understood; available evidence suggests that pre- and postsynaptic mechanisms are involved (10). The stimulation of receptors for N-methyi-D-aspartate (called NMDA receptors) has been shown to be necessary for the induction of LTP (2) and furtbermore, the high concen~ation of NMDA receptors in the hip~~p~ fo~ation has been related with the remarkable plasticity of this brain area
I_
^
‘Requestsfor rePrmts should be addressed to 0. A. Raminx 61, 5016 Cordoba, Argentina.
NMDA
Synaptic plasticity
(3). Blockade of NMDA receptors with o-phosphonic acid homologues, which prevents the induction of hippocampal LTP, also impairs different types of learning tasks (4,11). In the present investigation we studied in HP and LP rats the sensitivity to blockade of LTP generation, using the NMDA receptor blocking drug amino-phosphonovalerate, in order to correlate the hippocampal synaptic plasticity with the functional availability of N receptors. METHOD
Synaptic function was evaluated in vitro in hippocampai slices using LTP as an end point. Male albino rats, 6-9 weeks old were used; they were descendents from HP or LP rats. These lines had been inbred for over 30 generations at the time of this study. HP rats were defined as those that made 70% or more conditioned responses (CRs); LP rats were those that made 15% or less CRs. The CR was a barrier crossing response (7). Testing involved 50 trials, one every 30 s, using a buzzer as a con-
Departamentode Farmacologia, Facultad de Ciencias Quimicas, Sucursal 16, CC
291
292
RAMIREZ,
GOMEZ AND CARRER
4 HP RATS 0
LP RATS
L FIG. 1. Oscillographic recordings of responses from granule cell layer of the gyms dentatus evoked by stimulation of the perforant path in a hippocampal slice. Pretetanus (left panel) was recorded with stimuli delivered at 0.2 Hz: posttetanus (right panel) shows the responses recorded after a train of high frequency stimulation. Calibration bars represent 8 ms and 160 ~LV; PS: population spike.
ditioning stimulus and electric shock to the floor grid of the box as unconditioned stimuli. Animals from the normal stock responded at 40% level of CRs. Experiments were performed in vitro in hippocampal slices prepared essentially as described by Yamamoto et al. (16). The animals were killed with a blow to the neck between 11:OO and 12:00 a.m. to prevent variations determined by circadian rhythms or nonspecific stressors (5). Transverse hippocampal slices approximately 400 pm thick were placed in a recording chamber perfused with a modified Krebs solution (16) saturated with 95% 0, and 5% CO,. Rate of perfusion was 2-3 ml/min; the washout time of the chamber was 3 min (14); the temperature of the bathing solution was kept at 30°C. Under visual control a stimulating electrode made of 2 twisted wires insulated except for the cut end (diameter 50 pm) was placed in the perforant path. A recording microelectrode with an impedance <3 MR, made with a micropipette filled with NaCl 3 M, tip 10 to 20 pm, was placed in the dentate granule cell body layer. Evoked field potentials were conventionaly amplified and displayed in an oscilloscope for monitoring and photography. Averaged evoked potentials were obtained using an averaging module that accumulates 16 responses at 0.2 Hz. Once the evoked response had been obtained, including the PS, at 0.2 Hz in a slice perfused with the modified Krebs’ solution, perfusion was initiated with a freshly prepared solution of 2-amino-phosphonopentanoic acid (10 or 20 PM; AP5). After 14 min of perfusion with AP5, induction of LTP was attempted using a train of pulses (0.5 ms) of 1 s duration and 200 Hz frequency delivered to the slice; these parameters were selected because previous work has shown that this frequency of stimulation induces LTP in most preparations (12). Intensity of stimuli was kept constant at approximately 50% above threshold. Fifteen to 20 rnin after the tetanus had been delivered, a new averaged evoked response was recorded; LTP was considered to have been produced when the amplitude of the evoked population spike (PS; Fig. 1) recorded 20 min after the tetanus, had increased at least 30%. In all experiments in which during perfusion with
