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2019
Partial depletion of septohippocampal cholinergic cells reduces seizure
susceptibility, but does not mitigate
hippocampal neurodegeneration in the kainate model of epilepsy
Soares, JI
Elsevier BV
Tieteelliset aikakauslehtiartikkelit
© Elsevier B.V.
CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/
http://dx.doi.org/10.1016/j.brainres.2019.04.027
https://erepo.uef.fi/handle/123456789/7605
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Accepted Manuscript
Research report
Partial depletion of septohippocampal cholinergic cells reduces seizure sus- ceptibility, but does not mitigate hippocampal neurodegeneration in the kainate model of epilepsy
Joana I. Soares, Catarina Da Costa, Maria H. Ferreira, Pedro A. Andrade, Gisela H. Maia, Nikolai V. Lukoyanov
PII: S0006-8993(19)30229-X
DOI: https://doi.org/10.1016/j.brainres.2019.04.027
Reference: BRES 46232
To appear in: Brain Research Received Date: 17 February 2019 Revised Date: 22 April 2019 Accepted Date: 23 April 2019
Please cite this article as: J.I. Soares, C. Da Costa, M.H. Ferreira, P.A. Andrade, G.H. Maia, N.V. Lukoyanov, Partial depletion of septohippocampal cholinergic cells reduces seizure susceptibility, but does not mitigate hippocampal neurodegeneration in the kainate model of epilepsy, Brain Research (2019), doi: https://doi.org/10.1016/j.brainres.
2019.04.027
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Partial depletion of septohippocampal cholinergic cells reduces seizure susceptibility, but does not mitigate hippocampal neurodegeneration in the kainate model of epilepsy
Joana I. Soaresa,b,c,d*, Catarina Da Costac, Maria H. Ferreirac, Pedro A. Andradee, Gisela H. Maiaa,b,c, Nikolai V. Lukoyanova,b,c
a Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal
b Instituto de Biologia Molecular e Celular da Universidade do Porto, Porto, Portugal
c Departamento de Biomedicina, Faculdade de Medicina da Universidade do Porto, Porto, Portugal
d Programa Doutoral em Neurociências, Universidade do Porto, Porto, Portugal
e University of Eastern Finland, A.I. Virtanen Institute, Kuopio, Finland
* Corresponding author at: Neuronal Networks Group Instituto de Investigação e Inovação em Saúde
Rua Alfredo Allen, 208 4200-135 Porto, Portugal
E-mail address: joana.isa.soares@gmail.com (Joana I. Soares).
Abstract
The brain cholinergic system may undergo structural and functional alterations both in human epilepsy and in respective animal models, but the causal relationships between these alterations and epilepsy remain to be established. In this study, we attempted to examine how the inhibition of epilepsy-related cholinergic plasticity may be reflected in seizure susceptibility and/or in the development of chronic epilepsy and its neurological consequences. For this purpose, adult Wistar rats received intrahippocampal injections of low doses of 192-IgG-saporin (SAP) to produce a moderate, but significant loss of septohippocampal cholinergic cells and to suppress their plasticity. Then, animals were treated with kainic acid to induce status epilepticus, which leads to the development of chronic epilepsy later in life. It was found that SAP-pretreatment was associated with longer latency to the onset of status epilepticus and with reduced mortality rate, suggesting that increased activity of septal cholinergic cells may potentiate seizures.
Interestingly, months later, a greater percentage of rats with intact septohippocampal cholinergic connections showed spontaneous seizures, when compared to SAP- pretreated rats. Treatment with kainic acid produced death of 40-50% of hippocampal neurons and this effect was not ameliorated by prior cholinergic depletion. Moreover, the kainate induced cognitive deficits were detected in both SAP-pretreated and sham- pretreated groups. These data suggest that seizure-induced plasticity of cholinergic cells may indeed enhance seizure susceptibility and contribute to epileptogenic processes.
They do not support the hypothesis that epilepsy-related hypertrophy of cholinergic neurons may potentiate hippocampal cell loss and respective behavioral impairments.
Keywords: medial septum and diagonal band of broca; saporin; kainate; stereology;
temporal lobe epilepsy.
Highlights
SAP prevents seizure-induced plastic alterations in cholinergic cells
Acetylcholine depletion reduces seizures susceptibility
Effects of kainate in the hippocampus is not SAP-dependent
1. Introduction
The central cholinergic pathways have their origin in either the basal forebrain, which includes the medial septum (MS), vertical/horizontal limbs of the diagonal band of Broca (DB) and nucleus basalis of Meynert (NB), or the mesopontine tegmentum, which includes the pedunculopontine (PPN) and laterodorsal (LDT) nuclei (Mesulam et al., 1983; Wainer et al., 1993). Neuroanatomical evidence suggests that neurons located in the basal forebrain preferentially project to telencephalic structures, whereas mesopontine cholinergic cells innervate the spinal cord, brainstem, thalamus and hypothalamus (Mesulam et al., 1983; Rye et al., 1988). However, the rostral diencephalon and basal ganglia, and part of the limbic cortex have an overlapping innervation from both nuclear groups (Mena-Segovia, 2016). Cholinergic afferents provide both tonic and phasic modulation of the activity of their target cells and are involved in a wide range of brain functions. In particular, the septohippocampal and basocortical cholinergic pathways have been implicated in various aspects of cognition and emotion (Drever et al., 2011; Gu and Yakel, 2011). The mesopontine cholinergic cells, in turn, constitute an important part of the reticular activating system (RAS), participating in the control of arousal, consciousness and sleep-wake cycles (Moruzzi and Magoun, 1949). Thus, dysfunctional states of central cholinergic pathways are believed to account, partly at least, for a number of cognitive and behavioral deficits
associated, in particular, with aging and different neuropathological conditions (Chin and Scharfman, 2013).
A growing body of evidence links changes in the ascending cholinergic pathways to seizure susceptibility and epilepsy (for review see (Friedman et al., 2007). Studies of surgical specimens obtained from patients with temporal lobe epilepsy (TLE), the most common form of focal epilepsy in humans, have shown altered activity of the cholinergic marker enzymes, acetylcholinesterase (AChE) and choline acetyltransferase, ChAT (Green et al., 1989; Kish et al., 1988). In addition, high-resolution MRI studies have revealed septal nuclei enlargement in TLE patients without hippocampal sclerosis (Butler et al., 2013; Butler et al., 2014). Similarly, it has been reported that induction of epilepsy in animal models produces hypertrophic changes in septal neurons immunoreactive to either ChAT or vesicular acetylcholine transporter (VAChT) accompanied by a profound reorganization of the cholinergic fiber network in the hippocampal formation (HF) (Soares et al., 2017).
