Spatial Learning and Memory Testing (Morris Water Maze Test)

At the end of the experiment (4 weeks), the mice in all groups were tested in Morris water maze for spatial learning and memory (n = 12/group). Before subjecting the mice to the water maze test, all mice were screened for their swimming ability. The mice were allowed to swim in the water maze tank (100 cm in diameter) for 90 s in each trial, and the distance traveled was measured with an EzVideoTM 5.70 Digital Video Tracking System. For each mouse, three trials were given at an inter-trial interval of 2 min. The swim speed (cm/s; distance traveled in centimeter/90 s) was calculated for each mouse. In all groups, only the mice exhibiting a swim speed of 13–17 cm/s were used for the behavioral study (Figure 1). The water maze consisted of a water tank (1.0 m in diameter). The area of the tank water surface was divided into four virtual quadrants. The depth of the water tank was 20 cm. In the target/platform quadrant, a circular platform was submerged 1.0 cm below the water level. The mice were trained in the water maze on five consecutive days (a total of nine sessions: one session on the first day and two sessions each on the second to the 5th day). Each session consisted of four trials; each trial was of 90-s duration. The inter-trial interval was 60 s. In each trial, the time taken to reach the hidden platform (escape latency) was measured and analyzed using an EzVideo^TM 5.70 Digital Video Tracking System (Accuscan Instruments, Inc., Columbus, OH, USA). At 24 h after the last learning session, the mice were subjected to a memory retention test. Each memory retention test (probe test) session was of 30 s in duration. During the probe/memory retention test, the platform quadrant entry latency, the distance traveled in the target/platform quadrant, and the time spent in the target quadrant were measured and analyzed using an EzVideoTM 5.70 Digital Video Tracking System.

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Figure 1

(A) Graph showing the swim speed of mice in the different groups during the screening test before the water maze learning sessions. Note that there is no significant difference between any groups (two-way ANOVA, P > 0.05; main interaction df-1, F_(1,44) = 0.02908, P = 0.8654; lesion type (L or S) df-1, F_(1,44) = 0.01663, P = 0.8980; treatment type (dBcAMP or PBS) df-1, F_(1,44) = 0.006245, P = 0.9374). (B) Representative video tracking of mice in the different groups during the screening test.

Passive Avoidance Test

After the Morris water maze test, the mice (n = 12/group) were subjected to a passive avoidance test to test the avoidance learning and memory. We have screened all mice for their locomotor activity in an open-field apparatus prior to the passive avoidance test. The mice were allowed to explore the open-field apparatus [100 cm (L) × 100 cm (B) × 20 cm (H)] for 8 min in each trial, and the distance traveled was measured with an EzVideoTM 5.70 Digital Video Tracking System. For each mouse, three trials were given, and the mean distance traveled in 8 min was calculated. In all groups, only the mice which traveled 1,000–1,500 cm in 8 min were used for the behavioral study (Figure 2). The passive avoidance apparatus was made of Plexiglass. It consisted of a larger bright compartment (40 cm × 40 cm × 40 cm) and a smaller dark compartment (15 cm × 10 cm × 10 cm). The apparatus was kept in a relatively dark room. The bright compartment was illuminated with a 15-W bulb. The dark compartment was dark relative to the bright compartment. Stainless steel rods spaced at equal distance from the floor of the dark compartment were connected to an electrical stimulator (DC current, maximum—500 mA, 10–100 V). The bright and the dark compartments were separated from each other by a sliding door. The apparatus was kept under a video camera of the Ezvideo570DV video tracking system. The mice were trained to explore both the bright and the dark compartments of the passive avoidance apparatus for 5 min. The movement of the mice was tracked by the Ezvideo570DV video tracking system (EzVideo^TM 5.70 Digital Video Tracking System). The time spent in the bright and the dark compartments by each mouse was recorded and analyzed by the Ezvideo570DV video tracking system. The trial was repeated three times with 5-min inter-trial interval. At the end of the third trial, the mouse was confined to the dark compartment of the passive avoidance apparatus (by closing the sliding door) and a foot shock from the stimulator (DC, 0.2–0.5 mA) was given for 3 s. At 24 h after the foot shock, the memory retention test was done. During the memory retention test, each mouse was allowed to explore the bright and the dark compartments for 3 min. The time spent in the dark compartment and the dark compartment entry latency were recorded for each mouse.

