1Department of Neuroscience, 2Department of Neurology, and 3Department of Neurosurgery, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908; and 4Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland 21228
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ABSTRACT |
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Denslow, Maria J., Tore Eid, Fu Du, Robert Schwarcz, Eric W. Lothman, and Oswald Steward. Disruption of Inhibition in Area CA1 of the Hippocampus in a Rat Model of Temporal Lobe Epilepsy. J. Neurophysiol. 86: 2231-2245, 2001. Previous studies have revealed a loss of neurons in layer III of the entorhinal cortex (EC) in patients with temporal lobe epilepsy. These neurons project to the hippocampus and may activate inhibitory interneurons, so that their loss could disrupt inhibitory function in the hippocampus. The present study evaluates this hypothesis in a rat model in which layer III neurons were selectively destroyed by focal injections of the indirect excitotoxin, aminooxyacetic acid (AOAA). Inhibitory function in the hippocampus was assessed by evaluating the discharge of CA1 neurons in response to stimulation of afferent pathways in vivo. In control animals, stimulation of the temporo-ammonic pathway leads to heterosynaptic inhibition of population spikes generated by subsequent stimulation of the commissural projection to CA1. This heterosynaptic inhibition was substantially reduced in animals that had received AOAA injections 1 mo previously. Stimulation of the commissural projection also elicited multiple population spikes in CA1 in AOAA-injected animals, and homosynaptic inhibition in response to paired-pulse stimulation of the commissural projection was dramatically diminished. These results suggest a disruption of inhibitory function in CA1 in AOAA-injected animals. To determine whether the disruption of inhibition occurred selectively in CA1, we assessed paired-pulse inhibition in the dentate gyrus. Both homosynaptic inhibition generated by paired-pulse stimulation of the perforant path, and heterosynaptic inhibition produced by activation of the commissural projection to the dentate gyrus appeared largely comparable in AOAA-injected and control animals; thus abnormalities in inhibitory function following AOAA injections occurred relatively selectively in CA1. Electrolytic lesions of the EC did not cause the same loss of inhibition as seen in animals with AOAA injections, indicating that the loss of inhibition in CA1 is not due to the loss of excitatory driving of inhibitory interneurons. Also, electrolytic lesions of the EC in animals that had been injected previously with AOAA had little effect on the abnormal physiological responses in CA1, suggesting that most alterations in inhibition in CA1 are not due to circuit abnormalities within the EC. Comparisons of control and AOAA-injected animals in a hippocampal kindling paradigm revealed that the duration of afterdischarges elicited by high-frequency stimulation of CA3, and the number of stimulations required to elicit kindled seizures were comparable. Taken together, our results reveal that the selective loss of layer III neurons induced by AOAA disrupts inhibitory function in CA1, but this does not create a circuit that is more prone to at least one form of kindling.
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INTRODUCTION |
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Recent studies have revealed an extensive loss
of neurons in layer III of the entorhinal cortex (EC) in patients with
temporal lobe epilepsy (TLE) (Du et al. 1993,
1995
). Layer III neurons project to the CA1 region of
the hippocampus via the temporo-ammonic (TA) pathway (Steward
1976
; Steward and Scoville 1976
; Witter 1993
), and so the loss of layer III neurons would
interrupt one of the important routes of cortical input to the hippocampus.
TA projections terminate as asymmetric (presumed excitatory) synapses
on dendrites within the stratum lacunosum-moleculare of CA1
(Desmond et al. 1994; Witter et al.
1992
). Physiological studies indicate that the synapses of the
TA pathway are excitatory (Colbert and Levy 1992
;
Leung 1995
; Stringer and Colbert 1994
). At the same time, however, stimulation of the TA pathway produces powerful inhibition of CA1 pyramidal neurons (Empson and
Heinemann 1995
; Soltesz and Deschenes 1993
). On
this basis, it has been proposed that TA projections primarily activate
inhibitory interneurons, so that the pathway is a substrate for
projected inhibition (Lacaille and Schwartzkroin 1988
;
Soltesz and Jones 1995
). If this is so, loss of the TA
pathway in temporal lobe epilepsy could disrupt inhibitory mechanisms
in CA1, contributing to the development of seizure-prone circuitry.
An opportunity to test several predictions of this hypothesis in an
animal model has come from the discovery that the pattern of neuron
loss in layer III of the EC seen in epileptic patients can be
reproduced in experimental animals by injecting the indirect excitotoxin, aminooxyacetic acid (AOAA), into the EC (Du and
Schwarcz 1992). AOAA is termed an indirect excitotoxin because
it appears to cause neuronal death by compromising mitochondrial energy
metabolism (Beal et al. 1991
). Injections of AOAA into
the entorhinal cortex trigger limbic seizures that continue for several
hours after the injections (Du and Schwarcz 1992
). Over
the course of several days, there is an extensive loss of neurons in
layer III of the EC on the side of the injection, whereas the layer II
stellate neurons that project to the dentate gyrus are relatively
spared (Du and Schwarcz 1992
). As a result, the TA
pathway is largely destroyed, whereas the temporo-dentate pathway (the
perforant path) is preserved.
There are several predictions of the hypothesis that the loss of layer
III neurons would disrupt inhibition in CA1, potentially producing a
seizure-prone circuit. First, if inhibitory interneurons lose their
excitatory drive, there might be a general increase in excitability of
CA1 neurons, which would be reflected by a tendency to exhibit greater
discharges in response to afferent activation. Second, such changes in
excitability should be present in CA1 but not in other hippocampal
subfields (since the TA pathway projects selectively to CA1)
(Steward and Scoville 1976). Third, complete lesions of
the EC should cause the same increases in excitability as AOAA-induced
lesions of layer III neurons (in both cases, inhibitory interneurons
would lose their excitatory drive). Finally, if the changes lead to a
seizure-prone circuit, it should be relatively easier to trigger
electrographic and behavioral seizures by stimulating hippocampal
afferent pathways.