Despite the existing evidence for the involvement of cholinergic transmission in seizures and epilepsy, the functional implications of the cholinergic fiber network reorganization in epileptic brain are not clear. It has been suggested that such a reorganization, namely sprouting of the cholinergic fibers into molecular layer of the dentate gyrus and their removal from the dentate hilus, would make hippocampal circuits hyperexcitable (Soares et al., 2017), a mechanism which has long been implicated in neuropathology of TLE (Tauck and Nadler, 1985; Zeng et al., 2009). This possibility is supported by the findings showing that application of acetylcholine to hippocampal slices from epileptic rats, but not from control rats, produces epileptic-like neuronal discharges (Zimmerman et al., 2008). On the other hand, complete or nearly complete cholinergic cell depletion using immunotoxin 192-IgG-saporin (SAP, saporin-
conjugated antibody against p75 neurotrophin receptor that is only expressed at high levels by the basal forebrain cholinergic neurons) facilitates the development of kindling in the rat (Kokaia et al., 1996) and exacerbates seizure-related loss of the somatostatin-containing cells in the dentate hilus (Jolkkonen et al., 1997). One way to address this controversy is to block the seizure-induced fiber sprouting in cholinergic cells, while preventing their severe depletion. This can be achieved by treating rats with rapamycin, known to block key biochemical processes involved in axonal growth (Zeng et al., 2009). However, rapamycin also suppresses fiber sprouting in other types of neurons, including mossy fiber sprouting of the dentate granule cells, which has been suggested to play an important role in epileptogenesis (Buckmaster and Lew, 2011;
Wong, 2013). In the present study, we hypothesized that intracerebral infusion of SAP at subtoxic levels would specifically inhibit or moderate, at least, fiber sprouting in cholinergic cells, because it does not react with other cell types and because its active part, saporin, is known to block protein synthesis required for axonal growth. Thus, in a series of pilot experiments we determined a lowest dose of SAP at which, when infused directly into the hippocampal formation (HF), it produces a very small, but significant loss of cholinergic cells in the MS/DB. Then, we treated a group of rats with this
“subtoxic” dose of SAP and evaluated their susceptibility to kainate-induced status epilepticus (SE). The long-term consequences of kainate treatment in these rats, that is, spontaneous seizures, behavioral deficits and hippocampal neurodegeneration were compared to those in rats treated alone with either kainate or vehicle.
2. Results
2.1 Seizures and mortality
After KA injection, 9 out of 10 rats in KA group and 9 out of 10 rats in SAP+KA group showed a typical pattern of status epilepticus, that is, continuous stage 3 to 5 seizures on the Racine scale. One rat in KA group demonstrated numerous wet-dog-shake seizures 15-120 min after the injection of KA, which however did not progress to stage 3 or 4 seizures. No seizures were observed in rats injected with saline alone (control and SAP groups). Statistical analysis showed that there was no effect of SAP injection on the development of SE. However, there was a significant effect of SAP injection on the latency to SE (F(1,16)=8.83, p<0.01; Table 1). In particular, rats in the KA group developed SE almost twice as rapidly as rats in the SAP+KA group (46.8±7.9 min vs.
75.7±5.6 min). In addition, status epilepticus in the rats with intact cholinergic system frequently progressed into prolonged tonic-clonic seizures, including full hindlimb extension, while no prolonged tonic-clonic seizures were observed in SAP-pretreated rats. Despite all the efforts made to reduce the mortality rate, five of the sham-treated rats died after KA injection: one – within 24h, three – during the first week, and one – eight weeks post SE. Thus, the final size of this group at the beginning of behavioral tests was 5 animals. No animals pretreated with SAP died after KA injection (final n=10). Statistical analysis revealed a significant protective effect of SAP treatment
against kainate-induced mortality (H(1,20)=6.33, p<0.01).
During the entire period of observation, at least two behavioral seizures of stage 3 to 5 were observed in 5 out of 6 rats from the KA group (83%) and in 4 out of 10 rats from the SAP+ KA group (40%). In addition, offline review of the video-EEG recordings revealed the presence of seizure-like EEG activity in 5 out of 5 KA rats (one rat showing particularly frequent motor seizures died before the initiation of EEG recordings) and in 5 out of 10 rats in the SAP+KA group (Table. 1). The electrographic seizures in these animals were often associated with behavioral seizure manifestations,
such as stereotypic orofacial movements, periods of sudden immobility, clonus and clonus with rearing. Non-parametric ANOVA failed to show a significant effect of SAP pretreatment on the presence of recurrent behavioral seizures in post-SE rats (H(1,16)=2.68, p=0.10). However, when considering recurrent behavioral seizures and EEG seizures together, there was a significant effect of SAP pretreatment on the number of post-SE rats that developed spontaneous seizures later in life (H(1,16)=4.09, p<0.05).
Fig. 1 shows a representative EEG recordings from all the different groups.
2.2 Step-through passive avoidance and fear conditioning
Two-way repeated measures ANOVA revealed a significant main effect of training (F(1,23)=26.60, p<0.001) on the performance of rats in the passive avoidance test, as well as a significant effect of KA treatment (F(1,23)=17.33, p<0.001). ANOVA did not detect a significant effect of SAP pretreatment (F(1,23)=1.70, p=0.20). However, there were the following significant interactions between these effects: SAP×KA (F(1,23)=14.41, p<0.001), and SAP×KA×training (F(1,23)=6.43, p<0.01). Examination of the results of this test (Fig. 2) shows that all groups, except KA group, had higher latency to enter the dark compartment on the retention trial when comparing to training trial, i.e., successfully learned the task. In contrast, rats in KA group did not improve their performance, showing the retention latencies significantly inferior to those of control rats and rats from SAP group (p<0.001). Interestingly, KA-injected rats that were pretreated with SAP (SAP+KA group) learned the task better than sham-pretreated rats (KA group; p<0.05). However, their memory was also impaired when comparing to control rats and rats treated with SAP alone (p<0.01).