PIG. 2. Blocking effect of AP5 (10 and 20 )LM) on induction of LTP in hippocampal slices from HP and LP rats. LTP was induced by a l-s train of 200 Hz delivered to the perforant path. Stepwise logistic regression p
AP5 LTP could not be obtained, the same slice was perfused with the Krebs’ solution for at least 20 min and induction of LTP was tested again; if LTP could not be induced under perfusion with Krebs’ solution and with the same parameters, the experiment was discarded. Previous data from our laboratory demonstrated that the incidence of LTP in slices from both strains was 7/l 1 for HP rats and 6/l 1 for LP rats. RESULTS
Figure 1 shows a typical example of the field potential response recorded in the granule cell body layer of the dentate gyrus evoked by stimuli (0.2 Hz) given to the perforant path. In 5 out of 5 slices taken from 5 HP rats, AP5 10 p,M did not block the generation of LTP, but in slices taken from 5 LP rats, the same concentration of AP5 effectively prevented generation of LTP in 3 out of 5 slices. In a different experimental series, a higher concentration of AP5 (20 p,M ) was used. In this condition, in 5 out of 5 slices taken from 5 LP rats, AP5 blocked the generation of LTP whereas in slices taken from HP rats, only 1 out of 6 was blocked. These results were statistically evaluated using the stepwise logistic regression (6). The model fits the data with a p
These results indicate that the different hippocampal synaptic plasticity demonstrated in HP and LP rats (12) coincides with a different sensitivity to the blocking action of AP5 on LTP induction in dentate gyrus. This finding was to be expected since NMDA receptors have been related with the remarkable plasticity of the hippocampus (13). Our results, indicating a different sensitivity to the blocking effect of AP5 in HP and LP rats, suggest changes in the number of affinity of NMDA receptors in this area. This conclusion does not preclude the existence of other factors that may explain the altered threshold for LTP induction as well as the inborn learning ability of these animals.
HIPPOCAMPAL SYNAPTIC PLASTICITY
293
ACKh’OWLEDGEMENTS The present work was made possible through
CONICOR
and Fundacion
grants from CONICET,
Perez Companc.
RE~RENCES 1. Bliss, T. V. P.; Lomo, T. Long lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J. Physiol. (Land.) 232:331356; 1973. 2. Collingridge, G. L. Long term potentiation in the hippocampus: Mechanism of initiation and modulation by ne~tmnsmitters. Trends Pharmacol. Sci. 6:407-t11; 1985. 3. Cdlingridge, B. L.; Kehl, S. J.; McLennan, H. Excitatory amino acids in synaptic transmission in the Schaffer collateral commissural pathway of the rat hippocampus. J. Physiol. (Land.) 334:3346 1983. 4. Danysz, W.; Wroblewski, J. T.; Costa, E. Learning impairment in rats by N-methyl-D-aspartate receptor antagonist. Neuropharmacology 27653-656; 1988. 5. Di Scenna, P. Method and myth in m~ntaining brain slices. In: Schurr, A.; Tyler, T. J.; Tseng, M. T., eds. Brain slices: Fundamentals, application and implications. Base]: Karger; 1987: 10-21. 6. Engelman, -L. Stepwise logistic regression. BMDP Programs. Berkeley: Universitv of California Press: 1981:330-342. 7. Evangelista, A.-M.; Izquierdo, I. The effect of pre- and post-trail amphetamine injections on avoidance responses of rats. Psychopharmacologia 20~42-47; 1971. 8. Izquierdo, 1.; Orsingher, 0. A. A physiological difference in the hippocampus of rats with a low inborn learning ability. Psychopharmacologia 23:386-396; 1972. 9. Izquierdo, 1.; Orsingher, 0. A.; Ogura, 0. A. Hippocampal facili-
10.
11.
12.
13.
14.
15. 16.