The present study tests these predictions. Extracellular recording techniques were used to assess responses to afferent stimulation in control animals and animals that had received AOAA injections 30-35 days previously. Experiments were carried out in CA1, which had lost its normal input from the TA pathway, and in the dentate gyrus, in which the projections from the EC were largely intact. Paired-pulse stimulation paradigms were used in which the first (conditioning) pulse produces inhibition that is reflected by decreases in the population spike generated by a second (test) pulse. This analysis revealed that CA1 neurons in AOAA-injected animals exhibited multiple spiking in response to afferent stimulation and substantially decreased paired-pulse inhibition.
To evaluate whether electrolytic lesions that destroyed the EC would produce comparable effects, similar analyses were carried out on animals that had received EC lesions 1 mo previously. These experiments revealed that electrolytic lesions of the EC did not cause the same loss of inhibition as AOAA injections. To explore the possibility that the physiological anomalies induced by AOAA were localized within the EC itself, we evaluated the consequences of destroying the EC that had been previously injected with AOAA, and found that these secondary lesions had minimal effect on the hyperexcitability in CA1. To assess whether AOAA-induced disruption of inhibitory function made the hippocampus more prone to seizures, we used a kindling paradigm to evaluate the duration of afterdischarges elicited by high-frequency stimulation of CA3 and the number of stimulations required to elicit fully kindled seizures. These experiments revealed no differences between control and AOAA-injected animals, indicating that disruption of inhibitory processes within CA1 did not enhance seizure susceptibility in this hippocampal kindling paradigm.
Taken together, these results indicate that local injections of AOAA into the EC disrupt inhibition in CA1, but this cannot be explained solely by the loss of projected inhibition mediated by the TA pathway. Instead, the abnormalities probably reflect an alteration in inhibitory function within the hippocampus itself. Nevertheless, the disruption of inhibition did not lead to a circuit in the hippocampus that is more prone to kindling. These results provide new insights into the physiological changes that occur following loss of layer III neurons, and provide clues about the possible role that such a selective lesion may play in circuit abnormalities in temporal lobe epilepsy.
This work was included in a dissertation submitted by M. Denslow in partial fulfillment of the requirements for the Ph.D. degree at the University of Virginia.
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METHODS |
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Intraentorhinal injection procedure
Male Sprague-Dawley rats (200-300 g) were anesthetized with
chloral hydrate (350 mg/kg ip) and positioned in a stereotaxic frame
(Kopf, Tujunga, CA). AOAA (Sigma, St. Louis, MO) was dissolved in 0.1 M
phosphate-buffered saline (PBS) at a concentration of 100 µg/µl of
PBS, pH 7.4. AOAA was injected into the EC of one hemisphere using a
10-µl Hamilton microsyringe at the following stereotaxic coordinates:
AP 1.2 mm behind lambda, ML 5.0 mm, with the nose bar set at 3.0 mm and
the needle holder set at an angle of 20° posteriorly in the sagittal
plane. A total of 0.75 µl of the AOAA solution was delivered over a
period of 10 min in 0.25-µl aliquots at each of three dorsoventral
levels (3, 4, and 5 mm deep from the cortical surface). Control rats
received an injection of the vehicle solution (0.1 M PBS, pH 7.4). The
behavior of all animals was observed for at least 4 h post
injection. As described previously (Du and Schwarcz
1992), AOAA-injected animals exhibited evidence of limbic
seizures (wet dog shakes, chewing, forelimb clonus, and rearing)
starting at 2 h post-AOAA administration. None of the control
animals exhibited seizures.
Unilateral lesions of the entorhinal cortex
Male Sprague-Dawley rats (200-270 g) were anesthetized with
pentobarbital sodium (Nembutal; 50 mg/kg ip) and positioned in a
stereotaxic frame, and the entorhinal area was unilaterally ablated as
described previously (Loesche and Steward 1977). An electrode was placed in the entorhinal area at the following
stereotaxic coordinates: 1.5 mm anterior to the transverse sinus: 3, 4, and 5 mm lateral to the midsagittal sinus, and 2, 4, and 6 mm below the
cortical surface with the nose bar set at
2.0 mm and the electrode
positioned at an angle of 10° away from the midline. One milliampere
of current was passed at each electrode position for a total of 45 s.
Secondary lesions of the entorhinal cortex that had previously been injected with AOAA
For the experiments involving secondary lesions of the EC that had previously been injected with AOAA, male Sprague-Dawley rats (200-300 g) were injected with AOAA as described above. Thirty to 60 days following the injection, animals were anesthetized with urethan (1.2 g/kg ip) for an initial in vivo recording session to assess the hyperexcitability of CA1. Immediately following the recording session, the AOAA-injected EC was ablated as described above. Animals were given subcutaneous injections of 0.9% saline, and a second set of recordings was taken 10-14 h following the electrolytic lesion.
In vivo electrophysiology
A total of 27 animals (7 AOAA-injected, 8 control, 6 EC-lesioned, and 6 AOAA/EC-lesioned animals) was evaluated using in vivo electrophysiological procedures. The control data were pooled from two vehicle-injected and eight naive animals since their responses were virtually identical. Animals were evaluated 30-35 days following the unilateral intra-entorhinal injection of AOAA.
For the physiological experiments, animals were anesthetized with
urethan (1.2 g/kg ip) and placed into a stereotaxic frame with the nose
bar set at 0 mm. Stereotaxic coordinates and physiological stimulation
paradigms were as described by Bekenstein et al.
