SEARCH

SEARCH BY CITATION

Keywords:

  • Comorbidity;
  • Cortical dysplasia;
  • Memory;
  • Synaptic plasticity

Summary

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References

Purpose:  Memory impairment is a common comorbidity in people with epilepsy-associated malformations of cortical development. We studied spatial memory performance and hippocampal synaptic plasticity in an animal model of cortical dysplasia.

Methods:  Embryonic day 17 rats were exposed to 2.25 Gy external radiation. One-month-old rats were tested for spatial recognition memory. After behavioral testing, short-term and long-term synaptic plasticity in the hippocampal CA1 region was studied in an in vitro slice preparation.

Key Findings:  Behavioral assessments showed impaired hippocampal CA1-dependent spatial recognition memory in irradiated rats. Neurophysiologic assessments showed that baseline synaptic transmission was significantly enhanced, whereas paired-pulse facilitation, long-term potentiation, and long-term depression of the field excitatory postsynaptic potential (fEPSP) slope at Schaffer collateral/commissural fiber-CA1 synapses were significantly reduced in the irradiated rats. Histologic observations showed dysplastic cortex and dispersed hippocampal pyramidal neurons.

Significance:  This study has shown that prenatally irradiated rats with cortical dysplasia exhibit a severe impairment of spatial recognition memory accompanied by disrupted short-term and long-term synaptic plasticity and may help to guide development of potential therapeutic interventions for this important problem.

Medically intractable epilepsy, accounting for at least 30% of all cases of epilepsy (Kwan & Brodie, 2000; Wheless, 2006), is commonly associated with cognitive and psychosocial comorbidities that can be as serious as the seizures themselves (Dodrill et al., 1984; Jacobs et al., 2009; Brooks-Kayal, 2011). Cortical dysplasia (CD) is a major cause of medically intractable epilepsy (Taylor et al., 1971; Palmini et al., 1991). Epilepsy patients with CD often have concurrent cognitive and psychosocial impairment (Barkovich & Kjos, 1992; Bigio et al., 1998; Leventer et al., 1999; Janszky et al., 2003). We have studied a rat model of prenatal irradiation-induced CD that mimics certain important aspects of human CD (Roper et al., 1995, 1997). Immunohistochemical studies showed a selective loss of both interneurons (Roper et al., 1999; Deukmedjian et al., 2004; Zhou & Roper, 2010, 2011) and vesicular γ-aminobutyric acid (GABA) transporter terminals in dysplastic cortex (Zhou & Roper, 2010). Electrophysiologic studies demonstrated reduced inhibitory postsynaptic currents in pyramidal neurons in neocortex and heterotopic gray matter (Zhu & Roper, 2000; Chen & Roper, 2003) and imbalanced synaptic input that results in decreased firing rates of cortical GABAergic interneurons in this model (Xiang et al., 2006; Zhou et al., 2009; Zhou & Roper, 2011). Fetal irradiation produces dispersion of neurons in the pyramidal cell layer of the hippocampus (Roper et al., 1995; Smith et al., 1999). Hippocampal pyramidal neurons of irradiated rats also exhibit higher excitability and lower synaptic inhibition than controls (Smith et al., 1999). The widespread loss, dysfunction, and disorganization of neuronal populations in the irradiated rat brain would be likely to cause co-morbidities such as impaired cognitive functions, and a recent study has confirmed significantly impaired nonspatial recognition memory and spatial navigation memory in these animals (Zhou et al., 2011). But the mechanisms responsible for impaired memory function in CD are poorly understood.