tation and RNA buildup in response to stimulation in rats with a low inborn learning ability. Behav. Biol. 7:69%707; 1972. Lynch, G.; Muller, D.; Seubert, P.; Larson, J. Long-term potentiation: Persisting problems and recent results. Brain Res. Bull. 21: 363-372; 1988. Morris, R. G. M.; Anderson, E.; Lynch, G. S.; Bat&y, M. Selective impai~ent of learning and blockade of long term ~tentia~on by an N-methyl-D-aspartate receptor antagonist, AP5. Nature (Lond.) 319774-776; 1986. Ramirez, 0. A.; Orsingher, 0. A.; Carrer, H. F. Differential threshold for long-term potentiation in the hippocampus of rats with inborn high or low learning capacity. Neurosci. Lett. 92:27.5-279; 1988. Ramirez, 0. A.; Caner, H. F. Correlation between threshold to induce long-term ~tentiation in the hip~ampus and performance in a shuttle box avoidance response in rats. Neurosci. I.&. R&152156; 1989. Ramirez, 0. A.; Comez, R. A.; Carrer, H. F. Gangliosides improve synaptic transmission in dentate gyrus of hippocampal rat slices. Brain Res. 506291-293; 1990. Teyler, T. J.; Di Scenna, P. Long-term potentiation. Annu. Rev. Neurosci. 10:131-161; 1987. Yamamoto, C.; Matsumoto, K.; Takagi, M. Potentiation of excitatory postsynaptic potentials during and after repetitive stimulation in thin hippocampal sections. Exp. Brain Res. 38:469-477; 1980.
in the UXA
0361-9230/91 $3.00 + .OTI
BRIEF COMMUNICATION
Differences in Hippocampal Synaptic Plasticity in Rats With Inborn High or Low Learning Ability May Be Related to Different Sensitivity of Aspartate Receptors OSCAR A. RAMIREZ,“’
RAUL A. GOMEZ” AND HUGO F. CARRERt
*Departamento de Farmacologia, Facultad de Ciencias Quimicas, Universidad National de Cordoba and i_Institutode Investigation Medica Mercedes y Martin Ferreyra, Cordoba, Argentina
Received 15 November 1990 RAMIREZ, 0. A., R. A. GOMEZ AND H. F. CARRBR. Differences in hippocampal synaptic plasticity in rats with inborn high or low learning ability may be related ro different sensitivity of aspartate receptors. BRAIN RBS BULL 27(2) 291-293, 1991, -
Rats with an inborn high (HP) or low (LP) learning capacity were used to study the sensitivity to the blocking effect of 2-aminophosphono~n~oic acid (AP5; 10 and 20 uM) on long-term ~ten&iation (LTP) produced in hip~ampal slices by a l-s tetanus at 200 Hz. The potential evoked by stimulation of the perfomnt path was recorded from the granule cell layer of the dentate gyms in 400 pm slices perfused with standard Krebs’ solution or the APS. Under perfusion with IO pM of AP5, in 100% of slices from HP rats, LTP generation was not blocked; when AP5 20 pM was used, in 85% of the cases LTP was not blocked. In 60% of slices from LP rats, AP5 10 +M and in 100% of the cases at 20 p,M AP5 blocked LTP generation. These results are coherent with the hypothesis that the different inborn learning ability of HP and LP rats is related to the different population or sensitivity of N-methyl-D-aspartate (NMDA) receptors. Long-term ~tentiation
Learning ability
Hip~~pus
Gyms dentatus
PREVIOUS work from this laboratory has shown that hippocampal function is different in rats with an inborn high (HP) or low (LP) learning capacity (8,9). Evidence has been obtained (12) indicating that the inborn learning ability of these animals is closely related with the plasticity of hippocampaf synaptic transmission. This phenomenon was demonstrated in HP rats by the lower threshold to induce long-lasting potentiation following a brief burst of high frequency stimulation (l), i.e., long-term potentiation (LTP). This phenomenon is considered a useful model of learning and memory (15). More recently, a good correlation between performance in a shuttle box avoidance response and the threshold to induce LTP has been demonstrated in normal rats (13). The mechanisms generating LTP are little understood; available evidence suggests that pre- and postsynaptic mechanisms are involved (10). The stimulation of receptors for N-methyi-D-aspartate (called NMDA receptors) has been shown to be necessary for the induction of LTP (2) and furtbermore, the high concen~ation of NMDA receptors in the hip~~p~ fo~ation has been related with the remarkable plasticity of this brain area
I_
^
‘Requestsfor rePrmts should be addressed to 0. A. Raminx 61, 5016 Cordoba, Argentina.