(1993) (see also Fig. 1). To
activate the entorhinal projection to the hippocampus and dentate gyrus
(the "EC pathway"), a bipolar stainless steel electrode (0.01 in.
diam) was placed in the angular bundle on the side of the AOAA
injection (AP
8.1 mm from bregma, ML 4.4 mm, DV
2.0). This
electrode was angled at 20° posteriorly in the sagittal plane. To
activate the commissural projection (the "Comm pathway"), a second
bipolar stimulating electrode (SNEX 200, tip diameter 100 µm, Rhodes
Medical Instruments) was placed in the CA3 region of the dorsal
hippocampus, contralateral to the site of the AOAA injection (AP
3.0
mm from bregma, ML 3.5 mm, DV
2.5 mm). A glass capillary
microelectrode (2-6 M
resistance) filled with 2 M NaCl, and 5%
Fast Green was placed in the dorsal hippocampus ipsilateral to the AOAA
injection site [AP
3.0 mm from bregma, ML 2.0 mm, DV
2.0 mm for
CA1 and DV
3.1 mm for dentate gyrus (DG)]. The final depths of the
recording and stimulating electrodes were adjusted to produce evoked
responses of maximal amplitude at minimum stimulus intensities.
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Stimuli were applied through a constant voltage stimulus isolation unit controlled by a digital timer (Winston T-10). Data were collected through an amplifier (Biodyne) and in parallel to a computerized data acquisition system (pCLAMP, Axon Instruments, Foster City, CA) and a digital oscilloscope. Data were analyzed using Axograph (Axon Instruments) and Sigma Plot (Jandell). For each response, the amplitude of the population spike evoked by the stimulation was determined automatically by the software.
CA1 recordings and stimulation paradigms
To evaluate afferent-induced inhibition in the CA1 region, two paired-pulse stimulation paradigms were used. The first was a heterosynaptic paradigm, in which the TA pathway was activated, followed by the commissural pathway (EC/Comm protocol). Inhibition resulting from stimulation of the TA pathway is reflected by a decrease in the amplitude of the population spike generated by subsequent stimulation of the commissural pathway (see Fig. 3). The second was a homosynaptic paradigm, in which paired-pulse stimuli were delivered to the commissural pathway.
Input/output curves were established by varying the stimulus intensity from threshold to an intensity that evoked the maximal amplitude population spike for the first (conditioning) response. This produced maximal inhibition of the second (test) response. The interval between the conditioning and test pulse (C-T interval) was then varied from 20 to 1,000 ms to define the time course of the inhibition generated by the first pulse.
DG recordings and stimulation paradigms
To determine whether the AOAA treatment affects inhibitory
processes in circuits in which layer III neurons are not involved, we
evaluated paired-pulse inhibition in the DG. Two paired-pulse stimulation paradigms were used. The first was a heterosynaptic paradigm, in which the commissural pathway to the DG was activated, followed by activation of the temporo-dentate projection (Comm/EC protocol). Again, inhibition resulting from activation of the commissural pathway is reflected by a decrease in the amplitude of the
population spike generated by the temporo-dentate pathway (Douglas and Goddard 1975). The second was a
homosynaptic paradigm in which paired stimuli were delivered to the
temporo-dentate pathway (EC/EC protocol).
After all recordings were taken, both DG and CA1 recordings sites were
marked by iontophoretic application of Fast Green from the recording
electrode using 20 µA of bipolar current for 20 min, and the
position of the stimulating electrodes was verified by placement of
small electrolytic lesions. Animal body temperature was maintained by a
heating pad throughout the experiment.
Histological analysis
Animals that were tested using acute neurophysiological techniques received an additional dose of urethan (0.5 g/kg ip) after the recording session and were then perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Brains were removed and placed in 4% paraformaldehyde in 0.1 M PBS and then in 20% sucrose in 0.1 M PBS at 4°C. The rostral pole of the brain was sectioned coronally on a freezing microtome, and the caudal pole was sectioned horizontally. Serial 30-µm sections were taken, and every third section was collected on a gelatin-subbed slide and stained with thionin. Sections from AOAA-injected and vehicle-injected animals were evaluated by two independent individuals blinded with respect to the experimental protocol.
Kindling procedure
A separate group of animals was prepared to evaluate
afterdischarge duration and development of kindling resulting from
high-frequency stimulation of the CA3 region. One month following
either an AOAA (n = 8) or a vehicle (n = 7) injection into the EC, animals were anesthetized with ketamine (50 mg/kg) and xylazine (12.5 mg/kg ip) and placed in a stereotaxic frame
with the nose bar set at +5.0 mm. Twisted, insulated bipolar stainless
steel (0.01 in. diam) electrodes were implanted into the left ventral
hippocampus, ipsilateral to the AOAA injection (electrode placement: AP
3.6 mm from bregma, ML 4.9 mm, DV
5.0 mm); a ground electrode was placed in the frontal sinus. The electrodes were attached to the skull
with jewelers' screws and dental acrylate.
AOAA-injected and control animals were subjected to a rapid kindling
acquisition and retest paradigm beginning 1 wk after electrode
implantation (Lothman and Williamson 1994). For kindling acquisition, an alternate-day protocol was used that entailed delivering 12 stimulus trains (at 50 Hz, 10-s train of 1 ms, biphasic, 200-µA peak-to-peak) for 4 days with a stimulus-free day interspersed between each stimulion day. Therefore the complete kindling acquisition period consisted of a total of 7 days with four stimulation days alternating with three intervening stimulus-free days. Another 7 days
following the kindling acquisition, a retest paradigm was used that
consisted of 12 stimulations with the same stimulus trains used for
kindling acquisition.
SCORING OF KINDLING.
For each stimulus, animals were assigned a behavioral seizure score,
based on a 5-point system (Racine 1972): 1 = wet
dog shakes, 2 = chewing and nodding, 3 = unilateral clonus,
4 = rearing and forelimb clonus, and 5 = rearing and falling.
The behavioral seizure score was assigned by an observer blinded to the
experimental protocol.