Synaptic plasticity, an ability to modify synaptic strength in an activity-dependent manner, is a fundamental feature of the central nervous system (CNS) that is critical for learning and memory (Beck et al., 2000). Therefore, disturbed synaptic plasticity in brain regions important in memory and cognition may be an important mechanism underlying memory impairment in epilepsy (Beck et al., 2000; Goussakov et al., 2000; Sgobio et al., 2010) and other neurologic disorders (Murphy et al., 2000; Costa et al., 2002; von der Brelie et al., 2006). Synaptic plasticity can be categorized as short-term and long-term plasticity; the latter includes long-term potentiation (LTP) and long-term depression (LTD). Both of these processes are widely recognized as important determinants of learning and memory (Bliss & Collingridge, 1993; Bear & Abraham, 1996). LTP is a persistent enhancement of synaptic transmission following a brief, tetanic stimulation of afferent pathways (Bliss & Lomo, 1973). LTD is a lasting functional decrease of synaptic transmission following low-frequency repetitive stimulation (Abraham & Goddard, 1983). LTD and LTP are easily demonstrated in the hippocampus, a structure widely thought to be important in memory (Lynch, 2004). In the present study, we examined spatial recognition memory, a hippocampal CA1-dependent process (Brun et al., 2002), assessed by a place recognition task in control and irradiated rats. After behavioral testing, we examined short-term and long-term plasticity in CA1 by recording field excitatory postsynaptic potentials (fEPSPs). Finally, we examined the histologic changes in cortex and hippocampus. We found that spatial recognition memory, short-term synaptic plasticity (PPF) and long-term synaptic plasticity (including LTP and LTD) are severely impaired in irradiated rats with CD.

Materials and Methods

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References

Animals and irradiation

Pregnant Sprague-Dawley rats were obtained from Harlan Sprague Dawley Inc. (Indianapolis, IN, U.S.A.). The day of insemination was designated as embryonic day 0 (E0). E17 rats received 2.25 Gy of external X-irradiation or sham irradiation. Offspring from sham-irradiated control rats were weaned on postnatal day 21 (P21) and offspring from irradiated rats were weaned at P28 due to development and growth delay. Beginning on P30, male offspring were subjected to behavioral testing. After testing, slices from one hemisphere were cut for electrophysiologic recordings. All rats were given free access to food and water and were maintained on 12-h light/dark cycles. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida.

Place recognition task

Place recognition task was used to test spatial recognition memory. The apparatus consisted of an arena (60 × 60 × 60 cm) and two identical objects (glass cylinders, 7.0 cm in diameter × 6.5 cm in height). The arena was surrounded by black curtains to avoid anxiety-provoking situations and cues in the room environment. The apparatus was cleaned between trials to remove olfactory cues. Rat behavior was monitored by video camera and recorded using an automated tracking system (Ethovision, Noldus, The Netherlands) for offline analysis. The task consisted of a sample phase and a test phase delivered 24 h later (Fig. 1A1,B1). In the sample phase, the rats were allowed to explore the arena and objects for 5 min and then removed from the arena for a 24-h retention delay. In the test phase, one of the objects was moved to a new location; the rats were allowed to explore the arena and objects for 3 min. The time that the rat spent exploring each object/location was measured. Exploration of an object was defined as directing the nose at a distance <2 cm from the object. The exploration ratio was defined as ratio of time spent exploring one object to total time spent exploring both objects.

image

Figure 1.   Total exploring time and the exploration ratio for the objects during place recognition task. Schematics illustrate the general procedure for the task during the sample (A1) and test phase (B1), time spent in exploring identical object 1 and 2 (O1 and O2) for 5 min during the sample phase (A2), and time spent in exploring object 1 (familiar location) and object 2 (novel location) for 3 min during test phases (B2). The exploration ratio, TO2/(TO1 + TO2), is shown for the sample phase (A3) and for the test phase (B3). The dashed line represents the chance level of performance (i.e., a ratio of 0.5). *p < 0.01 versus control; #p < 0.01 versus O1; &p < 0.01 in control group, one sample t-test was performed.