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Figure 2

(A) Graph showing the distance traveled by mice in the different groups during the screening test before the passive avoidance test. Note that there is no significant difference between any groups (two-way ANOVA, P > 0.05; main interaction df-1, F_(1,44) = 0.05647, P = 0.8133; lesion type (L or S) df-1, F_(1,44) = 0.003144, P = 0.9555; treatment type (dBcAMP or PBS) df-1, F_(1,44) = 0.001009, P = 0.9748). (B) Video tracking of the mice in the different groups during the screening test.

Tissue Fixation and Processing for Immunostaining

The mice in all groups (n = 6/group) were anesthetized with a ketamine–xylocane cocktail (ketamine 40 mg/kg and xylocaine 5 mg/kg) 1 day after the completion of the behavioral tests. The heart was exposed and perfused with 50 ml of heparinized saline, followed by 200 ml of 4% paraformaldehyde. The brain was dissected and post-fixed for 48 h in the same fixative. The tissues were cryoprotected in 10, 20, and 30% sucrose solution (24 h in each). Then, the tissues were embedded in an optimum cutting temperature compound (Sigma Chemicals, St. Louis, MO, USA). Thirty-micron-thick serial, coronal cryostat sections were taken and stored in phosphate buffer-filled 24-well culture plates. The serial brain sections were stained for Cresyl violet, doublecortin (for neurogenesis), and NeuN (for assessing the neurons).

Cresyl Violet Staining

The general morphology and the neurodegeneration in the hippocampus were assessed by Cresyl violet staining. The structure of the different regions of the hippocampus was studied. Brain sections mounted on gelatinized slides were air-dried and stained with 0.1% aqueous Cresyl violet stain (Sigma Chemicals, St. Louis, MO, USA) for 20 min at 60°C. Then, the slides were washed in distilled water, differentiated in 70% ethyl alcohol and dehydrated in ascending grades of ethyl alcohol, cleared in xylene, and mounted with DPX (Sigma Chemicals, St. Louis, MO, USA). Observations were done on six serial sections (150 μm apart) from each animal.

Doublecortin and NeuN Immunostaining and Quantification

Thirty-micron-thick frozen serial, coronal sections were immunostained for doublecortin and NeuN. Endogenous peroxidase activity was reduced by treating the sections with 3% hydrogen peroxide. Nonspecific binding of the antibodies was blocked with 5% normal goat/rabbit serum (Vector Laboratories, Burlingame, CA, USA). The sections were then incubated with goat polyclonal anti-DCX (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) or mouse monoclonal anti-NeuN (1:500, Abcam, Cambridge, MA, USA) antibodies overnight at 4°C. The sections were incubated with biotinylated anti-goat IgG (1:20) or with biotinylated anti-mouse IgG (1:20, Vector Laboratories, Burlingame, CA, USA) for 1 h, followed by incubation with avidin biotin complex (1:20, ABC kit, Vector Laboratories, Burlingame, CA, USA) for another 1 h. The color was developed using diaminobenzidine (DAB) as chromogen for NeuN staining and vector gray for DCX staining. The sections were mounted on gelatin-coated slides, air-dried, dehydrated in ascending grades of ethyl alcohol, cleared in xylene, and coverslipped with DPX. The number of NeuN-positive neurons and the number of DCX-positive newly formed neurons were counted in six randomly selected fields (photographs taken with ×40 objective) in each section from the DG and the CA3 regions in the kainic acid lesion model. From each mouse, six sections (150 μm apart) were selected for quantification. Finally, the number of new neurons (DCX-positive) and the number of mature neurons (NeuN-positive) per cubic millimeter of tissue were calculated for each mouse (Beauquis et al., 2014; Abd-El-Basset and Rao, 2018). Briefly, the total number of DCX-positive neurons and the total number of NeuN-positive neurons per cubic millimeter of tissue were quantified by using the data collected from coronal brain sections using the formula T = N*V/t, where N is the numerical cell density, V is the volume of the tissue used for quantification (CA3 region, DG, and dentate hilus), and t is section thickness (30 μm). The numerical cell density (N) was calculated by measuring the area of the section used for quantification with the NIS-Elements software (NIS-Elements-D2.20) and the total cell counts in six randomly selected fields. The volume (V) was calculated by measuring the area of the section used for quantification [CA3, DG (DG+dentate hilus), and dentate hilus region] with the NIS-Elements software (NIS-Elements-D2.20) and multiplying by the section thickness (30 μm), the inter-section distance (60 μm), and the number of sections (6). All of the brain sections were examined with an Olympus BX51 TF upright transmitted light/fluorescence microscope using ×20 (aperture is 0.50) and ×40 (aperture is 0.75) objectives.