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RESULTS |
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As expected based on previous studies (Du and Schwarcz
1992), there was a conspicuous loss of layer III pyramidal
neurons in animals that had received AOAA 30-35 days previously. A
representative case is illustrated in Fig.
2. On the injected side, there were few
neuronal cell bodies in the medial portion of layer III, and instead,
the layer was filled with small cell bodies that had the appearance of
glial cells (Fig. 2, A and C). In addition to the
decrease in neuron density in layer III, the overall thickness of layer
III was reduced in comparison to control (compare Fig. 2, C
and D). The contralateral side appeared normal in all
animals (Fig. 2, B and D). In four animals,
neuronal loss was largely restricted to the medial portion of the EC,
as in the case illustrated in Fig. 2. In three other animals, there was
also a loss of neurons in the lateral part of layer III of the EC, and
in two of these animals there was also some loss of layer II neurons
and subicular neurons. The neuron loss was more extensive in the
ventral portions of the EC, whereas there was typically a greater
degree of preservation of neurons in the layer in more dorsal regions.
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Afferent-induced inhibition in CA1
In normal or vehicle-injected control animals, stimulation of the
Comm pathway without prior activation of the EC elicited a field
excitatory postsynaptic potential (EPSP) with a single prominent
population spike (Fig. 3A,
top trace). Stimulation of the EC elicited a small positive
response with no apparent population spike (Fig. 3A,
bottom trace, 1st response). When Comm stimulation was
preceded by stimulation of the EC, the population spike evoked by
stimulation of the commissural pathway was substantially reduced or
eliminated (Fig. 3A, bottom trace). This is
consistent with previous reports that activation of the TA pathway
produces substantial inhibition (Colbert and Levy 1992;
Empson and Heinemann 1995
; Lacaille and
Schwartzkroin 1988
; Soltesz and Deschenes 1993
).
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The extent of inhibition of the population spike produced by prior activation of the commissural pathway varied as a function of the intensity of stimulation of the EC. Figure 3C illustrates the ratio of the population spike amplitude evoked without prior activation of the commissural pathway (unconditioned) versus population spike amplitude following prior activation of the commissural system (conditioned). The graph plots this unconditioned versus conditioned response ratio across a range of stimulus intensities in both control and AOAA-injected animals. In control animals, higher intensity stimulation produced greater inhibition. Using such plots, the extent of inhibition produced by EC stimulation could be compared in control animals and animals with the AOAA-induced lesions.
The responses elicited in CA1 by stimulation of the commissural pathway were highly abnormal in animals with AOAA-induced lesions (Fig. 3B). In the first place, multiple population spikes were seen, rather than the single spike seen in control animals. Moreover, paired-pulse inhibition was minimal. Comparison of intensity versus inhibition plots of control and AOAA-injected animals revealed that activation of the EC projection produced less inhibition across the entire range of stimulation intensities (Fig. 3C). This difference in the extent of inhibition was highly significant (F = 5.23, P < 0.02).
The extent of heterosynaptic inhibition in CA1 at different conditioning-test (C-T) intervals was determined by varying the C-T interval from 20 to 1,000 ms (Fig. 4). In control animals, heterosynaptic inhibition was seen at C-T intervals between 15 and 100 ms (Fig. 4C). In the example shown in Fig. 4A, the population spike was completely inhibited at a C-T interval of 20 ms. In contrast, in the animals with AOAA-induced lesions, heterosynaptic paired inhibition was substantially reduced. In the example shown in Fig. 4B, there was no apparent heterosynaptic inhibition even at a C-T interval of 20 ms, at which inhibition was prominent in control animals. An analysis of covariance of the values over the entire set of C-T intervals (Fig. 4C) revealed that the difference in the population spike ratios between the control and AOAA-injected animals was highly significant (F = 22.9, P < 0.0001).
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The above results demonstrate that, as expected, the physiological effect of stimulation of the TA pathway (heterosynaptic inhibition) is substantially reduced in animals in which the cells of origin of this pathway have been destroyed by AOAA. The physiological responses induced by single-pulse stimulation of the commissural pathway were also highly abnormal, i.e., stimulation elicited multiple population spikes. To further explore this finding, we evaluated responses to paired-pulse stimulation of the commissural pathway.
Paired-pulse activation of the commissural projection to CA1
In control animals, paired-pulse activation of the commissural projection to CA1 (Comm/Comm) at high intensities (50-60 V) produced a field EPSP with a prominent population spike in response to the first stimulus. The second (test) stimulus produced a field EPSP with either no or a considerably reduced population spike (Fig. 5A). In this homosynaptic paradigm, AOAA-injected animals also exhibited paired-pulse inhibition at the 20-ms C-T interval (Fig. 5B). The inhibition is not the result of a larger population spike in response to the conditioning pulse in AOAA-injected animals (which would produce greater feedback inhibition) because the maximal population spike amplitude was comparable in AOAA and control animals (Fig. 5C). Nevertheless, evaluation of population spike amplitude across a range of stimulus intensities revealed larger population spikes in AOAA-injected animals at low stimulus intensities (Fig. 5C). These results suggest either enhanced excitability or diminished ongoing inhibition in CA1.
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When the extent of inhibition at the 10-ms C-T interval was compared across a range of stimulus intensities, there was no significant difference between the ratios of population spikes elicited by conditioning and test responses in AOAA-injected versus control animals (F = 1.853, P > 0.05). Nevertheless, at all of the higher stimulus intensities (25 V and above), the population spike ratios were lower in control animals, suggesting a greater degree of inhibition.