Fig. 3A shows the amount of freezing time observed in rats when they were placed into the training chamber 1 day after fear conditioning. Two-way ANOVA
applied on these data revealed a significant main effect of KA injection (F(1,22)=65.62, p<0.001). However, no significant effect of SAP pretreatment and no interaction
between these two effects were found, suggesting that both KA-treated groups froze to the familiar context significantly less than control and SAP groups, but did not differ between them. Similarly, the tone presentation in the novel context induced increased levels of freezing in control and SAP groups, but not in KA and SAP+KA groups (Fig.
3B). Consistent with this, two-way ANOVA of these data showed a significant effect of KA treatment (F(1,22)=49.45, p<0.001), but no significant effect of SAP pretreatment and no significant interactions. Together, these data indicate that KA-treated rats, either pretreated with SAP or not, were equally impaired in both fear conditioning tests. One animal in the SAP+KA group was excluded from the fear conditioning task due to a tail lesion.
2.3 Total number and somatic volume of VAChT-immunoreactive cells
Visual inspection of representative images obtained from immunostained brain sections (Fig. 4) showed a greater number of VAChT-positive cells in MS/DB region of KA- treated rats (Fig. 4B) when compared to rats in the other three groups (Fig. 4A,C,D).
The stereological estimates of the total number of these cells in all groups are illustrated in Fig. 5. Two-way ANOVA of these data revealed a significant effect of KA injection (F(1,23)=30.12; p<0.001), a significant effect of SAP pretreatment (F(1,23)=82.47;
p<0.001), and a significant interaction between the two effects (F(1,23)=20.82;
p<0.001). Consistent with our previous report (Soares et al., 2017), KA-treated rats had
almost as twice as many VAChT-stained neurons in the MS/DB comparing to controls (p<0.001 for post-hoc test). Interestingly, SAP-pretreated rats had somewhat (25%) lower number of VAChT-IR neurons when compared to control (p<0.05) and SAP-
pretreatment prevented the KA-induced increase in the number of VAChT-IR septal cells (p<0.001 for comparison between the KA group and SAP+KA group).
The mean somatic volume of the VAChT-stained cells measured in the MS/DB of control, KA- and SAP-treated rats are shown in Fig. 6. Statistical comparison of these estimates revealed the effect of KA injection and the effect of SAP pretreatment both just failed to reach significant level (F(1,23)=3.44, p=0.076 and F(1,23)=4.13, p=0.054, respectively). However, two-way ANOVA revealed a significant interaction between these two effects (F(1,23)=10.43; p<0.05). Post-hoc comparisons confirmed that the VAChT-stained cells of KA-treated rats had approximately 25% larger cell bodies when compared to control group (p<0.05) and to SAP+KA group (p<0.01). However, the somatic volumes in control and SAP-treated rats were statistically indistinguishable.
2.4 Areal density of VAChT-immunoreactive fiber varicosities
Fig. 7 shows representative photomicrographs of the dentate gyrus taken from the VAChT-immunostained brain sections of rats in different treatment groups used in this study. These images suggest that either SAP alone (Fig. 7G,H) or KA alone (Fig. 7D,E) treatments were associated with reduced density of VAChT-stained fibers and fiber varicosities in the dentate hilus when compared to controls (Fig. 7A,B). However, the lowest density of hilar fibers across all groups was revealed in rats from the SAP+KA group (Fig. 7J,K). In the inner molecular layer, in contrast, the density of VAChT- positive varicosities appeared increased in the rat treated with KA alone (Fig. 7D,F), but it was noticeably decreased in rats from the SAP group (Fig. 7G,I) and SAP+KA group (Fig. 7J,L). The means of the areal densities of fiber varicosities in the dentate hilus are shown in Fig. 8A. Two-way ANOVA of these data revealed both significant SAP-effect (F(1,23)=10.58; p<0.01) and significant KA effect (F(1,23)=6.27; p<0.05). However,
the interaction between the two effects was not observed (F(1,23)=0.41; p=0.5), suggesting that the profound loss of hilar VAChT-IR varicosities represents a simple net effect of SAP pretreatment and kainate-induced SE.
The means of the areal densities of fiber varicosities in the inner molecular layer are shown in Fig. 8B. Statistical comparison of the density estimates obtained in this area showed a significant effect of SAP pretreatment (F(1,23)=39.13; p<0.001), but the effect of KA did not reach significance (F(1,23)=1.90; p=0.18). However, ANOVA revealed a significant interaction between the SAP and KA effects (F(1,23)=12.49;
p<0.01). Post-hoc comparisons confirmed that KA treatment was associated with
increased density of VAChT-IR varicosities in the inner molecular layer (p<0.01 vs.
control; Fig. 8B) and that SAP-pretreated rats that were injected with KA had less varicosities than control rats (p<0.01) and rats treated with KA alone (p<0.001).
However, post-hoc tests did not confirm that SAP pretreatment alone produced a significant reduction in the density of VAChT-stained varicosities in this area (p=0.26 vs. control, but p<0.001 vs. KA group).
Because part of animals in the SAP+KA group developed spontaneous seizures, while another part did not, we were interested to analyze whether these two subgroups show different degrees of cholinergic fiber sprouting into the inner molecular layer. For this purpose, individual sprouting scores from 1 to 10 were attributed to each of the animals in these subgroups based on the varicosity density estimates. Although the nonparametric Kruskal-Wallis ANOVA failed to reveal a significant relationship between the presence of spontaneous seizures and the sprouting score, it detected a clear tendency in this direction (H(1,10)=2.50, p=0.11). Thus, it is possible that the lack of statistical significance in this analysis is due to the small sample size (n=5 in each of the two subgroups).
2.5 Total number of hippocampal cells
Representative photomicrographs of Nissl-stained brain sections containing the HF are shown in Fig. 9. As illustrated on these images, animals treated with KA (KA and SAP+KA groups) had reduced density of neurons in all principal subdivisions of the HF, typically dispersed granule cell layer and deformed anatomical organization of hippocampal layers. The stereological estimates of the total numbers of neurons in the hilus of the dentate gyrus, and CA3 and CA1 fields of the hippocampus are shown in Fig. 10. Two-way ANOVA of these estimates revealed that there was a significant main effect of KA treatment on neuron numbers in all three hippocampal subdivisions:
dentate hilus (F(1,23)=70.19, p<0.001), pyramidal CA3 field (F(1,23)=10.66, p<0.01) and pyramidal CA1 field (F(1,23)=59.92, p<0.001). However, the main effect of SAP pretreatment and the interaction between the effects of SAP and of KA were statistically nonsignificant.
