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Aging affects transcranial magnetic modulation of hippocampal evoked potentials
Neurobiology of Aging 22 (2001) 255-263
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Aging affects transcranial magnetic modulation of hippocampal evoked potentials Y. Levkovitz, M. Segal* Department of Neurobiology, The Weizmann Institute, Rehovot 76100, Israel Received 25 January 2000; received in revised form 19 July 2000; accepted 19 July 2000
Abstract Transcranial magnetic stimulation (TMS) is being proposed as a method of choice for the treatment of clinical depression, yet its action in the brain is still not well understood. In previous studies we found that TMS has a long-term effect on reactivity of the hippocampus to perforant path stimulation. Since the efficacy of antidepressants is highly age-dependent, we studied possible age-related effects of TMS on hippocampal evoked responses. Young adult (3 months), aging (10 months) and aged (24 –26 months) awake rats were subjected to daily TMS for one week, followed by measurements of several parameters of reactivity to perforant path stimulation in the anesthetized rat. TMS did not affect responses of the hippocampus to single perforant path stimulation, but reduced drastically paired-pulse and frequency dependent depression in the young and aging but not the old rats. Likewise, TMS increased LTP expression in the young but not the old rats, and reduced the efficacy of serotonin modulation of reactivity of the hippocampus, in the young but not the old rats. Thus, long term effects of chronic TMS on local GABAergic inhibition are highly age dependent. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Transcranial magnetic stimulation; aging rats; dentate gyrus; serotonin; LTP; GABAergic inhibition
1. Introduction Depression is a prominent psychiatric disorder in the elderly [7,13,21,23,38]. Serotonin-specific reuptake inhibitors (SSRI) and tricyclic antidepressant (TCA) are in general less effective in treating depression in aged patients than in their younger counterparts [31]. In the elderly patient, the time required for the improvement of depressive symptoms with antidepressant therapy has been reported to be as long as 6 –12 weeks [3,10]. This pattern of reduced therapeutic efficacy is characteristic of SSRI and TCA and involves an age-related decline in the serotonergic neuronal system [8,38]. Age-related alterations in the synthesis, release, and turnover of serotonin [16,39,40] 5-HT receptor density [2,11,30], density of the 5-HT transporter [1,42] as well as physiological sensitivity to serotonergic agonists [6,26 –27] have also been described. While several brain regions have been associated with emotional symptoms of depression, one of the most fre-
* Corresponding author. Tel.: ⫹972– 89342553; fax: ⫹972– 89344140. E-mail address: [email protected] (M. Segal).
quent symptoms, cognitive dysfunction, was repeatedly associated with hippocampal atrophy [24,35–37]. The volume loss in the hippocampus is related to memory function and significantly correlated with total lifetime duration of depression (34 –37). The cognitive deficits are features commonly associated with aging. Thus, the hippocampus, heavily innervated by serotonergic fibers arising from the raphe nuclei, is an attractive target for the analysis of the effects of antidepressive treatments on elderly brain physiology. Transcranial magnetic stimulation (TMS) is a new noninvasive, fairly safe method for stimulation of the brain [4,5]. Magnetic stimulation of the human brain is increasingly used for functional cortical mapping of primary motor areas and for the investigation of cortical function related to cognition in both health and disease states [5,14,19]. TMS has been suggested recently for the treatment of psychiatric disorders of mood and emotional dysfunction. Clinical studies [15,17,20,22] suggest an antidepressant efficacy for TMS in depressed human patients. In earlier studies we found long-lasting effects of TMS on reactivity of the hippocampus to stimulation of its main excitatory afferent pathway arising from the entorhinal cortex, the perforant path [29]. Chronic TMS, similar to two
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other antidepressive drugs, desipramine and mianserine, did not affect single population spikes but caused an increase in paired -pulse potentiation, which was still evident 3 weeks after the last of a series of daily TMS treatments. Serotonergic and noradrenergic modulation of evoked synaptic activity may be related to these long-term TMS effects on synaptic transmission in the hippocampus. The objectives of the present study are to test the hypothesis that aging retards the efficacy of TMS in modulating inhibitory circuits of the hippocampus, and their regulation by monoamines.