Polyacrylamide Gel Electrophoresis and Immunoblotting

Mice (n = 6/group) were perfused with 50 ml of cold saline. The hippocampus was dissected and snap-frozen in liquid nitrogen and stored at −80°C until Western blot analysis for doublecortin. The tissue was thawed and incubated in an ice-cold radioimmunoprecipitation assay lysis buffer [sodium orthovanadate (0.5 mM) and the protease inhibitors, phenylmethanesulfonyl fluoride (1 mM), aprotinin (10 μg/ml), and leupeptin (1 μg/ml)] for 10 min. The tissue was homogenized for 3–5 min at 4°C and centrifuged for 5 min at 14,000 rpm at 4°C. The supernatant was collected for analysis. The protein concentration in the samples was determined using an Epoch^TM Multi-volume spectrophotometer system (Biotek, Winooski, VT, USA). The protein standards were made by serial dilution of a concentrated stock of bovine serum albumin (Sigma-Aldrich, Saint Louis, MO, USA, PN-A3294) in double-distilled water. The protein standard curve was made by measuring the absorbance at 280 nm. The protein concentration in the samples was measured by loading 2 μl of the undiluted sample on the Take3^TM plate and measuring the absorbance at 280 nm. The proteins in all samples (75 μg protein/well) were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Laemmli, 1970). The proteins were transferred to the nitrocellulose membrane (Towbin et al., 1979). After transfer, the membranes were incubated with 5% skim milk in Tris-buffered saline-Tween 20 (TBST) for 1 h. The immunoblots were probed with goat anti-doublecortin antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-glyceradehde-3-phosphate dehydrogenase (endogenous sample loading control, Sigma Chemicals, St. Louis, MO, USA) diluted in 5% dry milk in TBST. The membranes were then incubated in rabbit anti-goat or goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA). Enhanced chemiluminescence system (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) was used to visualize the immunoreactive bands. The differences in doublecortin band intensities in the different groups were determined by scanning the bands and quantifying the density in the Image-J image analysis software.

Passive Avoidance Test

The passive avoidance learning session showed no significant difference (P > 0.05) in the time spent in the bright or the dark compartment between the groups [bright compartment: main interaction—df-1, F_(1,44) = 0.1149, P = 0.7362; lesion type (L or S)—df-1, F_(1,44) = 0.2208, P = 0.6407; treatment type (dBcAMP or PBS)—df-1, F_(1,44) = 0.2342, P = 0.6308); dark compartment: main interaction—df-1, F_(1,44) = 0.003214, P = 0.9550; lesion type (L or S)—df-1, F_(1,44) = 0.03687, P = 0.8486, treatment type (dBcAMP or PBS)—df-1, F_(1,44) = 0.3926, P = 0.5342)]. The mice in all groups explored both the dark and the bright compartments (Figures 5A,B). The video tracking of mice during the learning sessions is shown in Figure 5C. Memory retention test at 24 h after the last learning session showed a significant memory deficit in the L+PBS mice compared to that in the S+PBS mice. The time spent in the dark compartment was significantly more in the L+PBS group compared to those in the S+PBS (P < 0.0001) and the S+dBcAMP groups (P < 0.0001, Figure 6A). The L+dBcAMP group spent significantly less time in the dark compartment compared to the L+PBS group [main interaction—df-1, F_(1,44) = 28.09, P < 0.0001; lesion type (L or S)—df-1, F_(1,44) = 35.47, P < 0.0001; treatment type (dBcAMP or PBS)—df-1, F_(1,44) = 28.68, P < 0.0001]. The dark compartment entry latency was significantly more in the S+PBS and the S+dBcAMP groups compared to that in L+PBS (P < 0.0001, Figure 6B). The dark compartment entry latency was significantly more in the L+dBcAMP group compared to that in L+PBS, indicating a better avoidance memory [main interaction—df-1, F_(1,44) = 19.22, P < 0.0001; lesion type (L or S)—df-1, F_(1,44) = 24.64, P < 0.0001; treatment type (dBcAMP or PBS)—df-1, F_(1,44) = 29.85, P < 0.0001, Figure 6B]. The video tracking of mice during the memory retention test is shown in Figure 6C.