3. Discussion
One of the main findings of this experiment is that intrahippocampal injections of SAP at reduced doses, while producing loss of a small number of cholinergic cells in the MS/DB, prevented hypertrophic changes in the remaining cholinergic cells that were expected to occur following the induction of SE. Importantly, SE in SAP-pretreated rats developed less rapidly than in sham-pretreated rats and was characterized by reduced mortality rate. The long-term consequences of KA-induced SE in SAP-pretreated rats and control rats were also different: both cognitive deficits and spontaneous seizures
were partly prevented by SAP injections. However, the neurodegenerative changes in this epilepsy model, i.e., death of hippocampal neurons, were not dependent on SAP treatment.
Consistent with prior studies (Holtzman and Lowenstein, 1995; Soares et al., 2017), the current results show that KA-induced SE produces hypertrophic changes in the MS/DB cholinergic cells, which are accompanied by significant reorganization of their efferent terminals, namely reallocation of cholinergic fibers from the dentate hilus to the inner molecular layer. Further, the low-dose intrahippocampal injections of SAP, in contrast, leads to a small, but significant overall net loss VAChT-IR terminals in the DG. More importantly, we found that post-SE rats that were pretreated with SAP show neither cholinergic cell hypertrophy nor abnormally high increase in the density of VAChT terminals in the inner molecular layer. Thus, these data support the idea that SAP pretreatment is capable of abolishing SE-induced fiber plasticity and somatic hypertrophy in the MS/DB cholinergic cells. Given that this immunotoxin selectively affect only the basal forebrain cholinergic cells, it is likely that plasticity of other types of fibers, e.g., mossy fiber sprouting, was not blocked in post-SE rats.
Abnormal excessive neuronal activity during seizures is believed to enhance activity of the brain cholinergic pathways (Hillert et al., 2014). For example, it has been reported that both the lithium/pilocarpine- and the KA-induced seizures cause strong and fast (1-3h) increases in the levels of choline and acetylcholine in the rat cortex and hippocampus (Jope and Gu, 1991). Similarly, studies of surgical specimens obtained from TLE patients have shown increased activity of ChAT and AChE in the spiking vs.
non-spiking cortex (Kish et al., 1988). It has been suggested that the acutely elevated levels of ACh may be neuroprotective against seizures, for example, due to the anti- inflammatory or neurotrophic effects of this neurotransmitter in the brain (Gnatek et al.,
2012). However, under conditions of a prolonged seizure activity, such as SE, elevated ACh release is capable of increasing neuronal depolarization, via its action on muscarinic M1 receptor (Mingo et al., 1997; Sheffler et al., 2009; Smolders et al., 1997), which may contribute to further genesis and maintenance of seizures (Zimmerman et al., 2008). The results of the present study are fully consistent with this possibility, because they show that progression of SE is slower in rats that have partly depleted septohippocampal cholinergic pathway. In addition, the course of SE in these animals was characterized by less severe signs than in control group, as none of them, unlike control rats, showed tonic-clonic seizures. Another indication for the reduced seizure severity in rats with blunted ACh plasticity was the fact that none of rats in this group died as a consequence of KA injection, whereas in rats with intact cholinergic activity the final mortality rate reached 50%.
At longer post-SE times, an increased expression of enzymes involved in ACh metabolism, namely of AChE, is likely to represent a compensatory response destined to neutralize the excessive amounts of ACh in the intercellular space (Kish et al., 1988).
However, seizure-induced plasticity events in the cholinergic system additionally include changes in the expression of other relevant enzymes, such as ChAT and VAChT (Friedman et al., 2007), which are responsible, respectively, for ACh synthesis (Van der Zee and Keijser, 2011) and loading to synaptic vesicles (Prado et al., 2013). For example, it has been previously shown that SE in animals is related to upregulation of AChE and VAChT in the septohippocampal and mesencephalic neurons (Holtzman and Lowenstein, 1995; Soares et al., 2018). Other studies reported downregulation of ChAT in the basal forebrain of post-SE rats (Biagioni et al., 2019). Together, these data indicate that prolonged seizures or chronic epilepsy state can cause significant alterations in the activity of cholinergic neurons. With respect to the model employed in
the present experiment, our data confirm prior studies (Holtzman and Lowenstein, 1995;
Soares et al., 2017) showing that SE induces long-lasting hypertrophic changes in the septohippocampal cholinergic cells, in particular, enlarged perikarya and increased number of cells immunoreactive to VAChT, as well as profound reorganization of VAChT-stained fibers within the dentate gyrus. It has been previously suggested that such a dysregulation of the septohippocampal cholinergic system would increase overall excitatory drive upon principal hippocampal cells, thereby facilitating local epileptiform phenomena (Holtzman and Lowenstein, 1995; Soares et al., 2017). Thus, in this study, we hypothesized that supressing the plasticity of cholinergic cells in response to prolonged seizures may decrease seizures susceptibility and, ultimately, improve long- term neurological outcome of SE. We found that, indeed, injections of the cholinergic immunotoxin SAP at low doses prevented the SE-induced plastic alterations in cholinergic neurons, including somatic hypertrophy, upregulation of VAChT in the MS/DB, and appearance of new VAChT-stained varicosities in the dentate molecular layer. However, this treatment was also associated with a small, but significant reduction of the total number of septohippocampal cholinergic cells and with partial retraction of VAChT-IR terminals from the dentate hilus, which is unlikely to be beneficial for normal physiology of the hippocampal formation. Yet, the present findings rather support our main hypothesis by showing that the absence of plastic hypertrophic changes in septal neurons after SE is linked not only to reduced susceptibility to acute seizures (see above), but also to a reduction of the number of rats that developed spontaneous seizures later in life. It is worth of note that the effects of SAP pretreatment on seizure susceptibility described in this study were quite moderate – indeed, despite the absence of any signs of cholinergic plasticity, at least half of the rats in SAP+KA group showed spontaneous seizures and had severe behavioral deficits.