2. Materials and methods 2.1. Animals. Experiments were conducted with young (3 months old), aging (10 month) and old (24 –26 months) Long-Evans rats of a local breeding colony. Each age group was divided randomly to control and chronic TMS-treated rats. The rats were housed in a temperature-controlled room, three-four rats to a cage, with 12 h light/dark cycle and free access to food and water. 2.2. TMS. One millisecond pulses were applied with a Cadwell (Kennewick, WA). Rapid Stimulator at a frequency of 25Hz for 2 s with a field intensity of 2.2 tesla (at 100% current intensity; flowing clockwise; calculated between 1–1.5 cm from the center coil [19]; estimated peak electric field strength of 660 V/m) through a 5 cm coil with a teardrop shape. Using these stimulation parameters, the hippocampus and posterior cortex are likely to be stimulated irrespective of the thickness of the skull [19]. The awake rat was held gently by hand while the coil was placed above the head, aligned with its center on the midline, equidistant between the bregma and lambda sutures along the longitudinal body axis. Rats were stimulated once daily for 7 days. Control rats were handled the same way as the treated ones and were exposed to the same noise produced by the stimulator. Two days after the last day of TMS stimulation rats were anesthetized with urethane (21% solution, 1.2 gm/kg, i.p.) and placed in a stereotaxic apparatus. A bipolar, 125 m concentric stimulating electrode was placed in the perforant path (PP) (coordinates: 7.5 mm posterior to bregma, 3.0 mm lateral to the midline, depth of 3.5 mm), and a single glass pipette (diameter of 2–3 mm) containing 2M NaCl was advanced into the dentate gyrus of the dorsal hippocampus using an hydraulic microdrive. Electrode positions were optimized to record maximal population spike (PS) in response to 100 sec pulse stimulation of the medial PP. Evoked responses were amplified and filtered at 1 Hz- 1 kHz and stored for later analysis. 2.3. Paired-pulse inhibition. A twin pulse PP stimulus was
delivered at three interpulse intervals (15, 30 and 60 msec), and averages of 5 successive response to a given intensity applied at a rate of 0.5 Hz were constructed. Paired-pulse response was quantified as the magnitude of the second over the first PS or the slope of the second EPSP over the first one. 2.4. Frequency dependent inhibition. Two series of stimuli were delivered, each series includes twenty stimuli at 1 Hz and twenty stimuli at 0.1 Hz, with baseline recorded again between the two series. PS and EPSP were measured as the average of forty stimuli at the same frequency relative to prior baseline condition. Differences in PS and EPSP between the two frequencies were quantified as the second minus the first PS (PS 0.1 Hz-PS 1 Hz) or the slope of the second minus the first slope (EPSP 0.1 Hz - EPSP 1 Hz). 2.5. LTP Induction. After electrode insertion, input-output relations, paired-pulse stimulation and frequency dependent inhibition were obtained, recording was made for 15 min followed by 10 min of baseline recording, before tetanic stimulation was applied. The LTP-inducing stimulation intensity was at 50% of the level that could evoke the maximum asymptotic spike amplitude. The parameters for the stimulation to induce LTP were 10 trains with an inter-train interval of 1 s and each train consisting of eight 0.4 ms 400 Hz pulses. LTP in each experiment was assessed as the change in response measured 35– 40 min after tetanus and expressed as a percentage of the mean of the 20 responses obtained during the 10 min preceding the tetanus. 2.6. Serotonergic modulation. Seventy minutes after the tetanic stimulation, serotonergic modulation of PS was measured using the serotonin releaser, fenfluramine (FFA, 7.5 mg/kg, i.p, see 34 for details) PS and EPSP were measured 15 and 25 min after FFA injection. 2.7. Analysis. Off-line measurements of the slopes of the EPSPs (in volts per second) and magnitudes of the maximal population spike (in millivolts) were made from averages of 5 successive responses to a given stimulation intensity applied at a rate of 0.1 Hz. PS size and EPSP slope were measured as describe previously [28]. To standardize the calculations and minimize cross-animal variability, the magnitudes or slopes of all responses were expressed as percentage of the asymptotic response obtained at control conditions with maximal stimulation (100%). The normalized data were analyzed statistically with SPSS 8.0 statistical package using three-way analysis of variance (ANOVA), with repeated measures, i.e. stimulus intensity or interpulse interval (IPI), on one of the variables (age and treatment), followed by Schaffer post-hoc multiple comparisons. Significance level was set at 5%. In some cases, t tests were used for the comparisons between specific groups.
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Fig. 1.: Reactivity to afferent stimulation in young, aging, and old rats following TMS treatment. A. Sample illustrations of population responses recorded in the granular layer to stimulation of the perforant path. Arrows indicate population spikes B & C Input-output relations, depicting the changes in population spike (B) and EPSP slope (C) as a function of changes in stimulation intensity, are the same in the different-age groups. Note, however, the lower EPSP in the old rats. (ANOVA, repeated measurement, population spike, age*treatment, dF ⫽ 5, F ⫽ 1.2, P ⫽ 0.327; ANOVA, EPSP, EPSP*age , df ⫽ 4, F ⫽ 4.2, P ⫽ 0.004, significant post-hoc Scheffe comparisons: old vs. young P ⫽ 0.005, old vs. aging P ⫽ 0.005, EPSP*treatment, df ⫽ 2, F ⫽ 29.7, P ⫽ 0.414, EPSP*treatment*age, df ⫽ 4, F ⫽ 1.87, P ⫽ 0.124).