Although inhibition did not differ significantly at the 10-ms C-T interval, varying the C-T interval revealed substantial differences in the duration of paired-pulse inhibition in AOAA-injected versus control animals (Fig. 6). In control animals, paired-pulse inhibition was seen at C-T intervals up to 100 ms. In AOAA-injected animals, inhibition was seen only at C-T intervals up to 40 ms. Moreover, at C-T intervals of 20-40 ms, there was less inhibition than in control animals. In fact, at C-T intervals longer than 80 ms, paired-pulse potentiation of the population spike was observed (Fig. 6C), and the amplitude of the second and third spike in the series of multiple spikes increased dramatically (see Fig. 6B, traces at C-T intervals of 200 and 400 ms). This paired-pulse potentiation at long C-T intervals was dramatically different from what was seen in control animals, in which paired-pulse potentiation was seen in some individual control animals (Fig. 6B, C-T interval of 200 ms), but was not evident in the group average of the control animals.
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Afferent-induced inhibition in the DG
To determine whether the disruption of inhibition occurred selectively in CA1, we evaluated paired-pulse inhibition in the DG. Stimulation of the EC in control animals produced a robust field EPSP and a large population spike (Figs. 7 and 8). When paired-pulse stimuli were delivered at an intensity sufficient to generate a moderate sized population spike, the population spike elicited by the second stimulus was reduced or eliminated, reflecting paired-pulse inhibition (Fig. 7). In animals that had received an AOAA injection 30-35 days previously, the evoked responses in the DG generated by the EC stimulation did not appear abnormal (there were no multiple population spikes). At the same time, however, the average amplitude of the population spike was smaller across the range of stimulus intensities (Fig. 8C). This may reflect the damage to layer II neurons seen in some animals. Nevertheless, there was no statistically significant difference between AOAA-injected and control animals in the extent of paired-pulse inhibition (Fig. 8D).
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It has been demonstrated that stimulation of the commissural projection
to the DG produces strong inhibition (Steward et al. 1990). Accordingly, when EC stimulation was preceded by
activation of the Comm pathway, the population spike generated by the
EC stimulation was substantially inhibited (Fig. 8A).
Interestingly, the extent of inhibition produced by stimulation of the
commissural projection was less in AOAA-injected animals across the
range of stimulus intensities when measuring at a C-T interval of 20 ms
(Fig. 7C). This difference was not apparent at longer C-T
intervals, however (Figs. 9, A
and B), and there was no statistically significant difference between control and AOAA-injected animals across the range
of C-T intervals.
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These results indicate that there are minimal if any difference in inhibitory function in the dentate gyrus of AOAA-injected animals at the same time that inhibitory function in CA1 is highly abnormal.
Relationship between AOAA-induced cell loss and hyperexcitability
As noted above, the AOAA-injected group included four animals in which the loss of layer III neurons was limited to the medial EC, and three animals with larger lesions. To assess whether the degree of hyperexcitability was related to the extent of cell loss, we compared the two measures of excitability (multiple spiking and the loss of paired-pulse inhibition) in animals exhibiting different degrees of cell loss. For this analysis, we subdivided the AOAA-injected animals into two groups, one of which was made up of the four animals that exhibited cell loss restricted to the medial portion of layer III, and the other of which included the three animals exhibiting more extensive cell loss (see above). All animals exhibited multiple population spikes in response to commissural stimulation whether the lesions were large or small (data not shown). Also, a comparison of the time course of paired-pulse inhibition revealed that both groups of AOAA-injected animals exhibited reduced inhibition at short C-T intervals (Fig. 10). Interestingly, however, animals with a smaller degree of cell loss did not exhibit the same anomalous paired-pulse potentiation at long C-T intervals as the animals with more extensive cell loss. These data suggest that the anomalous paired-pulse potentiation may be related to the larger lesions. This conclusion should be considered provisional, however, because the number of animals in the group with large lesions were small, and the variability in the measure was high.
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Electrolytic lesions of the entorhinal cortex do not disrupt inhibitory function in CA1
If the loss of inhibitory function is related to the loss of TA projections, lesions of the EC should produce similar effects. To test this hypothesis, we evaluated whether electrolytic lesions of the EC caused the same disruption of inhibition within CA1 as AOAA injections. One month following a unilateral electrolytic lesion, we recorded from CA1 (ipsilateral to the lesion) and delivered paired stimuli to the commissural pathway. As illustrated in Fig. 11, the responses evoked by stimulation of the commissural pathway in animals with EC lesions were not noticeably different from control (there were no multiple population spikes). Moreover, there was no significant difference in paired-pulse inhibition at any C-T interval. Together, these results demonstrate that a more extensive, electrolytic destruction of the EC (which would also destroy the cells of origin of the TA pathway) does not lead to the same disruption of inhibition as the comparatively minor EC lesion produced by AOAA.
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Destruction of the AOAA-injected EC does not eliminate altered inhibitory function in CA1
The fact that electrolytic lesions of the EC do not cause any of the anomalies in CA1 found in the AOAA-injected animals suggested two new hypotheses about the possible mode of action of AOAA. One possibility is that either the cell loss or the acute seizures caused by AOAA might create a circuit abnormality within the EC itself. Alternatively, AOAA-induced neuron loss or seizures might produce a circuit or functional abnormality in the hippocampus. One important prediction of the first hypothesis is that physical destruction of the AOAA-injected EC should eliminate the abnormal responses (multiple population spikes and loss of paired-pulse inhibition) within CA1. The second hypothesis would predict that the abnormalities found in the CA1 region of AOAA-injected animals should persist when the EC is destroyed.
To test these hypotheses, we documented the AOAA-induced abnormal physiological responses in a new group of animals. This group exhibited the multiple population spikes, loss of paired-pulse inhibition, and anomalous paired-pulse potentiation in response to commissural stimulation described above. After documenting the responses, we electrolytically ablated the EC that had previously been injected with AOAA and reevaluated responses generated by paired-pulse stimulation of the commissural pathway 10-14 h later. As illustrated in Fig. 12, multiple population spikes and the loss of paired-pulse inhibition were still present after electrolytic lesions of the EC. Interestingly, however, the anomalous paired-pulse potentiation that had previously been seen at long C-T intervals was greatly reduced. Together, these results indicate that the anomalous paired-pulse potentiation, but not the other reflections of altered inhibitory function, are dependent on the presence of the EC that had previously been injected with AOAA.
