The relationship between tetanus intensity and the magnitude of hippocampal long-term potentiation in vivo

Abstract

In this study, we assessed the effects of varying tetanus and test-pulse intensity on the magnitude of long-term potentiation (LTP) in the perforant path-dentate gyrus projection of urethane-anaesthetized rats. We developed a novel within-subjects procedure in which test-pulse-stimulation intensity (60-1000 μA) was varied quasi-randomly under computer control throughout the recording period. After a baseline period, we applied a high-frequency tetanus, the intensity of which was varied over the same range as test-pulse intensity, but between subjects. The time-course of LTP was thus monitored continuously across a range of test-pulse intensities in each rat. Intense high-frequency tetanization at 1000 μA resulted in a paradoxical depression of the dentate field excitatory post-synaptic potential (fEPSP) slope at the lowest test intensity used (60 μA), but caused a potentiation at higher test intensities in the same animal. Moreover, intense tetanization induced less LTP than a moderate tetanus over most of the test-intensity range. Explanations for this pattern of data include a potentiation of feed-forward inhibition in conjunction with LTP of excitatory neurotransmission, or local tissue damage at the stimulation site. To address this issue, we conducted an additional experiment in which a second stimulating electrode was placed in the perforant path at a site closer to the dentate, in order to activate a common population of afferents at a location 'downstream' of the original stimulation site. After 1000-μA tetanization of the original ('upstream') site, fEPSPs were again depressed in response to test stimulation of the upstream site, but only potentiation was observed in response to stimulation of the downstream site. This is consistent with the idea that the depression induced by intense tetanization results from local changes at the stimulation site. In conclusion, while tetanus intensity must exceed the LTP induction threshold, intensities above 500 μA should be avoided; in the present study, tetanization at 250-500 μA yielded maximal levels of LTP.

Fig. 1

(A) Schematic diagram of the placement of electrodes in experiment 1—a stimulating electrode (S) in the perforant path and a recording electrode (R) in the hilus of the dentate gyrus. (B) Relationship between fEPSP and test-pulse stimulation intensity in a single rat. Examples of fEPSPs elicited by stimulation at each of the intensities sampled are shown.

Fig. 2

The time course of LTP across the full range of tetanus intensities (columns), and test intensities (rows). All data are normalized to the mean value obtained over the 20-min baseline period. In each panel, data from the non-tetanized control group at the corresponding test intensity (white circles) are plotted alongside the data from the relevant tetanized group (grey circles) for comparison.

Fig. 3

(A) Input/output curves relating fEPSP slope to test intensity both before and after tetanization. Data from each rat were normalized to the mean value obtained following test stimulation at 1000 μA before tetanization (arbitrarily designated 100%), and group means were calculated. In the top-right panel, examples of fEPSPs recorded before (dotted line) and after tetanization at 1000 μA (solid line) are shown at test intensities of 60 μA (left-hand side; scale bar = 1 mV (vertical) and 5 ms (horizontal)) and 1000 μA (right-hand side; scale bar = 5 mV (vertical) and 5 ms (horizontal)). (B) Relationship between mean LTP recorded 40–60 min after tetanization and test intensity across the range of tetanus intensities studied. (C) Relationship between LTP and tetanus intensity across the range of test intensities sampled. In both B and C, the significance level of potentiation or depression at each point, relative to baseline (100%; dotted line), is indicated on the graphs: ⁺0.1 > p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01.

Fig. 4

(A) The amplitude of the stimulus artefact (mean of positive and negative components) is plotted as a function of test-pulse intensity and time for the 1000-μA-tetanus group only, and normalized to the pre-tetanus value at each intensity. The stimulus artefact remained stable over time and, at low test intensities, was unaffected by tetanization. A small, transient decrease in the size of the artefact was observed at high test intensities (^∗∗p < 0.01; post hoc one-sample t-tests with Bonferroni correction). (B–D) Photomicrographs of the stimulation site (black arrows) in Cresyl-Violet-stained brain sections. Representative examples are shown from animals that received (B) a 1000-μA tetanus, (C) no tetanus, and (D) a small marking lesion. Scale bar = 0.5 mm.

Fig. 5

Experiment 2. (A) Schematic diagram of the placement of upstream (S1; white circle at tip) and downstream (S2; grey circle at tip) stimulating electrodes within the perforant path in experiment 2. Typical examples of fEPSPs elicited by 1000-μA test stimulation at both sites are shown. (B) The time course of LTP at each test intensity elicited by a 1000-μA upstream tetanus. Responses to stimulation of both upstream (filled circles) and downstream (open circles) pathways are shown; dotted lines indicate baseline (i.e. 100%). (C) The effects of a small marking lesion delivered via the upstream stimulating electrode on fEPSP slope in upstream (filled circles) and downstream (white circles) pathways at each test intensity. (D) Relationship between normalized fEPSP slope and test intensity in both upstream and downstream pathways after 1000 μA tetanization. Examples of fEPSPs recorded in both pathways are shown (below graph = upstream pathway; above graph = downstream pathway) before (dotted lines) and after tetanization of the upstream site (solid lines), in response to stimulation at 60 μA (left-hand side; scale bar = 1 mV (vertical) and 5 ms (horizontal)) and 1000 μA (right-hand side; scale bar = 5 mV (vertical) and 5 ms (horizontal)). (E) Relationship between normalized fEPSP slope and test intensity in both upstream and downstream pathways after marking stimulation. Examples of fEPSPs recorded in both pathways before and after marking stimulation are shown (details as in D). In both D and E, the significance level of potentiation or depression at each point, relative to baseline (100%; dotted line), is indicated on the graphs: ⁺0.1 > p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01. Symbols above the graph refer to the downstream pathway, and symbols below the graph refer to the upstream pathway. (F) Example of PPF following successive stimulation of S2 followed by S1 at an interval of 50 ms. fEPSPs were recorded from the dentate molecular layer. Stimulation of S1 alone resulted in a mean fEPSP slope of −0.46 mV/ms; prior stimulation of S2 resulted in a mean slope of −0.62 mV/ms in response to subsequent stimulation of S1, an increase of 35.1%.