3. Results 3.1. Input-output relation The old rats expressed a significantly smaller EPSP slope in response to perforant path stimulation than the young ones for all stimulus intensities (Fig. 1c), with the middleaged rats showing close values to those of the young ones, and not significantly different from them. However, for the same stimulation intensity, the old hippocampus produced the same population spike as the young and the aging ones. (Fig. 1b) indicating that in the old rats, the spike/EPSP ratio, which reflects the excitability of the cells is actually larger
than that of the young rats. TMS did not affect EPSP slopes in the young rats and the difference between the young and the old EPSP’s slope was maintained. 3.2. Paired-pulse inhibition Application of the paired-pulse stimulation protocol in young and aging rats caused a suppression of the response to the second stimulus of the pair when their interpulse interval was 15 and 30 ms (Fig. 2). The pairedpulse inhibition was replaced by a nearly 150% potentiation at 60 msec interpulse interval. A significant reduc-
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Fig. 2.: Paired pulse depression is markedly abolished in young and aging but not in old TMS treated group. A. Illustrations of paired pulse responses to stimulation applied with a 15 msec interpulse interval, in young and old, non-treated and TMS treated rats. B. Summary of the paired pulse responses in the six groups, using three interpulse intervals. (ANOVA, ps2/ps1, age*treatment, df ⫽ 6, F ⫽ 81.39, P ⫽ 0.005; treatment , df ⫽ 1, F ⫽ 241.28, P ⫽ 0.005, age, df ⫽ 2, F ⫽ 23.4, P ⫽ 0.0005, significant post-hoc Scheffe comparisons: old vs. young P ⫽ 0.005, old vs. aging P ⫽ 0.005, ANOVA, EPSP2/EPSP1, age*treatment, df ⫽ 6, F ⫽ 23.6, P ⫽ 0.001; treatment, df ⫽ 1, F ⫽ 105, P ⫽ 0.001, age, df ⫽ 2, F ⫽ 105, P ⫽ 0.007, significant post-hoc Scheffe comparisons: old vs. young P ⫽ 0.05, old vs. aging P ⫽ 0.05), (ANOVA, ps2/ps1, age*treatment, df ⫽ 6, F ⫽ 81.39, P ⫽ 0.005; treatment, df ⫽ 1, F ⫽ 241.28, P ⫽ 0.005, age, df ⫽ 2, F ⫽ 23.4, P ⫽ 0.0005, significant post-hoc Scheffe comparisons: old vs. young P ⫽ 0.005, old vs. aging P ⫽ 0.005, EPSP2/EPSP1, age*treatment, df ⫽ 6, F ⫽ 23.6, P ⫽ 0.001; treatment, df ⫽ 1, F ⫽ 105, P ⫽ 0.001, age df ⫽ 2, F ⫽ 105, P ⫽ 0.007, significant post-hoc Scheffe comparisons: old vs. young P ⫽ 0.05, old vs. aging P ⫽ 0.05).
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Fig. 3.: Frequency dependent inhibition is reduced in young and aging rats but not in the old group after TMS. A. Illustration of frequency dependent responses in young and old treated rats. B. A clear difference is seen between the population spikes of young, aging and old controls and TMS-treated groups. (ANOVA for the PS, age, df ⫽ 2. F ⫽ 25.5, P ⫽ 0.005; treatment, df ⫽ 1, f ⫽ 18.25, P ⫽ 0.005; age*treatment, df ⫽ 2, f ⫽ 38.4, P ⫽ 0.005, post-hoc significant Scheffe test: old vs. young, P ⫽ 0.005, ANOVA for the EPSP, age, df ⫽ 2. F ⫽ 3.4, P ⫽ 0.05; treatment, df ⫽ 1, f ⫽ 30.5, P ⫽ 0.001; age*treatment, df ⫽ 2, f ⫽ 18.1, P ⫽ 0.001, post-hoc significant Scheffe test: old vs. young, P ⫽ 0.05)
tion in paired-pulse inhibition at 15 and 30 msec was found in old rats (Fig 2). In young and aging rats, TMS caused a significant reduction in paired pulse inhibition at 15 and 30 msec. This reduction in inhibition did not occur when TMS was applied to the old rats The potentiating effect observed in the control at 60 msec interpulse interval was significantly enhanced by the TMS treatments (Fig. 2) in young and aging but not in old rats.
spike amplitude, caused approximately 50% suppression of the population spike in young rats (Fig. 3). Significantly less suppression was found in aging and old rats. TMS reduced the population spike inhibition by 25% and 15% in young and aging rats, respectively. In old rats the effect of TMS was opposite to that of the young rats; it increased the inhibition of population spike. 3.4. Long term potentiation
3.3. Frequency-dependent-inhibition Increasing stimulation frequency from 0.1 Hz to 1 Hz, at an intensity which evokes 50% of the maximum asymptotic
Tetanic stimulation of the perforant path caused a persistent increase in population spike amplitude and EPSP slope. In young and aging TMS groups the same stimulation
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Fig. 4.: Time-dependent changes in hippocampal responses to perforant path stimulation following a tetanic stimulation. A. Sample illustration of evoked responses before and after tetanic stimulation producing LTP in treated and non treated rats. B&C. Tetanic stimulation is applied at the arrowhead, and a clear difference between young and aging but no changes after TMS- is seen both with the population spike, and the EPSP slopes. (ANOVA for PS, repeated measurements: LTP*age*treatment: df ⫽ 20, F ⫽ 8.8, P ⬍ 0.005, LTP*age: df ⫽ 20, F ⫽ 11.2, P ⬍ 0.005, LTP*treatment, df ⫽ 10, F ⫽ 12.7, P ⬍ 0.005, significant Scheffe comparisons, P ⬍ 0.05, were found between the old rats and each of the young and aging, old vs. young P ⬍ 0.001, old vs. aging P ⬍ 0.005, aging vs. young P ⫽ 0.55, for EPSP: ANOVA, repeated measurement: LTP*age*treatment: df ⫽ 20, F ⫽ 7.2, P ⬍ 0.005, LTP*age: df ⫽ 20, F ⫽ 13.2, P ⬍ 0.001, LTP*treatment, df ⫽ 10, F ⫽ 14.2, P ⬍ 0.005, significant Scheffe comparisons, P ⬍ 0.05, were found between the groups, old vs. young P ⬍ 0.001, old vs. aging P ⬍ 0.005, aging vs. young P ⬍ 0.007).
produced significantly greater increase in population spike and EPSP (Fig. 4). No changes and even a reduction in population spike and EPSP were observed in the old rats after TMS treatment (Fig 4.).
non-treated rats (Fig. 5). In contrast, FFA injected into old TMS- treated rats did cause an increase in PS with no change in EPSP slope (Fig. 5). The increase in PS in young and aging non-treated rats was not accompanied by EPSP changes.