NMDA
Synaptic plasticity
(3). Blockade of NMDA receptors with o-phosphonic acid homologues, which prevents the induction of hippocampal LTP, also impairs different types of learning tasks (4,11). In the present investigation we studied in HP and LP rats the sensitivity to blockade of LTP generation, using the NMDA receptor blocking drug amino-phosphonovalerate, in order to correlate the hippocampal synaptic plasticity with the functional availability of N receptors. METHOD
Synaptic function was evaluated in vitro in hippocampai slices using LTP as an end point. Male albino rats, 6-9 weeks old were used; they were descendents from HP or LP rats. These lines had been inbred for over 30 generations at the time of this study. HP rats were defined as those that made 70% or more conditioned responses (CRs); LP rats were those that made 15% or less CRs. The CR was a barrier crossing response (7). Testing involved 50 trials, one every 30 s, using a buzzer as a con-
Departamentode Farmacologia, Facultad de Ciencias Quimicas, Sucursal 16, CC
291
292
RAMIREZ,
GOMEZ AND CARRER
4 HP RATS 0
LP RATS
L FIG. 1. Oscillographic recordings of responses from granule cell layer of the gyms dentatus evoked by stimulation of the perforant path in a hippocampal slice. Pretetanus (left panel) was recorded with stimuli delivered at 0.2 Hz: posttetanus (right panel) shows the responses recorded after a train of high frequency stimulation. Calibration bars represent 8 ms and 160 ~LV; PS: population spike.
ditioning stimulus and electric shock to the floor grid of the box as unconditioned stimuli. Animals from the normal stock responded at 40% level of CRs. Experiments were performed in vitro in hippocampal slices prepared essentially as described by Yamamoto et al. (16). The animals were killed with a blow to the neck between 11:OO and 12:00 a.m. to prevent variations determined by circadian rhythms or nonspecific stressors (5). Transverse hippocampal slices approximately 400 pm thick were placed in a recording chamber perfused with a modified Krebs solution (16) saturated with 95% 0, and 5% CO,. Rate of perfusion was 2-3 ml/min; the washout time of the chamber was 3 min (14); the temperature of the bathing solution was kept at 30°C. Under visual control a stimulating electrode made of 2 twisted wires insulated except for the cut end (diameter 50 pm) was placed in the perforant path. A recording microelectrode with an impedance <3 MR, made with a micropipette filled with NaCl 3 M, tip 10 to 20 pm, was placed in the dentate granule cell body layer. Evoked field potentials were conventionaly amplified and displayed in an oscilloscope for monitoring and photography. Averaged evoked potentials were obtained using an averaging module that accumulates 16 responses at 0.2 Hz. Once the evoked response had been obtained, including the PS, at 0.2 Hz in a slice perfused with the modified Krebs’ solution, perfusion was initiated with a freshly prepared solution of 2-amino-phosphonopentanoic acid (10 or 20 PM; AP5). After 14 min of perfusion with AP5, induction of LTP was attempted using a train of pulses (0.5 ms) of 1 s duration and 200 Hz frequency delivered to the slice; these parameters were selected because previous work has shown that this frequency of stimulation induces LTP in most preparations (12). Intensity of stimuli was kept constant at approximately 50% above threshold. Fifteen to 20 rnin after the tetanus had been delivered, a new averaged evoked response was recorded; LTP was considered to have been produced when the amplitude of the evoked population spike (PS; Fig. 1) recorded 20 min after the tetanus, had increased at least 30%. In all experiments in which during perfusion with
PIG. 2. Blocking effect of AP5 (10 and 20 )LM) on induction of LTP in hippocampal slices from HP and LP rats. LTP was induced by a l-s train of 200 Hz delivered to the perforant path. Stepwise logistic regression p
AP5 LTP could not be obtained, the same slice was perfused with the Krebs’ solution for at least 20 min and induction of LTP was tested again; if LTP could not be induced under perfusion with Krebs’ solution and with the same parameters, the experiment was discarded. Previous data from our laboratory demonstrated that the incidence of LTP in slices from both strains was 7/l 1 for HP rats and 6/l 1 for LP rats. RESULTS
Figure 1 shows a typical example of the field potential response recorded in the granule cell body layer of the dentate gyrus evoked by stimuli (0.2 Hz) given to the perforant path. In 5 out of 5 slices taken from 5 HP rats, AP5 10 p,M did not block the generation of LTP, but in slices taken from 5 LP rats, the same concentration of AP5 effectively prevented generation of LTP in 3 out of 5 slices. In a different experimental series, a higher concentration of AP5 (20 p,M ) was used. In this condition, in 5 out of 5 slices taken from 5 LP rats, AP5 blocked the generation of LTP whereas in slices taken from HP rats, only 1 out of 6 was blocked. These results were statistically evaluated using the stepwise logistic regression (6). The model fits the data with a p
These results indicate that the different hippocampal synaptic plasticity demonstrated in HP and LP rats (12) coincides with a different sensitivity to the blocking action of AP5 on LTP induction in dentate gyrus. This finding was to be expected since NMDA receptors have been related with the remarkable plasticity of the hippocampus (13). Our results, indicating a different sensitivity to the blocking effect of AP5 in HP and LP rats, suggest changes in the number of affinity of NMDA receptors in this area. This conclusion does not preclude the existence of other factors that may explain the altered threshold for LTP induction as well as the inborn learning ability of these animals.