Download figure to PowerPoint

Electrophysiology

Two weeks after behavioral testing, transverse hippocampal slices from one hemisphere (400-μm thickness) were cut in an ice-cold cutting solution using a Vibratome (Leica VT1000 s; Leica Microsystems, Wetzlar, Germany). The cutting solution contained (in mm) 220 sucrose, 3.0 KCl, 1.25 NaH2PO4, 25 NaHCO3, 0.5 CaCl2, 7 MgCl2, and 13 d-glucose, and was oxygenated with 95% O2 and 5% CO2 (pH 7.4 was adjusted with NaOH, and osmolarity was maintained at 350–360 mOsm). Slices were allowed to recover in oxygenated extracellular solution at room temperature (approximately 23°C) for at least 1 h before transferring to a recording chamber. The extracellular solution contained (in mm) 125 NaCl, 3.0 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2.5 CaCl2, 1.3 MgCl2, 13 d-glucose (pH 7.4 was adjusted with NaOH, and osmolarity was maintained at 305–315 mOsm). Individual slices were transferred to a submerged recording chamber perfused with oxygenated extracellular solution at constant flow rate of 3 ml/min. Brain regions, neurons, and electrodes were visualized through a 4× or 40× lens using an upright microscope equipped with infrared-differential interference contrast optics (Nikon Eclipse E600FN, Nikon Instruments Inc., Melville, NY, U.S.A.). Glass electrodes were pulled in four stages on a horizontal pipette puller (Model P-87 Flaming/Brown Micropipette Puller; Sutter Instruments, Novato, CA, U.S.A.) from Wiretrol II capillary glass (Drummond Scientific, Broomall, PA, U.S.A.). Recordings of fEPSPs were made in the stratum radiatum of the hippocampal CA1 region at 30°C. Electrodes for recording and stimulation were filled with 2 m NaCl (approximately 5 MΩ) and extracellular solution (approximately 1.5 MΩ), respectively. The stimulating electrode was placed in the stratum radiatum to activate Schaffer collateral/commissural afferents. The recording and stimulating electrodes were placed approximately 600 μm away from each other. Monopolar stimulation was applied with a stimulus isolator (WPI, Sarasota, FL, U.S.A.). To obtain input–output curves, we applied different stimulation intensities ranging from 0–400 μA in steps of 50 μA (50 μs for stimulation duration, 0.05 Hz). Short-term plasticity was examined using pairs of stimuli that were delivered at varying interstimulus intervals (30–500 msec) and intensities (0–400 μA) at 0.05 Hz. The baseline stimulation intensity was adjusted to elicit a fEPSP slope of 35% of the maximal response (Kluge et al., 2008). After establishing a stable baseline for 20 min (stimulation parameters: 0.05 Hz; duration, 50 μs; intensity, approximately 100 μA in control and approximately 80 μA in irradiated rats), LTP was induced with a single train of high-frequency stimulation (100 Hz for 1 s) and LTD was induced by low-frequency stimulation (1 Hz for 900 s) in separate slices. Stimulation intensity for LTP and LTD was same as the baseline stimulation intensity. Poststimulation responses were recorded for at least 60 min, with stimuli using baseline stimulation parameters. One recording for LTP and another one for LTD were obtained from two slices of each rat. Potentials were amplified using a multiClamp 700B (Molecular Devices, Union City, CA, U.S.A.). Amplifier control and data acquisition were performed using CLAMPEX 10.1 software via 16-bit data acquisition system Digidata 1320A (Molecular Devices). Signals were digitized at 10 kHz and analyzed off-line.

The slope of fEPSP was measured off-line using the clampfit analysis program (Molecular Devices). For analyzing paired-pulse facilitation (PPF), the slope ratio of the second to the first fEPSP was calculated, and plotted for different interstimulus intervals or stimulation intensities. For analyzing LTP or LTD (the increase or decrease of fEPSP slope after stimulation, respectively), values obtained after stimulation were expressed as percentage of the baseline values; the magnitude of LTP or LTD was compared between control and irradiated rats.