3.5. Serotonergic modulation 4. Discussion TMS treatment blocked the potentiating effects of fenfluramine (FFA) on population spikes in young and aging rats, compared with the typical 40 –50% increase in PS seen in
The present study demonstrates that chronic TMS treatment has a differential age-dependent effect on reactivity of
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Fig. 5.: FFA applied intraperitoneally produced a typical increase in population spike in non-treated rats, an increase in old, treated rats and no effects in the young and aging, TMS treated rats. (ANOVA for PS, repeated measurements: FFA*age*treatment: df ⫽ 2, F ⫽ 8.2, P ⬍ 0.003, FFA*age: df ⫽ 2, F ⫽ 6.0, P ⬍ 0.01, FFA*treatment, df ⫽ 1, F ⫽ 41.23, P ⬍ 0.001, significant Scheffe comparisons, P ⬍ 0.05, between the old rats and each of the young and aging, old vs. young P ⬍ 0.03, old vs. aging P ⬍ 0.015, aging vs. young P ⫽ 0.905. ANOVA for EPSP, repeated measurement: FFA*age*treatment: df ⫽ 2, F ⫽ 0.39, P ⫽ 0.99, FFA*age: df ⫽ 2, F ⫽ 1.39, P ⫽ 0.87, FFA*treatment, df ⫽ 1, F ⫽ 3.4, P ⫽ 0.84).
the hippocampus to stimulation of the perforant path. While in none of the different age groups TMS affected basal population spike size and EPSP, it did affect paired-pulse and frequency-dependent inhibition, indicating that it has a primary action on local inhibitory circuits activated in these stimulation protocols. TMS reduced paired-pulse and frequency-dependent inhibition in young and aging rats, but not old rats. In addition, TMS caused a large suppression of the reactivity of the hippocampus to the serotonin-releasing drug FFA, in young and aging rats, but not in old rats. The antidepressant drugs, especially serotonin-specific reuptake inhibitors and tricyclic antidepressants, are in general less effective in treating depression in old than in young patients. However, the influence of aging on serotonergic functions and antidepressant drugs is not clear. Recently, an age-related decrease in hippocampal serotonin transporter and their upregulation following chronic amitriptyline administration in rats was found [41]. The increase in the transporter density following tricyclic treatment may contribute to the general lower effectiveness of tricyclic antidepressants with aging and could explain the ability of the
serotonin-releaser FFA to increase excitability in TMStreated old rats. Whether the decreased serotonin transporter sites in the hippocampus represent a loss of serotonergic nerve terminals with aging or changes in transporter gene transcription and/or translation is not known. One other possibility to consider is that in the aged rat the input/output relations is such that for a given EPSP there is already a larger population spike, compared to the young rat. This ceiling effect may reduce the efficacy of TMS in further increasing the blocked paired pulse depression seen in the young rats. Regardless of the molecular mechanism, the changes in serotonergic release in the hippocampus of chronic TMStreated rats may have important implications for understanding the biologic basis of TMS treatment and depression in the elderly. The neuronal sites of action of TMS are not entirely clear. Obviously, it affects local inhibitory circuits more than the main excitatory afferent to the hippocampus. The modulation of local inhibition can either be a direct action, by reducing the efficacy of inhibition pre- or postsynapti-
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cally, or indirect, by reducing the efficacy of GABAergic modulators, e.g. serotonin. Since evidence for both actions was found in our experiments, if is not clear which of them is the main one. A selective loss of interneurons as opposed to preserved number of principle cells in old rats could also contribute to differential effects of TMS on evoked responses in the young and old rats. TMS has a potentiating effect on LTP in the young and aging rats, ages at which it also has a marked blocking action on paired pulse depression and frequency dependent inhibition. These correlated effects indicate that LTP is modulated by local inhibitory circuits in the hippocampus. Thus, a reduction in inhibition is assumed to allow the cells further depolarization, enhanced calcium influx and larger LTP. Patients with major depression typically show cognitive dysfunction including memory impairment and poor attention, features commonly associated with aging [12]. Previously, chronic amitriptyline treatment improved spatial memory in young but not in aged rats [29]. We demonstrated that TMS causes enhancement of population spike and EPSP after tetanic stimulation in young and aging rats, above that produced normally, while it did not affect old rats. Interestingly, one of the well-known adverse sideeffect of the tricyclic antidepressant, SSRI and electroconvulsive shock therapy in elderly patients is a temporary memory impairment that is related to cholinergic/monoaminergic changes caused by the antidepressant treatment. A reduction in long-term potentiation was found previously in the dentate gyrus of rats following selective depletion of monoamines [9,29]. In conclusion, the observed age-related effects of TMS on hippocampal excitability may contribute to the understanding of the reduced antidepressant efficacy with aging.
Acknowledgments We would like to thank Dr. N. Grisaru for help with the TMS equipment. Supported by a grant from the Israel Center for Psychobiology in Israel and by the Abramson Family Foundation.