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Comparison of afterdischarge duration and the rate of kindling in control and AOAA-injected animals
Because inhibitory processes in CA1 are disrupted in animals that had received AOAA, especially the inhibition that is generated by activation of the commissural pathway from the contralateral CA3 region, the affected hippocampus might be more prone to epileptiform activity, particularly if triggered by stimulation of the CA3 region. Specifically, the loss of inhibition might be expected to increase the duration of the afterdischarge following high-frequency stimulation, and to enhance the rate of kindling as a result of the increased stimulation-induced epileptiform activity. To evaluate these questions, we compared afterdischarge duration elicited by high-frequency stimulation of CA3 and the rate of development of kindling in AOAA-injected and control animals. For these experiments, the stimulating electrodes used to induce kindling were positioned in the CA3 region of the hippocampus ipsilateral to the AOAA injection.
Histological analyses revealed that the animals used for the kindling experiment exhibited lesions that were comparable to those of the group assessed neurophysiologically (data not shown). However, there were no differences between AOAA-treated and control animals in the duration of the afterdischarges elicited by the first 12 stimulations (see Table 1). Moreover, the rate of kindling acquisition was indistinguishable in AOAA-treated and control animals (Table 1). There were also no differences in the duration of the afterdischarges across trials, or in the total number of stimulations required to achieve stage 5 seizures, and AOAA-treated animals did not differ from control animals in the retest paradigm (data not shown).
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DISCUSSION |
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The present study was undertaken to evaluate whether selective loss of layer III neurons in the EC, which frequently occurs in temporal lobe epilepsy, disrupts inhibitory processes in the hippocampus. Assessment of inhibitory function using paired-pulse stimulation paradigms in the hippocampus in vivo revealed several physiological abnormalities that suggest altered inhibition. Specifically, animals with AOAA-induced EC lesions exhibited 1) multiple population spikes in response to afferent activation, 2) decreased paired-pulse inhibition in response to stimulation of the commissural projection system, and 3) enhanced paired-pulse potentiation of the population spike at long C-T intervals. Inhibitory processes in the DG appeared to be largely unaffected, suggesting that the alterations in inhibitory function occurred selectively in CA1. Despite the alterations in hippocampal inhibitory function, animals with unilateral AOAA-induced lesions did not exhibit longer afterdischarges or more rapid kindling in response to high-frequency stimulation of the affected hippocampus. These findings provide important clues about the possible physiological abnormalities that might result from the loss of layer III neurons.
Our experiments also revealed that complete electrolytic lesions of the EC did not cause any of the changes in inhibition that were seen following AOAA. Also, secondary lesions of the EC that had suffered AOAA-induced damage did not affect two key measures of disrupted inhibition (multiple population spikes and reduced paired-pulse inhibition), but did reduce the anomalous paired-pulse potentiation seen at long C-T intervals. These findings indicate that the principal indexes of disrupted inhibition in CA1 (multiple population spikes and reduced paired-pulse inhibition) are not due to aberrant circuitry within the AOAA-injected EC but may reflect abnormalities intrinsic to CA1. The implications of these findings are discussed below.
Key indexes of altered inhibitory function in CA1
Two of the three alterations seen after AOAA strongly suggest a disruption of inhibitory function in CA1: 1) multiple population spikes in response to afferent activation and 2) decrease in paired-pulse inhibition.
Multiple population spikes are the expected consequence of any process
that disrupts inhibition. In normal animals, single-pulse stimulation
of the commissural pathway produces a monosynaptic EPSP in pyramidal
cells, which is reflected in extracellular recordings as a
"population EPSP." At sufficiently high stimulus intensities, this
EPSP generates action potentials that are reflected by a single
population spike. Collaterals from pyramidal cells innervate inhibitory
interneurons within the hippocampus, so that the massive discharge of
the pyramidal cells produces feedback inhibition (Andersen et
al. 1971). Afferents also contact interneurons directly, leading to feed-forward inhibition (Alger and Nicoll
1982
). The inhibition produced by afferent stimulation is
thought to play an important role in limiting repetitive cell firing,
and to be the reason that only one population spike is seen regardless
of the intensity of afferent stimulation. Thus when inhibition is intact, only one population spike is generated.
The strong inhibition generated by afferent stimulation also accounts for the decreases in amplitude of the population spike generated by the second of two stimuli in a paired-pulse stimulation paradigm. It is not possible to distinguish whether the inhibition generated by activation of a particular afferent system is mediated by feedback or feed-forward circuits or a combination of both when the stimulation strongly activates the principal neurons (as in the case of stimulation of the commissural pathway). In the case of activation of the TA pathway, however, the stimulation does not strongly activate the pyramidal neurons, so that it is likely that the inhibition is mediated primarily by feed-forward circuitry (see next section).
Consistent with this interpretation, manipulations that disrupt
GABAergic inhibition cause multiple spiking and a loss of paired-pulse
inhibition. For example, following blockade of GABAergic neurotransmission with bicuculline, a single stimulus generates multiple spikes, and paired-pulse inhibition is eliminated
(Kapur et al. 1989; Sloviter and Brisman
1995
; Thiels et al. 1994
). Inhibitory function
in CA1 is also disrupted following prolonged high-frequency stimulation
of the perforant pathway, and an important reflection of this is the
appearance of multiple population spikes in response to afferent
stimulation and a loss of paired-pulse inhibition (Sloviter
1991
). Thus the most likely explanation of the multiple population spikes and disruption of paired-pulse inhibition following AOAA injections is that local inhibitory processes in CA1 are disrupted.