This may be due to the fact that a portion of the cholinergic cells were lost in these animals, which was accompanied by marked removal of their fibers from the dentate hilus. Indeed, the retraction of cholinergic afferents from the dentate hilus, by itself, can render dentate neurons hyperexcitable and has been previously described in post-SE rats with spontaneous seizures (Soares et al., 2017). It is also reasonable to suggest that the low efficacy of SAP pretreatment is related to the epilepsy model employed in our study, in which SE was induced using very strong convulsive stimulus, i.e., practically sublethal dose of KA. In this context, it would be of interest to test this approach using less severe seizure models. Further, the present data should be confirmed in future studies using other ACh related enzymes. In particular, it would be of interest to examine the possible compensatory metabolic response of AChE in post SE models, as well as the expression of ChAT in the seizure-induced plasticity events.
Complete depletion of cholinergic neurons or pharmacological manipulations with central cholinergic neurotransmission are known to provoke considerable cognitive and behavioral impairments (Craig et al., 2008; Gibbs, 2007). In this study, despite loss of approximately 20% of septal cholinergic cells, the performance of SAP-pretreated rats was comparable to that of control rats in the passive avoidance test and in the context retention and tone retention tests of the fear conditioning task. The results of post- mortem histological analysis depicted no death of hippocampal neurons in this group, suggesting that loss of a small amount of septohippocampal cholinergic cells alone is not sufficient to cause easily detectable cognitive deficits. In contrast, rats treated with KA, depending on cell population, had 40-50% reductions in hippocampal neuron numbers relative to control rats and showed marked behavioral impairments in all tests.
Interestingly, pretreating rats with SAP did not mitigate the neurotoxic effects of KA and did not rescue most of the long-lasting cognitive deficits in these animals. The lack
of neuroprotective effect of SAP pretreatment can be explained by the fact that KA exerts its seizurogenic and neurotoxic actions via distinct mechanisms, that is, membrane depolarization in a large number of neurons versus fragmentation of the Golgi complex and translocation of the high mobility group box 1 protein from nucleus to cytosol with subsequent post-translational abnormalities (Kaneko et al., 2016;
Kaneko et al., 2017). Somewhat surprisingly, the performance of rats in the SAP+KA group on the passive avoidance task was superior to that of rats in the KA group, in spite of the fact that the degrees of hippocampal neurodegeneration were identical in both groups. There are several explanations for this effect. Firstly, the excessive cholinergic fiber sprouting in the dentate ML, absent in SAP-KA rats, can provide an additional contribution to stronger behavioral deficits in rats treated with KA alone.
Secondly, along with hippocampal neuron loss and cholinergic neurotransmission, there could be other, so far unidentified neuroanatomical or physiological factors that can account for the relatively normal performance of SAP+KA rats in the passive avoidance retention test. Finally, it should be kept in mind that a half of the rats in the SAP+KA group were non-epileptic, while all of the animals in the KA group showed spontaneous seizures. Therefore, it is possible that the performance of some rats in the KA group was additionally affected by subclinical seizure events that were undetected by experimenters. Indeed, in this study, we excluded or delayed behavioral trials only in rats showing signs of motor seizure activity, but no EEG monitoring was performed during behavioral assessments.
The present findings on seizure-induced cholinergic plasticity/fiber sprouting in the KA model of SE may be a target of interest for developing novel treatments of epilepsy. Although very little is known about the molecular or cellular mechanisms triggering fiber sprouting, several possibilities appear viable. Firstly, it is likely that this
process can be initiated due to the loss of either hippocampal GABAergic interneurons (Sloviter, 1987), which would prompt axon outgrowth aimed to establish connections with new target cells, or septal parvalbumin-containing cells (also GABAergic) (Sanabria et al., 2006), which would disinhibit local circuits within the MS/DB. Should this be the case, implementation of recently developed novel treatment strategies based on stem cell therapy, in particular transplantation of GABAergic precursor cells (Lippert et al., 2018; Rao et al., 2017; Yasuhara et al., 2013), may lead to restoration of the septohippocampal projection system, thereby removing excessive cholinergic drive upon dentate molecular layer. Another possibility is that fiber sprouting can be triggered by neuroinflammatory signalling. Indeed, considerable evidence suggest that at the initial stages of epileptogenesis axonal sprouting is commensurate with increased oxidative stress and inflammation and that treating post-SE animals with drugs possessing robust antioxidant and anti-inflammatory activities, such as resveratrol (Castro et al., 2017), lovastatin (Lee et al., 2012) and simvastatin (Xie et al., 2011), suppresses neuroinflammatory processes and restrains both mossy fiber sprouting and development of chronic epilepsy. In this respect, it is worth noting that the use of soluble epoxide hydrolase inhibitors, known for its potent anti-inflammatory effects, has been recently shown to be very promising therapeutic approach for treatment of a number of neurological disorders, including epilepsy (Zarriello et al., 2019). However, the effects of these compounds on SE-induced fiber sprouting have not yet been assessed. Further, it is not clear whether the results of the studies analysing SE-induced mossy fiber sprouting can be extrapolated to axon sprouting in other neuronal populations. Thus, further studies are warranted to demonstrate clinical relevance of the data presented in this study.
In summary, to the best of our knowledge, this study is the first addressing the importance of cholinergic plasticity for seizure susceptibility, epileptogenesis, and long- term sequela of status epilepticus. These first data suggest that seizure-induced plasticity of septohippocampal cholinergic cells may indeed enhance seizure susceptibility and contribute to epileptogenic processes. However, they do not support the hypothesis that hypertrophy of cholinergic neurons or sprouting of their fibers may potentiate epilepsy-related neurodegenerative and respective functional alterations in the hippocampal formation. Being valid only for the present experimental setting, these conclusions require thorough examination in future studies employing distinct models of epilepsy.
4. Experimental Procedure
4.1 Ethical Statement
The handling and care of the animals were conducted according to the “Principles of laboratory animal care” (NIH publication No. 86–23, revised 1985) and Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. The experimental protocol has been approved by the Ethics Committee of the Faculty of Medicine of Porto and the General Veterinary Direction (03.04.2012) for the FCT application grant PTDC/SAU- NSC/115506/2009. All efforts were made to minimize the number of animals used and their suffering.