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Aging affects transcranial magnetic modulation of hippocampal evoked potentials Y. Levkovitz, M. Segal* Department of Neurobiology, The Weizmann Institute, Rehovot 76100, Israel Received 25 January 2000; received in revised form 19 July 2000; accepted 19 July 2000
Abstract Transcranial magnetic stimulation (TMS) is being proposed as a method of choice for the treatment of clinical depression, yet its action in the brain is still not well understood. In previous studies we found that TMS has a long-term effect on reactivity of the hippocampus to perforant path stimulation. Since the efficacy of antidepressants is highly age-dependent, we studied possible age-related effects of TMS on hippocampal evoked responses. Young adult (3 months), aging (10 months) and aged (24 –26 months) awake rats were subjected to daily TMS for one week, followed by measurements of several parameters of reactivity to perforant path stimulation in the anesthetized rat. TMS did not affect responses of the hippocampus to single perforant path stimulation, but reduced drastically paired-pulse and frequency dependent depression in the young and aging but not the old rats. Likewise, TMS increased LTP expression in the young but not the old rats, and reduced the efficacy of serotonin modulation of reactivity of the hippocampus, in the young but not the old rats. Thus, long term effects of chronic TMS on local GABAergic inhibition are highly age dependent. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Transcranial magnetic stimulation; aging rats; dentate gyrus; serotonin; LTP; GABAergic inhibition
1. Introduction Depression is a prominent psychiatric disorder in the elderly [7,13,21,23,38]. Serotonin-specific reuptake inhibitors (SSRI) and tricyclic antidepressant (TCA) are in general less effective in treating depression in aged patients than in their younger counterparts [31]. In the elderly patient, the time required for the improvement of depressive symptoms with antidepressant therapy has been reported to be as long as 6 –12 weeks [3,10]. This pattern of reduced therapeutic efficacy is characteristic of SSRI and TCA and involves an age-related decline in the serotonergic neuronal system [8,38]. Age-related alterations in the synthesis, release, and turnover of serotonin [16,39,40] 5-HT receptor density [2,11,30], density of the 5-HT transporter [1,42] as well as physiological sensitivity to serotonergic agonists [6,26 –27] have also been described. While several brain regions have been associated with emotional symptoms of depression, one of the most fre-
* Corresponding author. Tel.: ⫹972– 89342553; fax: ⫹972– 89344140. E-mail address: [email protected] (M. Segal).
quent symptoms, cognitive dysfunction, was repeatedly associated with hippocampal atrophy [24,35–37]. The volume loss in the hippocampus is related to memory function and significantly correlated with total lifetime duration of depression (34 –37). The cognitive deficits are features commonly associated with aging. Thus, the hippocampus, heavily innervated by serotonergic fibers arising from the raphe nuclei, is an attractive target for the analysis of the effects of antidepressive treatments on elderly brain physiology. Transcranial magnetic stimulation (TMS) is a new noninvasive, fairly safe method for stimulation of the brain [4,5]. Magnetic stimulation of the human brain is increasingly used for functional cortical mapping of primary motor areas and for the investigation of cortical function related to cognition in both health and disease states [5,14,19]. TMS has been suggested recently for the treatment of psychiatric disorders of mood and emotional dysfunction. Clinical studies [15,17,20,22] suggest an antidepressant efficacy for TMS in depressed human patients. In earlier studies we found long-lasting effects of TMS on reactivity of the hippocampus to stimulation of its main excitatory afferent pathway arising from the entorhinal cortex, the perforant path [29]. Chronic TMS, similar to two
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other antidepressive drugs, desipramine and mianserine, did not affect single population spikes but caused an increase in paired -pulse potentiation, which was still evident 3 weeks after the last of a series of daily TMS treatments. Serotonergic and noradrenergic modulation of evoked synaptic activity may be related to these long-term TMS effects on synaptic transmission in the hippocampus. The objectives of the present study are to test the hypothesis that aging retards the efficacy of TMS in modulating inhibitory circuits of the hippocampus, and their regulation by monoamines.