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Figure 5

(A,B) Graph showing the time spent in the bright and in the dark compartments of the passive avoidance apparatus by mice in the different groups during the passive avoidance learning session. Note that there is no significant difference between any groups. (C) Video tracking of mice in the different groups during the last passive avoidance learning session. BC, bright compartment; DC, dark compartments of the passive avoidance apparatus. Note that the mice in all groups explored both the bright and the dark compartments.

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Figure 6

(A,B) Graph showing the time spent in the dark compartment and dark compartment entry latency in the different groups during the passive avoidance memory retention test. Note that L+PBS mice spent significantly more time in the dark compartment compared to mice in the S+PBS and the S+dBcAMP groups (P < 0.0001). However, L+dBcAMP mice spent significantly less time in the dark compartment compared to L+PBS mice (P < 0.0001), like the S+PBS and the S+dBcAMP groups, suggesting significant memory retention. Similarly, dark compartment entry latency was significantly less in L+PBS compared to S+PBS and S+dBcAMP (P < 0.0001), and it is increased significantly in the L+dBcAMP group. (C) Video tracking of mice in the different groups during the passive avoidance memory retention test 24 h after the last learning session. Note that mice in L+PBS entered the dark compartment (DC) as soon as the test started, suggesting poor memory retention. However, mice in S+PBS, S+dBcAMP, and L+dBcAMP explored only the bright compartments (BC) except for one entry to the dark compartment (DC).

Morphological Changes in the Hippocampus

Cresyl Violet Staining

Cresyl violet staining of the hippocampal sections showed decreased neurons in the CA3 and the dentate hilus (DH) regions in the L+PBS group compared to those in the S+PBS and the S+dBcAMP groups. Treatment with dBcAMP (L+dBcAMP) protected the neurons from degeneration both in the CA3 and the DH regions compared to that in the L+PBS group (Figures 7A–C).

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Figure 7

Photomicrographs of the hippocampus (A), dentate gyrus (DG)—hilus region (B), and CA3 region (C) stained with Cresyl violet to analyze the hippocampal morphology in the different groups. Note the significant degenerative changes in the CA3 and the dentate hilus (DH) regions in the L+PBS group compared to the S+PBS and the S+dBcAMP groups. Treatment with dBcAMP (L+dBcAMP) decreased the degenerative changes and protected the neurons from degeneration both in the CA3 and the DH regions compared to the L+PBS group. Scale bar = 100 μm in (A), scale bar = 10 μm in (B,C).

NeuN Immunostaining

NeuN immunostaining of the hippocampal section confirmed the decreased neurons in the DH and the CA3 regions in the L+PBS group compared to those in the S+PBS and the S+dBcAMP groups. Further, treatment with dBcAMP (L+dBcAMP) protected the neurons from degeneration both in the DH and the CA3 regions compared to that in the L+PBS group (Figures 8A–C). Quantification of neurons in the dentate hilus and the CA3 regions showed significantly decreased neurons in the dentate hilus (P < 0.0001) and CA3 regions (P < 0.0001) in the L+PBS group compared to those in the S+PBS and the S+dBcAMP groups (Figures 9A,B). Treatment with dBcAMP (L+dBcAMP) significantly increased the number of neurons in both the DH (P < 0.05) and the CA3 (P < 0.001) regions compared to that in the L+PBS group (DH: main interaction—df-1, F_(1,20) = 4.545, P = 0.0456; lesion type (L or S)—df-1, F_(1,20) = 48.3, P < 0.0001; treatment type (dBcAMP or PBS)—df-1, F_(1,20) = 4.211, P = 0.0535; CA3: main interaction—df-1, F_(1,20) = 10.55, P = 0.0040; lesion type (L or S)—df-1, F_(1,20) = 77.35, P < 0.0001; treatment type (dBcAMP or PBS)—df-1, F_(1,20) = 12.55, P = 0.0020, Figures 9A,B).

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Figure 8

Photomicrographs of the hippocampus (A), DG–hilus region (B), and CA3 region (C) immunostained for NeuN to analyze the number of neurons in the CA3 and in the dentate hilus regions. Note the decreased number of neurons in the CA3 and in the dentate hilus (DH) regions in the L+PBS group compared to the S+PBS and the S+dBcAMP groups. Treatment with dBcAMP (L+dBcAMP) protected the neurons from degeneration, both in the CA3 and in the DH region compared to the L+PBS group. Scale bar = 100 μm in (A), scale bar = 10 μm in (B,C).