Cresyl violet staining

After recording, slices were fixed with 4% paraformaldehyde and resectioned into 50-μm sections with a Vibratome (Lancer Vibratome Series 1000, Diversified Equipment Company, Inc., Lorton, VA, U.S.A.). Sections were transferred through 70% ethanol, 95% ethanol, 95% acid ethanol (95% ethanol:100% glacial acetic acid = 100:0.25), 100% ethanol, 2 × xylene, 100% ethanol; 95% acid ethanol; 95% ethanol, 70% ethanol, and then washed in distilled H2O. Sections were immersed in 0.5% cresyl violet acetate for 5 min and moved through distilled H2O, 70% ethanol, 90% ethanol, 95% acid ethanol, 100% ethanol, 2 × xylene, and finally mounted with Permount mounting medium (Fisher Scientific, Fair Lawn, NJ, U.S.A.).

Statistical analysis

All values are expressed as mean ± standard error of the mean (SEM). Analysis of variance (ANOVA) or other specific methods of statistical analysis (described in the text) were used. For all tests, p < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References

Spatial recognition memory is impaired in irradiated rats

Behavioral testing was performed in 13 controls and 12 irradiated rats. One-way ANOVA showed no significant difference in the amount of total time spent exploring each object in either control or irradiated rats (all p > 0.05, Fig. 1A2), indicating no object or location preference during the sample phase in each group. During the test phase when one of the objects was changed to a new location, ANOVA indicated that control rats spent more time exploring the novel location than the familiar one (p < 0.01, Fig. 1B2), but the irradiated rats did not (p > 0.05, Fig. 1B2); the analysis also revealed that control rats spent more time exploring the novel location (p < 0.01, Fig. 1B2) than the irradiated. We used the exploration ratio to estimate differential exploration activity. Control and irradiated rats had a similar exploration ratio during the sample phase (p > 0.05, ANOVA, Fig. 1A3); however, irradiated rats exhibited a significantly lower exploration ratio compared with controls during the test phase (p < 0.01, Fig. 1B3). Finally, one-sample t-tests were used to determine whether the exploration ratios were significantly different from chance level (0.5). The ratio was significantly above chance in controls (p < 0.01, Fig. 1B3) but not in irradiated rats (p > 0.05, Fig. 1B3) during the test phase. These results indicated that hippocampal CA1-dependent spatial recognition memory was significantly impaired in irradiated rats.

Enhancement of basal synaptic transmission in the irradiated rats

Basal synaptic transmission was assessed by input–output curves constructed by plotting fEPSP slope versus stimulation intensity of 26 recordings from controls and 24 recordings from irradiated rats. As shown in Fig. 2A,B, fEPSP slope increased with increasing stimulation intensities in control and irradiated rats (both p < 0.01, repeated-measures one-way ANOVAs) until the maximum slope of fEPSP was recorded at 350–400 μA; the maximum fEPSP slope was not significantly different between the two groups (p > 0.05, ANOVA). The input–output curve obtained in the irradiated rats was shifted toward lower stimulation intensities, indicating that basal synaptic transmission was increased in irradiated rats.

image

Figure 2.   Left shift in input–output curves in irradiated rats. (A) Representative traces of fEPSPs at different stimulation intensities in control and irradiated rats. (B) Input–output curves of fEPSP slope versus stimulation intensities 0–400 μA.

Download figure to PowerPoint

Reduction in paired-pulse facilitation in irradiated rats

To further test any abnormal presynaptic function, we examined the differences in PPF in slices of control (n = 26) and irradiated rats (n = 24). PPF, a well-known form of short-term plasticity, is produced by applying a pair of stimuli at short interstimulus intervals (<1 s). It is thought to depend primarily on initial release probability in the presynaptic terminals (Schulz et al., 1994; Dobrunz & Stevens, 1997). At a given stimulation intensity (approximately 100 μA in control and approximately 80 μA in irradiated rats) with varying short interstimulus intervals (30–500 ms), paired-pulse ratios increased with interstimulus intervals, became maximal at intervals 50–70 msec, and then declined at intervals ranging from 70–500 msec in control and irradiated rats (Fig. 3B), but the irradiated rats showed significantly lower ratios at interstimulus intervals ranging from 30–500 msec (50 msec in Fig. 3A; all p < 0.01, Fig. 3B, ANOVA). When paired-pulse stimulation with an interstimulus interval of 50 msec was performed with varying stimulation intensities (0–400 μA), the ratios were unchanged by intensities in the control and irradiated rats, consistent with a previous study (Goussakov et al., 2000). But the irradiated rats showed significantly lower ratios compared with controls at all stimulation intensities (all p < 0.01, Fig. 3C, ANOVA). These results indicated that irradiation resulted in a reduction of PPF.