2. Materials and methods 2.1. Animals. Experiments were conducted with young (3 months old), aging (10 month) and old (24 –26 months) Long-Evans rats of a local breeding colony. Each age group was divided randomly to control and chronic TMS-treated rats. The rats were housed in a temperature-controlled room, three-four rats to a cage, with 12 h light/dark cycle and free access to food and water. 2.2. TMS. One millisecond pulses were applied with a Cadwell (Kennewick, WA). Rapid Stimulator at a frequency of 25Hz for 2 s with a field intensity of 2.2 tesla (at 100% current intensity; flowing clockwise; calculated between 1–1.5 cm from the center coil [19]; estimated peak electric field strength of 660 V/m) through a 5 cm coil with a teardrop shape. Using these stimulation parameters, the hippocampus and posterior cortex are likely to be stimulated irrespective of the thickness of the skull [19]. The awake rat was held gently by hand while the coil was placed above the head, aligned with its center on the midline, equidistant between the bregma and lambda sutures along the longitudinal body axis. Rats were stimulated once daily for 7 days. Control rats were handled the same way as the treated ones and were exposed to the same noise produced by the stimulator. Two days after the last day of TMS stimulation rats were anesthetized with urethane (21% solution, 1.2 gm/kg, i.p.) and placed in a stereotaxic apparatus. A bipolar, 125 m concentric stimulating electrode was placed in the perforant path (PP) (coordinates: 7.5 mm posterior to bregma, 3.0 mm lateral to the midline, depth of 3.5 mm), and a single glass pipette (diameter of 2–3 mm) containing 2M NaCl was advanced into the dentate gyrus of the dorsal hippocampus using an hydraulic microdrive. Electrode positions were optimized to record maximal population spike (PS) in response to 100 sec pulse stimulation of the medial PP. Evoked responses were amplified and filtered at 1 Hz- 1 kHz and stored for later analysis. 2.3. Paired-pulse inhibition. A twin pulse PP stimulus was
delivered at three interpulse intervals (15, 30 and 60 msec), and averages of 5 successive response to a given intensity applied at a rate of 0.5 Hz were constructed. Paired-pulse response was quantified as the magnitude of the second over the first PS or the slope of the second EPSP over the first one. 2.4. Frequency dependent inhibition. Two series of stimuli were delivered, each series includes twenty stimuli at 1 Hz and twenty stimuli at 0.1 Hz, with baseline recorded again between the two series. PS and EPSP were measured as the average of forty stimuli at the same frequency relative to prior baseline condition. Differences in PS and EPSP between the two frequencies were quantified as the second minus the first PS (PS 0.1 Hz-PS 1 Hz) or the slope of the second minus the first slope (EPSP 0.1 Hz - EPSP 1 Hz). 2.5. LTP Induction. After electrode insertion, input-output relations, paired-pulse stimulation and frequency dependent inhibition were obtained, recording was made for 15 min followed by 10 min of baseline recording, before tetanic stimulation was applied. The LTP-inducing stimulation intensity was at 50% of the level that could evoke the maximum asymptotic spike amplitude. The parameters for the stimulation to induce LTP were 10 trains with an inter-train interval of 1 s and each train consisting of eight 0.4 ms 400 Hz pulses. LTP in each experiment was assessed as the change in response measured 35– 40 min after tetanus and expressed as a percentage of the mean of the 20 responses obtained during the 10 min preceding the tetanus. 2.6. Serotonergic modulation. Seventy minutes after the tetanic stimulation, serotonergic modulation of PS was measured using the serotonin releaser, fenfluramine (FFA, 7.5 mg/kg, i.p, see 34 for details) PS and EPSP were measured 15 and 25 min after FFA injection. 2.7. Analysis. Off-line measurements of the slopes of the EPSPs (in volts per second) and magnitudes of the maximal population spike (in millivolts) were made from averages of 5 successive responses to a given stimulation intensity applied at a rate of 0.1 Hz. PS size and EPSP slope were measured as describe previously [28]. To standardize the calculations and minimize cross-animal variability, the magnitudes or slopes of all responses were expressed as percentage of the asymptotic response obtained at control conditions with maximal stimulation (100%). The normalized data were analyzed statistically with SPSS 8.0 statistical package using three-way analysis of variance (ANOVA), with repeated measures, i.e. stimulus intensity or interpulse interval (IPI), on one of the variables (age and treatment), followed by Schaffer post-hoc multiple comparisons. Significance level was set at 5%. In some cases, t tests were used for the comparisons between specific groups.
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Fig. 1.: Reactivity to afferent stimulation in young, aging, and old rats following TMS treatment. A. Sample illustrations of population responses recorded in the granular layer to stimulation of the perforant path. Arrows indicate population spikes B & C Input-output relations, depicting the changes in population spike (B) and EPSP slope (C) as a function of changes in stimulation intensity, are the same in the different-age groups. Note, however, the lower EPSP in the old rats. (ANOVA, repeated measurement, population spike, age*treatment, dF ⫽ 5, F ⫽ 1.2, P ⫽ 0.327; ANOVA, EPSP, EPSP*age , df ⫽ 4, F ⫽ 4.2, P ⫽ 0.004, significant post-hoc Scheffe comparisons: old vs. young P ⫽ 0.005, old vs. aging P ⫽ 0.005, EPSP*treatment, df ⫽ 2, F ⫽ 29.7, P ⫽ 0.414, EPSP*treatment*age, df ⫽ 4, F ⫽ 1.87, P ⫽ 0.124).