4.2 Animals
A total of 32 male Wistar rats maintained individually under standard laboratory conditions were used in this study. At 10 weeks of age, they were randomly divided into two groups: SAP-treated group (n=16) and sham group (n=16).
4.3 Cholinergic lesions
The immunotoxin SAP (Advanced Targeting Systems, Inc.) was used to inhibit protein synthesis in cholinergic neurons of the MS/DB. Rats were firstly placed in a Kopf stereotaxic apparatus, under isoflurane anesthesia, and the scalp was incised along the midline and retracted to the side. Using a hand drill, three small openings were made in each parietal bone at respective coordinates given bellow. Bilateral lesions of cholinergic cells were made by infusing 0.5 µl of SAP (0.08 g/l saline solution) into the hippocampus at the following coordinates: AP (from bregma) -3.5 mm, DV (from brain surface) -3.3 mm, ML (from midline) ±2 mm; AP -4.8 mm, DV -5.0 mm, ML
±4.8 mm; AP -5.6 mm, DV -6.5 mm, ML ±5 mm. All infusions were delivered over a 3- minute period using a 1-µl Hamilton syringe equipped with a 33-gauge blunt-tip needle.
Five minutes of diffusion time were allowed before the needle was retracted. It has been previously shown that SAP, when injected into the hippocampus, is retrogradely transported by septohippocampal cholinergic fibers and, therefore, it produces selective lesions of cholinergic cells in MS/DB complex. Sham-treated rats underwent an identical procedure but received vehicle alone rather than SAP solution.
4.4 Kainic acid (KA) treatment
Six weeks after SAP administration, ten of the SAP-treated rats (n=10) and ten of the sham-treated rats (n=10) were injected with KA (9.5 mg/kg, i.p, Sigma) to induce convulsive SE. Remaining SAP-treated (n=6) and sham-treated (n=6) rats received
saline injection alone. After the injection, rats were observed for the presence of spontaneous seizures, classified into five stages according to the modified Racine scale (Racine, 1972), in which stage 1 corresponds to behavioral arrest and/or orofacial movements, including “wet-dog shakes”, stage 2 – head nodding, stage 3 – forelimb clonus, stage 4 – clonus with rearing, and stage 5 – rearing with falling. The onset of SE was defined as the appearance of continuous behavioral symptoms of stage 3-5 seizures, which lasted for at least 20 min. KA-treated rats demonstrated numerous wet-dog-shake seizures 20–60 min after the injection and lasted, in majority of the animals, for 3-4 hr.
During the first 48 hours of the recovery period, the animals were injected with saline (s.c.) and sliced apples were introduced as a supplement to their diet. The rats that refused to eat or drink were hand-fed using a plastic syringe.
By the end of the treatments, all the animals were grouped as follows: sham pretreatment followed by saline injection (control, n=6), sham pretreatment followed by KA injection (KA, n=10), SAP pretreatment followed by saline injection (SAP, n=6), SAP pretreatment and KA injection (SAP+KA, n=10).
4.5 Behavioral evaluation
All animals were submitted to the step-through passive avoidance and fear conditioning tests, which were performed 14 weeks after SE induction.
4.5.1 Step-through passive avoidance test
The apparatus for this behavioral test consisted of two adjacent compartments separated by a guillotine door (Lukoyanov and Lukoyanova, 2006). The larger compartment (45 cm × 45 cm × 45 cm; opaque acrylic walls) had open top and was brightly lit. The smaller compartment (30 cm × 16 cm × 16 cm) had black acrylic walls and top and was
dark. The floor of both compartments was composed of stainless steel bars, 0.5 cm in diameter and spaced 1.2 cm apart; but only the floor of the dark compartment was wired to the stimulus generator (Hugo-Sachs Elektronik, Germany).
On the first day, the rats were allowed to explore the apparatus with the guillotine door open for 5 minutes. The following day, each rat was placed into the brightly lit compartment facing away from the closed door. When it turned around to face the dark compartment, the door was manually raised and the latency for the rat to enter the dark compartment was recorded. Upon entry into the dark compartment, the door was lowered and a 1-mA, 1-s footshock was delivered 3 times at 5-s intervals. Ten seconds after the last shock, the rat was removed from the apparatus and returned to its home cage. Twenty four hours later, this procedure was repeated, with the exception that no footshock was delivered, and the latency to enter the dark compartment was again recorded (up to a maximum of 300 s).
4.5.2 Fear conditioning
Five days later, the rats were given a single session of fear conditioning. The conditioning chamber (San Diego Instruments, USA) consisted of a clear Plexiglas box (26 × 26 × 18 cm) equipped with a metal grid floor, wired to a stimulus generator, and acoustic stimulus unit. The grid floor was composed of stainless steel bars, 0.6 cm in diameter, spaced 1.4 cm apart.
On the first day, the apparatus was located in a quiet, dimly illuminated behavioral testing room and scented with 1% acetic acid solution in order to provide a context odor. Rats were placed inside the apparatus and left undisturbed for 3 minutes. During next 3-min period, they received five tone-footshock conditioning trials, with 30-s intervals. The conditioning trials consisted of a 10-s tone conditioned stimulus (80 dB,
2.8 kHz), which co-terminated with a 1-s footshock unconditioned stimulus (0.8 mA) delivered by the grid floor. Thirty seconds after the last trial, the animals were removed from the apparatus and the grid floor was cleaned with 1% acetic acid. Twenty four hours later, the rats were tested for retention of the conditioned context by repeating the procedure employed during training and placing the rats into the same conditioning chamber for 3 minutes in the absence of the tone or footshock. The following day, the rats were tested for retention of the conditioned tone. In this test, the rats were placed into a novel chamber, which was located in a novel, brightly lit behavioral room. The new chamber was composed of black acrylic, except for the top, which was translucent, and for the floor, which was composed of a piece of a black carpet. In addition, the chamber was scented with lemon instead of acetic acid. The animals remained in the chamber for a period of 6 minutes and the conditioned stimulus (10-s tone) was presented 5 times during the last 3 minutes of this period.