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Figure 9

Graph showing the number of NeuN-positive neurons in the dentate hilus (DH; A) and in the CA3 region (B). Note the significantly decreased number of neurons in the L+PBS group compared to the S+PBS and the L+dBcAMP groups (P < 0.0001) in both the DH and the CA3 regions. However, the number of neurons increased significantly in the L+dBcAMP group compared to that in the L+PBS group (P < 0.05). Two-way ANOVA, Bonferroni’s test, n = 6 in all groups.

Hippocampal Neurogenesis

Doublecortin immunostaining showed a markedly decreased generation of new neurons in the hippocampal DG of the L+PBS group compared to those of the S+PBS and the S+dBcAMP groups; treatment with dBcAMP enhanced the generation of the new neurons in L+dBcAMP (Figures 10, 11A–C). The quantification of new neurons in the hippocampal DG revealed a significant decrease in the generation of new neurons in the L+PBS group compared to those in the S+PBS and the S+dBcAMP groups (P < 0.05, Figure 10). Treatment with dBcAMP (L+ dBcAMP) significantly (P < 0.0001) increased the number of new neurons in the hippocampal DG compared to that in the L+PBS group [main interaction—df-1, F_(1,20) = 69.46, P < 0.0001; lesion type (L or S)—df-1, F_(1,20) = 16.9, P = 0.0005; treatment type (dBcAMP or PBS)—df-1, F_(1,20) = 87.97, P < 0.0001, Figure 12A].

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Figure 10

Photomicrographs of the entire hippocampal DG immunostained for doublecortin (DCX) to analyze the neurogenesis. Crest (CREST), suprapyramidal blade (SPB), and infrapyramidal blade (IPB) regions indicated in boxes in all groups are shown at higher magnification in Figures 11A–C. Note the noticeable decrease in DCX-positive neurons in the L+PBS group compared to those in the S+PBS and the S+dBcAMP groups in all regions. Treatment with dBcAMP (L+dBcAMP) enhanced the hippocampal neurogenesis in all regions compared to that in the L+PBS group. DH, dentate hilus. Scale bar = 20 μm.

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Figure 11

Photomicrographs of the hippocampal DG immunostained for doublecortin (DCX) to analyze the neurogenesis (regions indicated by boxes in Figure 10 are shown at higher magnification). (A) Crest (CREST), (B) suprapyramidal blade (SPB), and (C) infrapyramidal blade (IPB) regions. Selected regions (rectangular box) of CREST, SPB, and IPB are shown at higher magnification in the adjacent photomicrograph. Note the noticeably decreased DCX-positive neurons in the L+PBS group compared to those in the S+PBS and the S+dBcAMP groups in all regions. Treatment with dBcAMP (L+dBcAMP) enhanced the hippocampal neurogenesis in all regions compared to that in the L+PBS group. Scale bar = 10 μm. DH, dentate hilus.

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Figure 12

(A) Graph showing the number of doublecortin (DCX)-positive neurons in the entire DG. (B) DCX immunoblot. (C) Mean gray value (DCX/glyceradehde-3-phosphate dehydrogenase). Note the significantly decreased number of new neurons (A) and DCX content (B,C) in the L+PBS group compared to those in the S+PBS and the L+dBcAMP groups (P < 0.05). However, the number of neurons and the DCX content increased significantly in the L+dBcAMP group compared to those in the L+PBS group (P < 0.0001). Two-way ANOVA, Bonferroni’s test, n = 6 in all groups.

dBcAMP and Learning and Memory

Memory retention test as assessed by the Morris water maze test and the passive avoidance test revealed significant memory impairment in the hippocampus-injured mice, and memory impairment was decreased by treatment with dBcAMP. Screening of mice for their swimming ability and locomotor activity before subjecting them to the behavioral tests did not show any swimming deficit or locomotor deficit among them. Hence, locomotor activity may not have contributed to the observed behavioral deficit. Our results were consistent with those of other studies. Sharifzadeh et al. (2007) showed the preventive role of dBcAMP in spatial memory impairment in the Morris water maze test by increasing the cAMP level. dBcAMP, a synthetic analog of cAMP, is highly permeable through the cell membrane and it mimics the activity of endogenous cAMP (Hosseini-Zare et al., 2011). dBcAMP is also reported to improve auditory/audio memory (Nassireslami et al., 2013). dBcAMP is reported to simulate the distinct components of long-term potentiation (population spike and population excitatory post-synaptic potential slope) in the CA1 region of rat hippocampus in culture (Slack and Walsh, 1995). A recent study showed that memory impairment was induced by beta-amyloid protein, and treatment with dBcAMP prevented such spatial memory impairment as evaluated by the Morris water maze test (Aghsami et al., 2018).