3. Results 3.1. Input-output relation The old rats expressed a significantly smaller EPSP slope in response to perforant path stimulation than the young ones for all stimulus intensities (Fig. 1c), with the middleaged rats showing close values to those of the young ones, and not significantly different from them. However, for the same stimulation intensity, the old hippocampus produced the same population spike as the young and the aging ones. (Fig. 1b) indicating that in the old rats, the spike/EPSP ratio, which reflects the excitability of the cells is actually larger
than that of the young rats. TMS did not affect EPSP slopes in the young rats and the difference between the young and the old EPSP’s slope was maintained. 3.2. Paired-pulse inhibition Application of the paired-pulse stimulation protocol in young and aging rats caused a suppression of the response to the second stimulus of the pair when their interpulse interval was 15 and 30 ms (Fig. 2). The pairedpulse inhibition was replaced by a nearly 150% potentiation at 60 msec interpulse interval. A significant reduc-
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Fig. 2.: Paired pulse depression is markedly abolished in young and aging but not in old TMS treated group. A. Illustrations of paired pulse responses to stimulation applied with a 15 msec interpulse interval, in young and old, non-treated and TMS treated rats. B. Summary of the paired pulse responses in the six groups, using three interpulse intervals. (ANOVA, ps2/ps1, age*treatment, df ⫽ 6, F ⫽ 81.39, P ⫽ 0.005; treatment , df ⫽ 1, F ⫽ 241.28, P ⫽ 0.005, age, df ⫽ 2, F ⫽ 23.4, P ⫽ 0.0005, significant post-hoc Scheffe comparisons: old vs. young P ⫽ 0.005, old vs. aging P ⫽ 0.005, ANOVA, EPSP2/EPSP1, age*treatment, df ⫽ 6, F ⫽ 23.6, P ⫽ 0.001; treatment, df ⫽ 1, F ⫽ 105, P ⫽ 0.001, age, df ⫽ 2, F ⫽ 105, P ⫽ 0.007, significant post-hoc Scheffe comparisons: old vs. young P ⫽ 0.05, old vs. aging P ⫽ 0.05), (ANOVA, ps2/ps1, age*treatment, df ⫽ 6, F ⫽ 81.39, P ⫽ 0.005; treatment, df ⫽ 1, F ⫽ 241.28, P ⫽ 0.005, age, df ⫽ 2, F ⫽ 23.4, P ⫽ 0.0005, significant post-hoc Scheffe comparisons: old vs. young P ⫽ 0.005, old vs. aging P ⫽ 0.005, EPSP2/EPSP1, age*treatment, df ⫽ 6, F ⫽ 23.6, P ⫽ 0.001; treatment, df ⫽ 1, F ⫽ 105, P ⫽ 0.001, age df ⫽ 2, F ⫽ 105, P ⫽ 0.007, significant post-hoc Scheffe comparisons: old vs. young P ⫽ 0.05, old vs. aging P ⫽ 0.05).
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Fig. 3.: Frequency dependent inhibition is reduced in young and aging rats but not in the old group after TMS. A. Illustration of frequency dependent responses in young and old treated rats. B. A clear difference is seen between the population spikes of young, aging and old controls and TMS-treated groups. (ANOVA for the PS, age, df ⫽ 2. F ⫽ 25.5, P ⫽ 0.005; treatment, df ⫽ 1, f ⫽ 18.25, P ⫽ 0.005; age*treatment, df ⫽ 2, f ⫽ 38.4, P ⫽ 0.005, post-hoc significant Scheffe test: old vs. young, P ⫽ 0.005, ANOVA for the EPSP, age, df ⫽ 2. F ⫽ 3.4, P ⫽ 0.05; treatment, df ⫽ 1, f ⫽ 30.5, P ⫽ 0.001; age*treatment, df ⫽ 2, f ⫽ 18.1, P ⫽ 0.001, post-hoc significant Scheffe test: old vs. young, P ⫽ 0.05)
tion in paired-pulse inhibition at 15 and 30 msec was found in old rats (Fig 2). In young and aging rats, TMS caused a significant reduction in paired pulse inhibition at 15 and 30 msec. This reduction in inhibition did not occur when TMS was applied to the old rats The potentiating effect observed in the control at 60 msec interpulse interval was significantly enhanced by the TMS treatments (Fig. 2) in young and aging but not in old rats.
spike amplitude, caused approximately 50% suppression of the population spike in young rats (Fig. 3). Significantly less suppression was found in aging and old rats. TMS reduced the population spike inhibition by 25% and 15% in young and aging rats, respectively. In old rats the effect of TMS was opposite to that of the young rats; it increased the inhibition of population spike. 3.4. Long term potentiation
3.3. Frequency-dependent-inhibition Increasing stimulation frequency from 0.1 Hz to 1 Hz, at an intensity which evokes 50% of the maximum asymptotic
Tetanic stimulation of the perforant path caused a persistent increase in population spike amplitude and EPSP slope. In young and aging TMS groups the same stimulation
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Fig. 4.: Time-dependent changes in hippocampal responses to perforant path stimulation following a tetanic stimulation. A. Sample illustration of evoked responses before and after tetanic stimulation producing LTP in treated and non treated rats. B&C. Tetanic stimulation is applied at the arrowhead, and a clear difference between young and aging but no changes after TMS- is seen both with the population spike, and the EPSP slopes. (ANOVA for PS, repeated measurements: LTP*age*treatment: df ⫽ 20, F ⫽ 8.8, P ⬍ 0.005, LTP*age: df ⫽ 20, F ⫽ 11.2, P ⬍ 0.005, LTP*treatment, df ⫽ 10, F ⫽ 12.7, P ⬍ 0.005, significant Scheffe comparisons, P ⬍ 0.05, were found between the old rats and each of the young and aging, old vs. young P ⬍ 0.001, old vs. aging P ⬍ 0.005, aging vs. young P ⫽ 0.55, for EPSP: ANOVA, repeated measurement: LTP*age*treatment: df ⫽ 20, F ⫽ 7.2, P ⬍ 0.005, LTP*age: df ⫽ 20, F ⫽ 13.2, P ⬍ 0.001, LTP*treatment, df ⫽ 10, F ⫽ 14.2, P ⬍ 0.005, significant Scheffe comparisons, P ⬍ 0.05, were found between the groups, old vs. young P ⬍ 0.001, old vs. aging P ⬍ 0.005, aging vs. young P ⬍ 0.007).
produced significantly greater increase in population spike and EPSP (Fig. 4). No changes and even a reduction in population spike and EPSP were observed in the old rats after TMS treatment (Fig 4.).
non-treated rats (Fig. 5). In contrast, FFA injected into old TMS- treated rats did cause an increase in PS with no change in EPSP slope (Fig. 5). The increase in PS in young and aging non-treated rats was not accompanied by EPSP changes.