image

Figure 3.   Reduced paired-pulse facilitation (PPF) in irradiated rats. (A) Representative traces of fEPSPs to paired-pulse stimulation with an interstimulus interval of 50 msec in control and irradiated rats. (B) PPF ratios at different interstimulus intervals of 30–500 msec. (C) The PPF ratio of fEPSP slope at different stimulation intensities of 0–400 μA with an interstimulus interval of 50 msec.

Download figure to PowerPoint

Impaired long-term potentiation in irradiated rats

We examined the changes in fEPSP slope after high-frequency stimulation. Application of a single train of high-frequency stimulation (100 Hz for 1 s) to Schaffer collateral/commissural afferents caused a significantly larger initial poststimulation potentiation of the fEPSP slope (244.6 ± 13.1% [n = 13] and 154.4 ± 9.7% [n = 12] of prestimulation baseline in control and irradiated rats, respectively, p < 0.01, Fig. 4A,B), and larger stable LTP of the fEPSP slope in control compared with the irradiated rats (157.6 ± 8.5% and 112.8 ± 5.6% of prestimulation baseline in control and irradiated rats, respectively; measured 1 h after stimulation; p < 0.01, Fig. 4A,B).

image

Figure 4.   Long-term potentiation (LTP) deficit in irradiated rats. (A) Representative fEPSPs elicited at the time points indicated by the numbers in control and irradiated rats. (B) LTP was elicited by high-frequency stimulation (HFS) and it was impaired in irradiated rats.

Download figure to PowerPoint

Impaired long-term depression in irradiated rats

We examined whether hippocampal LTD induced by low frequency stimulation was changed in irradiated rats. Application of 900 stimuli at 1 Hz induced a significantly larger initial poststimulation depression of the fEPSP slope (52.6 ± 3.6% [n = 13] and 82.9 ± 3.5% [n = 12] of prestimulation baseline in controls and irradiated rats, respectively; p < 0.01, Fig. 5A,B) and larger stable long-term depression after 1 h (78.2 ± 2.5% and 95.6 ± 2.6% of prestimulation baseline in control and irradiated rats, respectively; p < 0.01, Fig. 5A,B).

image

Figure 5.   Long-term depression (LTD) deficit in irradiated rats. (A) Representative fEPSPs elicited at the time points indicated by the numbers in control and irradiated rats. (B) LTD was elicited by low-frequency stimulation (LFS) and it was impaired in irradiated rats.

Download figure to PowerPoint

Taken together, these results show that induction and late phases of both LTP and LTD were markedly impaired in irradiated rats, indicating that hippocampal synapses may have lost much of their normal capacity for activity-dependent, long-term synaptic modification.

Histologic observations in irradiated rats

Neuronal populations have well-defined regional and laminar distribution in the neocortex and hippocampus in control rats (Fig. 6A). Consistent with previous reports (Roper et al., 1995), irradiated rats showed loss of lamination in the neocortex, subcortical gray matter heterotopia, and dispersion and disorganization of the normally compact CA1 pyramidal layer (Fig. 6B).

image

Figure 6.   Cresyl violet–stained section of control and irradiated rats. (A) Low- (left) and high-power (right) images show well-defined neocortex and hippocampus. (B) The low-power (left) and high-power image (right) shows dyslamination of the neocortex and a dispersed and disorganized hippocampal CA1 pyramidal layer. High-power images are indicated by the boxes in the low-power images. Scale bars for low-power images equal 300 μm and for high power images equal 150 μm.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References