The rat’s behavior on the testing trials was recorded with a digital video camera Sony DCR-SR58E (Sony Corporation, Japan) for subsequent analysis. Freezing behavior, used as the measure of fear conditioning, was identified by the absence of any movement except that required for breathing. Measurements were performed by observers blinded with respect to experimental group, and freezing was only scored if the rat remained inactive for at least 3 seconds. The percentage of accumulated time spent freezing was then calculated.
4.6 Behavioral monitoring and electroencephalographic (EEG) recording
Starting from the sixth week after KA or vehicle treatments, including during the behavioral experiments, the rats were daily (except weekends) observed for spontaneous behavioral seizures during 2-h intervals between 09:00 a.m. and 11:00 a.m. by a person
blind to treatment groups. Upon completion of the fear conditioning tests, the animals were subjected to video-EEG recording in order to confirm or exclude the presence of electrographic seizures. Rats were anesthetized with isoflurane and placed in a stereotaxic apparatus. After the scalp was incised and retracted to the side, two epidural stainless steel electrodes (E363/20 Plastics One Inc., Roanoke, VA, USA) were implanted above the left and right parietal cortex overlaying the hippocampus (4.3 mm posterior to bregma, 2.0 mm lateral to midline). Two additional screw electrodes were placed over the cerebellum to serve as a reference electrode and as a ground. The electrodes were connected to a plastic pedestal (Plastics One, Inc.) that was cemented to the skull using dental acrylic. The recordings were performed one week after the surgery. Recordings were simultaneously performed in pairs of rats and were 48h in duration. For this purpose, rats were placed in acrylic cages where they could move freely (one rat per cage). EEG activity was continuously registered from the epidural electrodes using the Truscan-32 acquisition system (Deymed Diagnostic, Hronov, Czech Republic) connected to computer via a universal serial bus port amplifier (Deymed Diagnostic). Recordings were sampled at 256Hz, high- and low-pass-filtered at 1Hz and 100Hz, respectively, and stored on the computer disk for offline seizure review using the TruScan Explorer software (Deymed Diagnostic). The behavior of the animals was also simultaneously recorded using a digital video camera Sony DCR- SR58E (Sony Corporation, Japan), which was positioned above the cages. The video- EEG recordings were analyzed by a study-blinded clinical neurophysiologist.
Electrographic seizures were defined by the presence of sustained spike and poly-spike activity longer than 3s. The numbers of both electrographic and motors seizures observed during the 24-h recording period were registered.
4.7 Tissue preparation
Six weeks after the behavioral tests, i.e., when the animals were approximately 8-month old, they were deeply anesthetized with pentobarbital (90 mg/kg) and perfused transcardially with 150 ml of 0.1 M phosphate buffer (pH 7.4) for vascular rinse, followed by 250 ml of a fixative solution (containing 4% paraformaldehyde in phosphate buffer). The brains were removed from the skulls, immersed in fixative for 2h and then infiltrated in 10% sucrose solution for 36h at 4oC. The brain blocks were dissected, mounted on a vibratome and sectioned in the coronal plane at 60 m, in order to obtain sections containing the medial septum and hippocampal formation. The sections were stored in cryoprotectant (30% sucrose, 30% ethylene glycol, 0.25 mM polyvinylpyrrolidone in PBS) at -20oC until use.
4.8 Immunohistochemistry and Nissl staining
For immunohistochemistry, every sixth section containing the medial septum region and the hippocampal region were systematically sampled and immunostained for vesicular acetylcholine transporter protein (VAChT). Firstly, the sections were rinsed three times in PBS and the endogenous peroxidases were removed by immersing the sections in 1%
H2O2 solution for 15 minutes. The sections were washed six times in PBS (5 minutes each) and blocked for nonspecific staining in a solution containing 10% normal goat serum (NGS) and 0.5% Triton X-100 in PBS. The sections were then incubated with a guinea-pig primary polyclonal antibody against VAChT (Merck Millipore AB1588;
1:1250 dilution in PBS,) for 72h at 4ºC. Subsequently, the sections were washed in 2%
NGS in PBS three times (10 minutes each) and incubated with a biotinylated anti- guinea-pig antibody (Vector Laboratories, BA7000, diluted 1:200 in 0.02M PBS containing 1% NGS and 0.3% Triton X-100). Sections were washed three times (10
minutes each) in PBS with 2% NGS and incubated with avidin-biotin-peroxidase complex (Vector laboratories, Vectastain Elite ABC Kit) for 45 minutes. After three washes, the sections were recycled in secondary antibody and ABC for 45 and 30 minutes respectively. The peroxidase reaction was obtained by incubation of sections in the solution of 3,3'-diaminobenzidine (DAB, 1 mg/ml) and H2O2 (0.08%) in PBS. The sections were washed two times in PBS (10 minutes each), mounted on gelatin-coated slides and dried overnight. The sections were dehydrated by immersion in a series of ethanol solutions (50%, 70%, 96% and 100%), cleared with xylene and coverslipped using Histomount (National Diagnosis, Atlanta, GA, USA).
For Nissl staining, another set of systematically sampled sections (1:6) was selected, mounted on gelatin-coated slides and air-dried. These sections were then stained with Giemsa, dehydrated in a series of ethanol solutions and coverslipped.
4.9 Density of varicosities
Six consecutive VAChT-stained sections were sampled from the middle part of the dorsal hippocampal formation of each brain. Across all animals included in the analysis, the sections were level-matched using the rat brain atlas (Paxinos and Watson, 1998).
The sections were visualized using an Axio Scope.A1 microscope (Zeiss, Germany) equipped with a Leica EC3 colour digital camera. The boundaries of the dentate gyrus and of its layers were defined with a 40× objective lens on the basis of the rat brain atlas. In each section, 2 photomicrographs of the central area of the hilus and 2 photomicrographs of the inner molecular layer (IML, adjacent to the suprapyramidal blade of the granular layer) were taken with a 100× objective lens. Two non- overlapping counting frames of 1274 µm2 each were applied on each image and the number and the cross-sectional area of all varicosities that fell within each frame were
measured using Fiji image-processing software (http://rsb.info.nih.gov/ij/). However, only fiber varicosities with the cross-sectional areas ranging between 0.11 µm2 and 0.21 µm2 were included in further analysis. The densities of varicosities thus obtained were normalized to an arbitrary chosen area of 30000 µm2 and averaged across all sections per animal.