3.5. Serotonergic modulation 4. Discussion TMS treatment blocked the potentiating effects of fenfluramine (FFA) on population spikes in young and aging rats, compared with the typical 40 –50% increase in PS seen in
The present study demonstrates that chronic TMS treatment has a differential age-dependent effect on reactivity of
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Fig. 5.: FFA applied intraperitoneally produced a typical increase in population spike in non-treated rats, an increase in old, treated rats and no effects in the young and aging, TMS treated rats. (ANOVA for PS, repeated measurements: FFA*age*treatment: df ⫽ 2, F ⫽ 8.2, P ⬍ 0.003, FFA*age: df ⫽ 2, F ⫽ 6.0, P ⬍ 0.01, FFA*treatment, df ⫽ 1, F ⫽ 41.23, P ⬍ 0.001, significant Scheffe comparisons, P ⬍ 0.05, between the old rats and each of the young and aging, old vs. young P ⬍ 0.03, old vs. aging P ⬍ 0.015, aging vs. young P ⫽ 0.905. ANOVA for EPSP, repeated measurement: FFA*age*treatment: df ⫽ 2, F ⫽ 0.39, P ⫽ 0.99, FFA*age: df ⫽ 2, F ⫽ 1.39, P ⫽ 0.87, FFA*treatment, df ⫽ 1, F ⫽ 3.4, P ⫽ 0.84).
the hippocampus to stimulation of the perforant path. While in none of the different age groups TMS affected basal population spike size and EPSP, it did affect paired-pulse and frequency-dependent inhibition, indicating that it has a primary action on local inhibitory circuits activated in these stimulation protocols. TMS reduced paired-pulse and frequency-dependent inhibition in young and aging rats, but not old rats. In addition, TMS caused a large suppression of the reactivity of the hippocampus to the serotonin-releasing drug FFA, in young and aging rats, but not in old rats. The antidepressant drugs, especially serotonin-specific reuptake inhibitors and tricyclic antidepressants, are in general less effective in treating depression in old than in young patients. However, the influence of aging on serotonergic functions and antidepressant drugs is not clear. Recently, an age-related decrease in hippocampal serotonin transporter and their upregulation following chronic amitriptyline administration in rats was found [41]. The increase in the transporter density following tricyclic treatment may contribute to the general lower effectiveness of tricyclic antidepressants with aging and could explain the ability of the
serotonin-releaser FFA to increase excitability in TMStreated old rats. Whether the decreased serotonin transporter sites in the hippocampus represent a loss of serotonergic nerve terminals with aging or changes in transporter gene transcription and/or translation is not known. One other possibility to consider is that in the aged rat the input/output relations is such that for a given EPSP there is already a larger population spike, compared to the young rat. This ceiling effect may reduce the efficacy of TMS in further increasing the blocked paired pulse depression seen in the young rats. Regardless of the molecular mechanism, the changes in serotonergic release in the hippocampus of chronic TMStreated rats may have important implications for understanding the biologic basis of TMS treatment and depression in the elderly. The neuronal sites of action of TMS are not entirely clear. Obviously, it affects local inhibitory circuits more than the main excitatory afferent to the hippocampus. The modulation of local inhibition can either be a direct action, by reducing the efficacy of inhibition pre- or postsynapti-
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cally, or indirect, by reducing the efficacy of GABAergic modulators, e.g. serotonin. Since evidence for both actions was found in our experiments, if is not clear which of them is the main one. A selective loss of interneurons as opposed to preserved number of principle cells in old rats could also contribute to differential effects of TMS on evoked responses in the young and old rats. TMS has a potentiating effect on LTP in the young and aging rats, ages at which it also has a marked blocking action on paired pulse depression and frequency dependent inhibition. These correlated effects indicate that LTP is modulated by local inhibitory circuits in the hippocampus. Thus, a reduction in inhibition is assumed to allow the cells further depolarization, enhanced calcium influx and larger LTP. Patients with major depression typically show cognitive dysfunction including memory impairment and poor attention, features commonly associated with aging [12]. Previously, chronic amitriptyline treatment improved spatial memory in young but not in aged rats [29]. We demonstrated that TMS causes enhancement of population spike and EPSP after tetanic stimulation in young and aging rats, above that produced normally, while it did not affect old rats. Interestingly, one of the well-known adverse sideeffect of the tricyclic antidepressant, SSRI and electroconvulsive shock therapy in elderly patients is a temporary memory impairment that is related to cholinergic/monoaminergic changes caused by the antidepressant treatment. A reduction in long-term potentiation was found previously in the dentate gyrus of rats following selective depletion of monoamines [9,29]. In conclusion, the observed age-related effects of TMS on hippocampal excitability may contribute to the understanding of the reduced antidepressant efficacy with aging.
Acknowledgments We would like to thank Dr. N. Grisaru for help with the TMS equipment. Supported by a grant from the Israel Center for Psychobiology in Israel and by the Abramson Family Foundation.
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