This study found alterations of hippocampal CA1-dependent spatial recognition memory and synaptic functions in area CA1 of the hippocampus in the irradiated rat model of CD. Behavioral assessments showed impaired spatial recognition memory. Neurophysiologic assessments showed that baseline synaptic transmission was significantly enhanced, whereas PPF, LTP, and LTD of the fEPSP slope were reduced in irradiated rats. Enhanced excitatory transmission as revealed by analysis of dendritic input–output functions is consistent with increased transmitter release probability. This interpretation is supported by the finding of reduced PPF in irradiated rats. LTP and LTD deficits suggest that basic mechanisms of information storage in the brain were impaired in irradiated rats. Together, these alterations in synaptic functions in CA1 may underlie the impairment of spatial recognition memory in irradiated rats with CD.

Impairment of spatial recognition memory

Patients with epilepsy often have cognitive impairment as a comorbidity (Lhatoo & Sander, 2001). Patients with intractable epilepsy due to CD often have severe persistent cognitive impairment (Barkovich & Kjos, 1992; Bigio et al., 1998; Leventer et al., 1999; Janszky et al., 2003). This study utilized the irradiated rat model of CD and found an impairment of spatial recognition memory using a place recognition task, which is hippocampal CA1-dependent (Brun et al., 2002). Irradiated rats demonstrate impaired nonspatial object recognition memory revealed by an object recognition task (Zhou et al., 2011), which is neocortex dependent (Brown & Aggleton, 2001). Irradiated rats also show impaired spatial navigation memory in the Morris water maze task (Zhou et al., 2011), which is hippocampus (subregions CA1, CA3, and dentate gyrus) dependent (Morris et al., 1982; Clark et al., 2007). Memory deficits are also present in methylazoxy-methanol (MAM)–treated rats, another model of diffuse CD that involves the hippocampus (Di Luca et al., 1995; Cattabeni & Di Luca, 1997).

Correlation of memory loss and impaired synaptic plasticity

Activity-dependent synaptic plasticity, a fundamental feature of most CNS synapses, is critical for memory and learning (Beck et al., 2000; Silva, 2003). Impaired synaptic plasticity has long been considered as a candidate mechanism underlying impaired memory performance in models of epilepsy (Beck et al., 2000; Goussakov et al., 2000; Sgobio et al., 2010). In a genetic model of epilepsy, impairment of hippocampal synaptic plasticity is accompanied by specific hippocampal-dependent memory deficits (Sgobio et al., 2010). In MAM-treated rats with diffuse CD, cognitive deficits are accompanied by impaired LTP (Di Luca et al., 1995; Cattabeni & Di Luca, 1997). In patients with epilepsy, impaired activity-dependent synaptic plasticity has been regarded as a contributor to deficient declarative memory (Beck et al., 2000; Goussakov et al., 2000).

In the present study, impaired LTP and LTD in irradiated rats was accompanied by an impairment of hippocampal CA1–dependent spatial recognition memory. Although we did not prove a causal relationship between alterations in synaptic plasticity and memory deficits, these results are consistent with the general notion that hippocampal synaptic plasticity underlies spatial memory in rodents, which is supported by studies using selective elimination of genes in specific hippocampal cell types (Tsien et al., 1996).