HIPPOCAMPAL SYNAPTIC PLASTICITY
293
ACKh’OWLEDGEMENTS The present work was made possible through
CONICOR
and Fundacion
grants from CONICET,
Perez Companc.
RE~RENCES 1. Bliss, T. V. P.; Lomo, T. Long lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J. Physiol. (Land.) 232:331356; 1973. 2. Collingridge, G. L. Long term potentiation in the hippocampus: Mechanism of initiation and modulation by ne~tmnsmitters. Trends Pharmacol. Sci. 6:407-t11; 1985. 3. Cdlingridge, B. L.; Kehl, S. J.; McLennan, H. Excitatory amino acids in synaptic transmission in the Schaffer collateral commissural pathway of the rat hippocampus. J. Physiol. (Land.) 334:3346 1983. 4. Danysz, W.; Wroblewski, J. T.; Costa, E. Learning impairment in rats by N-methyl-D-aspartate receptor antagonist. Neuropharmacology 27653-656; 1988. 5. Di Scenna, P. Method and myth in m~ntaining brain slices. In: Schurr, A.; Tyler, T. J.; Tseng, M. T., eds. Brain slices: Fundamentals, application and implications. Base]: Karger; 1987: 10-21. 6. Engelman, -L. Stepwise logistic regression. BMDP Programs. Berkeley: Universitv of California Press: 1981:330-342. 7. Evangelista, A.-M.; Izquierdo, I. The effect of pre- and post-trail amphetamine injections on avoidance responses of rats. Psychopharmacologia 20~42-47; 1971. 8. Izquierdo, 1.; Orsingher, 0. A. A physiological difference in the hippocampus of rats with a low inborn learning ability. Psychopharmacologia 23:386-396; 1972. 9. Izquierdo, 1.; Orsingher, 0. A.; Ogura, 0. A. Hippocampal facili-
10.
11.
12.
13.
14.
15. 16.
tation and RNA buildup in response to stimulation in rats with a low inborn learning ability. Behav. Biol. 7:69%707; 1972. Lynch, G.; Muller, D.; Seubert, P.; Larson, J. Long-term potentiation: Persisting problems and recent results. Brain Res. Bull. 21: 363-372; 1988. Morris, R. G. M.; Anderson, E.; Lynch, G. S.; Bat&y, M. Selective impai~ent of learning and blockade of long term ~tentia~on by an N-methyl-D-aspartate receptor antagonist, AP5. Nature (Lond.) 319774-776; 1986. Ramirez, 0. A.; Orsingher, 0. A.; Carrer, H. F. Differential threshold for long-term potentiation in the hippocampus of rats with inborn high or low learning capacity. Neurosci. Lett. 92:27.5-279; 1988. Ramirez, 0. A.; Caner, H. F. Correlation between threshold to induce long-term ~tentiation in the hip~ampus and performance in a shuttle box avoidance response in rats. Neurosci. I.&. R&152156; 1989. Ramirez, 0. A.; Comez, R. A.; Carrer, H. F. Gangliosides improve synaptic transmission in dentate gyrus of hippocampal rat slices. Brain Res. 506291-293; 1990. Teyler, T. J.; Di Scenna, P. Long-term potentiation. Annu. Rev. Neurosci. 10:131-161; 1987. Yamamoto, C.; Matsumoto, K.; Takagi, M. Potentiation of excitatory postsynaptic potentials during and after repetitive stimulation in thin hippocampal sections. Exp. Brain Res. 38:469-477; 1980.