Hypothesis of "projected inhibition"
One possible explanation of the alterations in inhibitory function
in CA1 derives from the hypothesis that the TA pathway activates
hippocampal interneurons and thus mediates projected inhibition.
Several physiological studies have shown that the monosynaptic
projection from the EC to CA1 produces excitatory responses in CA1
(Andersen et al. 1965; Doller and Weight
1982
; Steward et al. 1973
; Yeckel
and Berger 1990
, 1995
). Nevertheless, activation
of the pathway does not trigger population spikes in CA1 (Leung
1995
). Other studies demonstrated that activation of the TA pathway produced only a weak excitatory response that is masked
by strong inhibition (Buszaki et al. 1995
;
Colbert and Levy 1992
; Empson and Heinemann
1995
; Jones 1995
; Soltesz and Deschenes
1993
; Stringer and Colbert 1994
). An inhibitory
effect is also indicated by the fact that activation of the
temporo-dentate pathway produces strong inhibition of cell firing in
response to subsequent stimulation of other pathways (Colbert
and Levy 1992
; Empson and Heinemann 1995
;
Soltesz and Deschenes 1993
). This latter effect was
confirmed in the present experiments.
If the temporo-dentate pathway mediates projected inhibition, damage to
the pathway would reduce the excitatory drive to hippocampal interneurons, producing disinhibition. The idea of a loss of excitatory drive of interneurons is similar to the "dormant basket cell
hypothesis" proposed by Sloviter (1991) to account for
the loss of inhibition following prolonged high-frequency stimulation
of the perforant path. This idea was advanced because there was no
detectable loss of interneurons in CA1 following prolonged perforant
path stimulation despite the loss of inhibition. Hence it was proposed
that the basket cells were present but "dormant." If inhibitory
interneurons in AOAA-injected animals are dormant because of a loss of
afferent drive, the resulting disinhibition could account for two of
the three response abnormalities discussed above, namely the multiple population spikes in response to afferent activation and the decrease in paired-pulse inhibition.
An important prediction of the disinhibition hypothesis is that physical destruction of the EC should produce the same disruption of inhibition in CA1 as seen following AOAA-induced degeneration of layer III neurons, because such lesions would also remove the excitatory drive provided by layer III neurons. However, our results demonstrated that electrolytic EC lesions do not lead to detectable alterations in inhibitory function in CA1.
Possible mechanisms at the circuit level
There are several possible explanations for our findings that are
based on circuit abnormalities that might result from the lesions.
Injections of AOAA into the EC result in the loss of layer III
pyramidal neurons while layer II neurons (the cells of origin of the
perforant pathway) are largely preserved (Du and Schwarcz
1992; Du et al. 1998
). Thus the physiological
abnormalities observed in the present study could be due to a
disruption of the balance between the inhibition generated by the TA
pathway and polysynaptic excitation of CA1 pyramidal cells (via the
trisynaptic circuit).
A related possibility is that the temporo-dentate pathway might have two components, one of which projects to interneurons, and another that projects directly to pyramidal neurons in CA1. In this scenario, AOAA might preferentially kill those layer III neurons in the EC that project to the interneurons that mediate projected inhibition, and spare those neurons that give rise to direct excitatory projections to CA1 pyramidal cells. Again, this would disrupt the balance between projected inhibition and excitation of CA1 pyramidal cells. In either of the above cases, complete electrolytic lesions of the EC would not have the same effect as a selective lesion because both projected inhibition and polysynaptic excitation would be lost.
A third possibility is that the loss of layer III neurons in the EC
could create a circuit abnormality within the EC itself. These neurons
probably participate importantly in intra-entorhinal circuitry, and
their loss could disrupt overall functional activity. The loss of layer
III neurons could also trigger synaptic reorganization within the EC,
modifying circuit function. In this regard, it is of interest that
physiological studies of AOAA-treated EC slices reveal important
physiological abnormalities (Scharfman et al. 1998) (see AOAA model below).
Also, multiple spiking and a loss of inhibition in CA1 is seen as early
as 7 days after the AOAA injections (Denslow et al.
1995a
,b
), indicating that the alterations in inhibitory
function can occur rather rapidly after the lesions.
An important prediction of all of these hypotheses is that secondary lesions of the EC that had previously been injected with AOAA should eliminate the physiological indexes of disrupted inhibition. This was not the case, however. Indeed, both multiple population spikes and diminished paired-pulse inhibition were still seen in the animals after secondary electrolytic lesions of the AOAA-injected EC. Only the late anomalous paired-pulse potentiation was disrupted by secondary lesions of the AOAA-injected EC. Thus anomalous paired-pulse potentiation could be related to circuit abnormalities within the EC, whereas the multiple spiking and loss of paired-pulse inhibition most likely reflects a process intrinsic to CA1.
Possible alterations in CA1
The explanation that is most consistent with the present data is
that the multiple population spikes and loss of paired-pulse inhibition
reflect a disruption of inhibitory mechanisms within CA1 itself.
Possible mechanisms include 1) decreased release of an
inhibitory neurotransmitter (see, for example, Gibbs et al. 1996), 2) altered receptor function, 3) a
loss of inhibitory synapses, and 4) loss of a population of
inhibitory interneurons. It remains to be established whether such
changes do occur and if they are in fact triggered by intra-entorhinal
AOAA injections. Such alterations could be a consequence of
AOAA-induced acute seizures or deafferentation, and/or synaptic
reorganization within CA1.
AOAA model
One of the strengths of the "AOAA model" is that
hyperexcitability in CA1 can be examined with little direct pathology
in the hippocampus (Du and Schwarcz 1992). This is
distinct from other models of epilepsy, such as those induced by kainic
acid or prolonged hippocampal stimulation, in which there is extensive neuron death in the hippocampus, as well as the parahippocampal region,
and other extrahippocampal areas. In those models it is therefore more
difficult to identify the cause of altered function that makes the
circuit more prone to seizures. The AOAA model involves a localized
lesion (layer III of the EC) and a resulting hyperexcitability in a
region (CA1) downstream from the affected site.