Enhanced basal synaptic transmission and reduced paired-pulse facilitation

PPF, one form of short-term plasticity and a measure of the ability of chemical synapses to increase the probability of transmitter release in response to the second pulse of a pulse pair (Dobrunz & Stevens, 1997), was reduced in irradiated rats. This suggests an increase of the probability of transmitter release in response to the first pulse of the pair such that the second pulse arrives at a time of diminished releasable vesicle pools. In the CA1 region, synapses with low initial release probability exhibit LTP when 2.5 mm [Ca2+]o/2.5 mm [Mg2+]o is used in the bath solution, whereas LTP is absent under conditions that support high initial release probability; that is, when 4.0 mm [Ca2+]o/4.0 mm [Mg2+]o is used (Larkman et al., 1992). Therefore, the level of the probability of transmitter release strongly influences the capacity of CA1 synapses for LTP induction and expression (Goussakov et al., 2000). Previous studies have demonstrated a low initial release probability (approximately 0.3) (Dobrunz & Stevens, 1997) and pronounced LTP in normal hippocampal CA3-CA1 synapses when 2.5 mm [Ca2+]o/1.3 mm [Mg2+]o is used (von der Brelie et al., 2006). In the present study, we found robust LTP in controls and impaired LTP in irradiated rats at 2.5 mm [Ca2+]o/1.3 mm [Mg2+]o, conditions that support low initial release probability in control tissue. The magnitude of PPF is inversely related to the initial synaptic release probability (Dobrunz & Stevens, 1997). Therefore, in the present study, the reduction of PPF indicates an increased initial release probability that could contribute to the LTP deficits seen in irradiated rats. Increased basal synaptic transmission was also observed in input–output analyses in slices from irradiated animals. Increased synaptic transmission could stem from changes in transmitter release properties. These presynaptic dysfunctions could disrupt information processing in the hippocampal neuronal network that plays an important role in the formation of synaptic plasticity and spatial memory. Reduced PPF has also been reported in excitatory synapses of the neocortex in irradiated rats (Chen et al., 2007).

LTP and LTD deficits

In the present study, two forms of long-lasting, activity-dependent synaptic modification, LTP and LTD, were assessed. We found significantly diminished LTP and LTD in CA1 in irradiated rats, suggesting that Schaffer collateral/commissural–CA1 synapses have lost much of their potential for bidirectional, activity-dependent synaptic modification. In MAM-treated rats with diffuse CD, LTP induced by high-frequency stimulation of the stratum radiatum afferent fibers is impaired in CA1 (Di Luca et al., 1995; Cattabeni & Di Luca, 1997). In another model of focal CD induced by a perinatal freeze-lesion, LTP could not be observed in dysplastic somatosensory cortex (Peters et al., 2004). Therefore, impaired long-term synaptic plasticity appears to be a consistent feature of several animal models of CD.

Mechanisms underlying the altered synaptic plasticity

Cellular mechanisms underlying the altered synaptic plasticity/memory in irradiated rats have not been defined, although several candidate mechanisms exist.

Imbalance of excitatory and inhibitory circuit elements

Enhanced GABAergic inhibition in a mouse model for the learning and memory deficits associated with neurofibromatosis type 1 (Costa et al., 2002) and decreased GABAergic inhibition in a mouse model of Rett syndrome (Chao et al., 2010) have both been reported to cause impairment of LTP. Reduced GABAergic inhibition in dentate gyrus can also facilitate LTP (Wigstrom & Gustaffson, 1985). Normal plasticity may occur when excitatory and inhibitory circuit elements reach an optimal balance (Fagiolini & Hensch, 2000) and any disruption of this balance (by enhanced or decreased GABAergic inhibition) may cause an impairment of plasticity and memory (Chao et al., 2010). In the current study, inhibition could be decreased due to the loss of GABAergic interneurons and presynaptic terminals similar to that seen in the neocortex (Roper et al., 1999; Deukmedjian et al., 2004; Zhou & Roper, 2010, 2011) and in hippocampus of irradiated rats (Gaiarsa et al., 1994; Smith et al., 1999). Therefore, decreased GABAergic inhibition could lead to deficits in both LTP and memory in irradiated rats.

Damage of neuronal circuits in hippocampus

Neural loss, dysfunction, and disorganization have been described in cortex and hippocampus in irradiated rodents (Brizzee et al., 1980; Roper et al., 1995, 1997, 1999; Smith et al., 1999; Deukmedjian et al., 2004; Zhou et al., 2009; Zhou & Roper, 2010, 2011) and epilepsy patients with dysplastic neurons (Barkovich & Kjos, 1992; Bigio et al., 1998; Leventer et al., 1999; Janszky et al., 2003). Both rodents and humans with CD have demonstrated impaired memory (Barkovich & Kjos, 1992; Bigio et al., 1998; Leventer et al., 1999; Janszky et al., 2003; Zhou et al., 2011). Pathologic changes in hippocampus and entorhinal cortex caused by in utero irradiation can reflect damage of both neural circuits (Gaiarsa et al., 1994) and the balance of inhibition and excitation (Smith et al., 1999) and may have severe consequences for synaptic plasticity. In addition, dendritic spines function as specialized postsynaptic structures, represent a primary locus of excitatory synaptic transmission, and are associated with experience-dependent plasticity (Engert & Bonhoeffer, 1999). Therefore, the lower density of dendritic spines of pyramidal cells in the hippocampus that has been demonstrated in prenatally irradiated squirrel monkeys (Brizzee et al., 1980) could account for the observed synaptic plasticity impairment in this study.