Neuroprotection by dBcAMP—Role of Astrocytes and Microglia

In the present study, we observed a significant neuroprotection in the hippocampal CA3 and dentate hilus regions in kainic acid-injured mice that were treated with dBcAMP. This neuroprotection may be mediated by astrocytes and microglia in the vicinity of the injury. Microglial activation after injury leads to the release of a variety of pro-inflammatory cytokines and free radicals. These cytokines and free radicals induce the degenerative process (Kim and de Vellis, 2005; Block et al., 2007). In gliosis, the impaired handling of extracellular glutamate by astrocytes in the regions of gliosis leads to excitotoxic neuronal damages. The microglia are recognized as a potent inducer of astrocyte activation and proinflammatory mediators. Tilleux et al. (2009) reported the enhanced expression of GLT-1 in astrocytes that are treated with dBcAMP or astrocytes that are exposed to soluble mediators released by the activated microglia. Matsuura et al. (2002) demonstrated a markedly increased glutamate transport activity, in a concentration- and time-dependent manner, in astrocytes in cultures that are treated with dBcAMP. Thus, the neuroprotection by dBcAMP may be attributed to enhanced gliosis and hence decreased the excitotoxicity and neuronal death. Indeed we have reported earlier enhanced astrocytes and microglial population in the CA3 and the DG regions of the hippocampus within a week after the excitotoxic lesion was induced by an intracerebro-ventricular injection of kainic acid (Abd-El-Basset and Rao, 2018).

dBcAMP and Neurogenesis

An analysis of hippocampal neurogenesis by labeling the newly formed neurons with anti-doublecortin antibody showed a significant decrease in hippocampal neurogenesis in the kainic acid-injured hippocampus at 4 weeks after the injury. However, hippocampal neurogenesis was found to be enhanced in those injured mice treated with dBcAMP. In our study, we have killed the mice on the 36th day of the experiment (i.e., 29 days after the last dose of dBcAMP or PBS treatment) and quantified the number of doublecortin-positive neurons in the hippocampal DG. In this study, we have not addressed the survival and the maturation of newly generated neurons. Several studies have shown the transition of newborn neurons into mature neurons (Rao and Shetty, 2004; Rao et al., 2005). We used DCX as a marker for new neurons in the DG. This is based on the fact that new neurons that are predominantly born during the 12 days prior to euthanasia can be visualized with DCX immunostaining (Rao and Shetty, 2004). Evidence from both in vivo and in vitro studies suggests that hippocampal neurogenesis is linked to an increase in the intracellular cAMP, a second messenger that regulates many functions, including metabolism, cell proliferation, and neuronal signaling, by altering gene expression (West et al., 2001; Nicot et al., 2002; Fujioka et al., 2004; Gabellini, 2004; Pandey, 2004). The cAMP cascade in the neuronal cells can be triggered by the same conditions known to influence hippocampal neurogenesis, such as neural injury (Nakagawa et al., 2002). Thus, in our study, the initial lesion by kainic acid itself might have triggered the cAMP cascade in the neural progenitor cells in the DG; further, dBcAMP injected for 7 days enhanced the intracellular cAMP, resulting in enhanced hippocampal neurogenesis.

Adenylyl cyclase (a potent stimulator of adenylyl cyclase) regulates neurogenesis in prenatal hindbrain and cortical neuroblasts (Lu and DiCicco-Bloom, 1997; Waschek et al., 1998; Suh et al., 2001). A recent study showed that Lot1 (a zinc finger transcription factor) expression in CGC is cAMP dependent as treatments with adenylyl cyclase activator increased Lot1 expression at both the mRNA and the protein levels, and dibutyryl cAMP mimicked the actions of Lot1 expression (Contestabile et al., 2005). In addition, dBcAMP is reported to increase the recruitment of neural stem cells into the brain parenchyma and the olfactory bulb in experimental autoimmune encephalomyelitis mice (Khezri et al., 2013). These evidences further support our finding that treatment with dBcAMP has enhanced the DG neurogenesis.

We acknowledge Dr. Muhammad Mostafa Abd-El-Barr, Assistant Professor, Department of Neurosurgery, Duke University, North Carolina, USA, for his valuable suggestions, critical reading, and editing of the manuscript. The authors would like to thank Ms. Amna Najem for technical assistance.