4.10 Total number of neurons and somatic volume
The VAChT-stained sections containing the MS/DB region were visualized using an Olympus BX-53 microscope equipped with a computer-controlled motorized stage system (MBF Bioscience, Williston, USA). The boundaries of the MS/DB complex were consistently defined using previously described cytoarchitectonic criteria (Cadete- Leite et al., 2003) and the rat brain atlas of Paxinos and Watson (Paxinos and Watson, 1998). In our material, we estimated the rostrocaudal boundaries of the MS/DB region at approximately the following coordinates (relative to bregma): from 1.6 to -0.3. In Nissl-stained sections, the boundaries of the HF and of its subdivisions (dentate hilus and hippocampal pyramidal cell layers) were defined at all levels along the rostrocaudal axis of the brain based on cell morphology and cytoarchitectonic criteria (Amaral and Witter, 1995). Estimations were carried out using the optical fractionator probe (West et al., 1991) of the Stereo Investigator software (MBF Bioscience). The regions of interest were delineated with a 10× objective lens and cell counting was carried out with a 100×
oil-immersion lens. Starting at an arbitrary position, visual fields were systematically sampled using a raster pattern procedure. At each counting frame the tissue thickness was estimated and guard zones of 2 µm were implemented. The nucleus of the neurons
was considered as the counting unit. The coefficient of error (CE) of the individual estimates was calculated according to Gundersen et al. (Gundersen et al., 1999) and it ranged between 0.08 and 0.12 for MS/DB, 0.03 and 0.09 for hilus, 0.04 to 0.07 for CA3, and 0.04 and 0.09 for CA1 fields. All the neurons belonging to the CA2 hippocampal field were included in the CA3 region.
The optical fractionator probe of the Stereo Investigator software was also used to sample the VAChT- and ChAT-stained cells to measure their somatic volumes. The measurements were carried out using the nucleator probe for isotropic systematically random sampled sections (Gundersen, 1988) and the nucleolus of each cell was used as a central point. In these measurements, the coefficients of error of the individual estimates were inferior to 0.04 in all animals.
4.11 Statistical analysis
The binary data on the presence of status epilepticus, mortality and spontaneous seizures were analyzed using nonparametric Kruskal-Wallis ANOVA with one independent variable, namely, the SAP/sham pretreatment. The same test was applied to analyze the variations in the density of varicosities (arbitrary scale of 1 to 10) as a function of the presence of spontaneous seizures in the SAP+KA group. The remaining data sets passed the Shapiro-Wilk’s W normality test (p>0.05) and were analyzed for statistical significance using parametric tests. Data derived from the passive avoidance task were analyzed using two-way repeated measures ANOVA. Latency to SE, fear conditioning data and morphometric estimates were compared using regular two-way ANOVA. When appropriate, ANOVA was followed by the by Tukey’s post-hoc test for multiple comparisons. All data are presented as the mean±SD. Differences were considered as significant at the p<0.05 level.
Acknowledgments
This work was supported by FEDER Funds through the Programa Operacional Factores de Competitividade – COMPETE, COMPETE 2020 – Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and National Funds through FCT − Fundação para a Ciência e a Tecnologia within the scope of the Project PTDC/SAU-NSC/115506/2009–FCOMP-01- 0124-FEDER-015919. The work was also supported by FCT (SFRH/BD/87886/2012) to Joana Soares.
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Table 1: Effects of the intrahippocampal infusion of 192-IgG-saporin on KA- induced status epilepticus (SE), mortality and recurrent seizures
Sham SAP Effect of SAP
Rats with SE
90%
(9/10)
90%
(9/10)
n.s.a
aNonparametric Kruskal-Wallis ANOVA by ranks with SAP-pretreatment as independent variables.
bParametric ANOVA with SAP pre-treatment as independent variable.
Figure captions:
Figure 1: Representative EEG recordings from a KA-treated rat (A), SAP+KA-treated rat with an electrographic seizure (B), SAP+KA-treated rat without an electrographic seizure (C), SAP-treated rat (D) and control rat (E), performed approximately 4 months after respective injections. The recording electrodes were located over the left parietal cortex overlaying the hippocampus. The recordings represented in (A) and (B) show high-frequency spiking activity, with black arrows indicating the beginning of seizures.
Latency to SE, min 46.8±7.9 75.7±5.6 F(1,16)=8.83, p=0.009b
Mortality
50%
(5/10)
0%
(0/10)
H(1,20)=6.33, p=0.012a
Recurrent motor seizures
83%
(5/6)
40%
(4/10)
H(1,16)=2.68, p=0.101a
Recurrent motor+EEG seizures
100%
(6/6)
50%
(5/10)
H(1,16)=4.09, p=0.043a
These electrographic seizures were typically accompanied by the Stage 3–5 behavioral seizures on the Racine scale, that is, unilateral and bilateral forelimb clonus or forelimb clonus with rearing and falling. No electrographic seizures were detected in the control group and SAP-group.
Figure 2: Results from the passive avoidance test showing the mean (±SD) step- through latencies during training as well as on the retention trial performed 24 h later.
Note that rats in the SAP+KA group had higher latency on the retention trial when comparing with KA rats. Also, the rats from control and SAP groups did not enter into the dark compartment. The performance of the rats in all groups were significantly different between training and retention sessions. Moreover, there was a significant interaction between training, KA-treatment and SAP-injection. *p<0.05 vs. training,
#p<0.01 vs. control and SAP, p<0.05 vs. SAP+KA and +p<0.01 vs. control and SAP.
Group size: control n=6, KA n=5, SAP+KA n=10, SAP n=6.
Figure 3: (A) represents the mean (±SD) percentage of freezing time in the 6 min of the context test. Note that there is a significant main effect of KA, with less freezing time for KA-treated groups. It is shown in (B) the mean (±SD) percentage of freezing time during the last 3 min period of the tone test in a novel context. In this session there was a significant main effect of KA, but no significant interaction between conditioning (not shown) and either KA or SAP was demonstrated. *p<0.001 vs. control and SAP. Group size: control n=6, KA n=5, SAP+KA n=9, SAP n=6.
Figure 4: Representative photomicrographs taken from MS/DB a control rat (A), kainic acid-treated rat (B), SAP-treated rat (C) and a SAP+KA-treated rat (D). The coronal