Change of receptor expression function and signaling for synaptic plasticity

The different N-methyl-d-aspartate receptor (NMDAR)–mediated intracellular signaling pathways that diversely regulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)–mediated synaptic transmission also mediate the induction and expression of LTP and LTD in the hippocampus (Zhu et al., 2002; Liu et al., 2004). Changes in the expression levels of these proteins could modulate the magnitude of LTP and LTP. Overexpressing NMDA receptor 2B (NR2B)–containing NMDARs in the hippocampus enhances LTP and memory (Tang et al., 1999; Wang et al., 2009) while overexpression of NR2A attenuates LTP (Barria & Malinow, 2005). Previous studies have shown an enhanced NR2A- and NR2B-containing NMDAR expression and an increased NR2B-containing NMDAR current in animal and human dysplastic neurons (Ying et al., 1998; Mikuni et al., 1999; DeFazio & Hablitz, 2000; White et al., 2001; André et al., 2004). Irradiation has been demonstrated to increase the expression of NR2A-containing NMDAR (Shi et al., 2006). One possible explanation for impaired synaptic plasticity and memory in irradiated rats is that, despite NR2 protein overexpression in the soma, NR2-containing receptors fail to be incorporated into the synaptic membrane (Philpot et al., 2001) and overexpression of these receptors may not function in the mediation of LTP and LTD. Another explanation is that the NR2A/NR2B ratio controls the degree of LTP (Matsuzaki et al., 2004) with a low NR2A/NR2B ratio favoring LTP (Yashiro & Philpot, 2008). NR2A/NR2B ratios in irradiated rats may be higher than in controls and contribute to the impaired LTP observed in the present study. In addition to the possible influence of NR2A- and NR2B-containing NMDAR, irradiation has been shown to modulate gene expression of signaling molecules including the RAS-GTPase family, p38, Jun N-terminal kinase (JNK), and ERK1/2 MAP kinases (Hwang et al., 2006; Verheyde et al., 2006), which are important downstream plasticity signaling molecules (Zhu et al., 2002; Liu et al., 2004). Therefore, altered expression of these molecules in irradiated rats may contribute to impaired synaptic plasticity. Reduced AMPAR subunit GluR1/2 expression in irradiated rats (White et al., 2001) could also contribute to impaired synaptic plasticity. In addition, some key trophic factor such as brain-derived neurotrophic factor (BDNF) (Lu et al., 2008), enzymes such as Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Lie et al., 1998), or proteins such as methyl CpG binding protein 2 (MeCP2) (Asaka et al., 2006) that play critical roles in signaling for synaptic plasticity are altered in irradiated rats (Dimberg et al., 1997; Pogribny et al., 2005; Verheyde et al., 2006), and this could adversely affect synaptic plasticity in irradiated rats.

In conclusion, this study has shown that prenatally irradiated rats with CD exhibit a severe impairment of spatial recognition memory accompanied by disrupted short-term and long-term synaptic plasticity in the hippocampus. These findings may lay the groundwork for future efforts to develop potential therapeutic interventions for this important problem.

Acknowledgments

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References

This work was supported by the Citizens United for Epilepsy Research (CURE), the McKnight Brain Research Foundation, the Densch Foundation, and the Wells Fund.

Disclosure

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References

The authors have no conflicts of interest to disclose. We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

References

  1. Top of page
  2. Summary
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References