A disadvantage of the present model is that it is difficult to assess the dorso-ventral gradient of the alterations in inhibition because it is complicated to evaluate the ventral hippocampus in vivo. Therefore further studies are needed where hippocampal slices are taken from AOAA-injected animals along the dorsal-ventral extent of the hippocampus to determine whether the more ventral hippocampus also exhibits disrupted inhibition.
Although a circuit abnormality within the EC cannot account for the
alterations in inhibitory function in CA1 (see above), this does not
imply the absence of an important circuit abnormality within the
AOAA-injected EC. Indeed, intracellular recordings from neurons in EC
slices from animals that had received AOAA in vivo revealed that
surviving neurons in superficial layers exhibited abnormal, prolonged
repetitive EPSPs in response to white matter stimulation
(Scharfman et al. 1998). Similar excitable repetitive
responses were seen in vivo following stimulation of the CA1/subicular
border. The circuitry that mediates these responses has not been
defined, and the neurons in superficial layers that exhibit abnormal
repetitive activity are likely to be those that project to the dentate
gyrus rather than to CA1 (Steward 1976
). Nevertheless,
similar excitatory activity may occur in other neuronal populations in
the EC, including any surviving cells of origin of the TA pathway.
Conceivably, abnormal physiological responses within the EC due to the
loss of layer III neurons could play a key role in triggering seizure
activity in temporal lobe epilepsy (see next section).
It is noteworthy that Scharfman et al. (1998) also
described hyperexcitability in the EC contralateral to the
AOAA-injected hemisphere, and we have also observed evidence of
disrupted inhibition in CA1 contralateral to the AOAA injection
(Denslow et al. 1995a
). Although the cellular basis of
these evoked responses was not completely characterized, those
abnormalities resembled the multiple population spikes described in the
present study. Given the commissural projections between the entorhinal
cortices in the two hemispheres (Blackstad 1956
), there
may be a common mechanism underlying the abnormal activity in
structures that receive synaptic input from the AOAA-injected EC.
Implications for seizure susceptibility
Given the disruption of inhibition in CA1, especially the
inhibition generated by stimulation of the commissural pathway, the
absence of detectable differences in afterdischarge duration or in the
rate of development of kindled seizures in animals with AOAA-induced
lesions was surprising. In many situations, disruption of inhibition is
associated with an increase in seizure susceptibility. For example,
prolonged electrical stimulation of the hippocampus creates an
epilepsy-prone state in which spontaneous electrographic seizures
continue after the stimulation has ceased (Lothman et al.
1995). Animals that exhibit such spontaneous seizures also exhibit physiological changes similar to those described here, including multiple population spikes in response to afferent
stimulation, and diminished paired-pulse inhibition (Lothman et
al. 1995
; Mangan et al. 1995
). At the same time,
however, there is evidence that a disruption of GABAergic inhibition
does not invariably lead to seizures. Apparently, inhibition must be
disrupted to some threshold level for spontaneous seizures to occur
(Kapur et al. 1989
). The loss of inhibition, which
results from the AOAA-induced lesion, may be below this threshold, or
there may be mechanisms that compensate for the loss of inhibition and
thus decrease susceptibility to seizures, at least in CA1. It is also
important to note that the AOAA-induced lesions in the present
experiments were unilateral. It remains to be seen whether there are
differences in afterdischarge duration or in the rate of development of
kindled seizures when the kindling involves pathways more closely
related to the affected EC.
Possible significance of loss of layer III neurons in temporal lobe epilepsy
Studies from patients with TLE provide evidence implicating the EC
as the source of seizure generation in the hippocampal region. For
example, EEG studies of patients with TLE suggest that the EC may be
the site of seizure initiation (Spencer and Spencer
1994). Also, in patients with epilepsy resulting from tumors in
the EC, seizures cease when the EC with the tumor is removed.
Our evaluations of afterdischarge threshold and propensity to kindling
suggest that the loss of layer III neurons in the EC produced by focal
AOAA injections is not sufficient to lead to a seizure-prone circuit in
the hippocampus. Nevertheless, increased excitability in the EC
(Scharfman et al. 1998) could serve as a focus for
seizure initiation that could then propagate through hippocampal
circuits in which inhibition was diminished. Thus, although loss of
layer III neurons is not sufficient to promote kindled seizures in the
hippocampus, it may still be an important component in the propagation
of seizure activity generated in the EC. In any case, our results
suggest that a loss of layer III neurons such as occurs in TLE does
have important consequences on hippocampal function. Because these
changes appear to be intrinsic to the hippocampus, it should be
possible to test for their presence in tissue that has been surgically
removed for seizure management.
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ACKNOWLEDGMENTS |
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This project was initiated in the laboratory of Dr. Eric Lothman, who was Dr. Denslow's dissertation advisor. For this reason, the work should be considered a product of his lab. We thank J. Williamson for technical help in the kindling protocol, M. Hofstadter for preparing the final illustrations, and Dr. Helen Scharfman for comments on an early version of the manuscript.
This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-25605 to E. W. Lothman, NS-32280 and NS-12333 to O. Steward, and NS-16102 to R. Schwarcz.
Present address of M. J. Denslow: Dept. of Pathology, Harvard Medical School, Boston, MA 02215.
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FOOTNOTES |
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Present address and address for reprint requests: O. Steward, Director, Reeve-Irvine Research Center, College of Medicine, 1105 Gillespie Neuroscience Research Facility, University of California, Irvine, CA 92697-4292 (E-mail: osteward{at}uci.edu).
Received 9 November 1999; accepted in final form 26 July 2001.
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