Learning and Memory
Behavioural Brain Research
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Neurocognitive deficits arising from anesthetic exposure have recently been debated, while studies have shown that the phosphorylation of cyclic AMP response element-binding protein (CREB) in the hippocampus is critical for long-term memory. To better understand the neural effects of inhalational anesthetics, we studied the behavioral and biochemical changes in aged rats that were exposed to sevoflurane (Sev) and nitrous oxide (N2O) for 4 h. Eighteen-month-old rats were randomly assigned to receive 1.3% sevoflurane and 50% nitrous oxide/50% oxygen or 50% oxygen for 4 h. Spatial learning and memory were tested with the Morris water maze 48 h after exposure, and the results showed that sevoflurane–nitrous oxide exposure induced a significant deficit in spatial learning acquisition and memory retention. Experiments revealed that the cAMP and pCREB levels in the dorsal hippocampus were decreased in rats with anesthetic exposure in comparison with control rats 48 h after anesthesia as well as 15 min after the probe trial, but there were no significant differences in CREB expression. Besides these, the current study also found the DG neurogenesis significantly decreased as well as neuronal loss and neuronal apoptosis increased in the hippocampus of rats exposed to Sev+N2O. The current study demonstrated that down-regulation of cAMP/CREB signaling, decrease of CREB-dependent neurogenesis and neuronal survival in the hippocampus contributed to the neurotoxicity and cognitive dysfunction induced by general anesthesia with sevoflurane–nitrous oxide.
The combination of sevoflurane with nitrous oxide is widely used in clinical anesthesia practice. However, recent studies have raised concerns about the neurotoxicity of inhalational anesthetics and their contribution to postoperative cognitive dysfunction (POCD) , , . Studies indicated that general anesthesia with a combination of nitrous oxide (N2O) and isoflurane (ISO) or sedation with 70% N2O produced lasting impairment in spatial working memory in rats , , , , . Exposure of neonatal mice to inhaled sevoflurane not only caused persistent learning deficits in fear conditioning later in adulthood, but also abnormal social behaviors resembling autism spectrum disorder . In addition, such exposure induced apoptosis, increased beta-amyloid protein levels  and tau phosphorylation through activation of specific kinases, which is considered a potential mechanism of cognitive dysfunction caused by anesthesia . Although detrimental effects of anesthetics on cognitive function have been reported, to our knowledge, no study has investigated the effects of the anesthetic sevoflurane combined with N2O on spatial learning and memory in aged rats.
A current “hot spot” of memory research involves the cyclic AMP response element-binding protein (CREB), which has been extensively implicated in learning and memory , long term potentiation(LTP) , and neuroprotection . It is fairly well established that hippocampus-mediated memory consolidation involves signaling cascades leading to gene transcription of the transcription factor CREB . Phosphorylation/activation of CREB (pCREB) on Ser 133 by cyclic AMP- or Ca2+-dependent protein kinase is critical for long-term memory consolidation, , . Inhibition of phosphodiesterase-4 (PDE4), an enzyme that catalyzes cAMP hydrolysis, increases cAMP and phosphorylation of CREB , , facilitates induction of hippocampal LTP  and enhances memory , . Consistent with this, several studies have shown that pCREB is also involved in hippocampal neurogenesis, influences the neurotrophic factor-dependent survival of culture neurons and regulates several steps of neurogenesis including proliferation, differentiation, and survival , , . To our knowledge, adult neurogenesis in the hippocampus plays a key role in spatial memory function, regulating acquisition of a spatial memory and the subsequent flexible use of spatially precise learning strategies , , . Besides the neurogenesis, CREB phosphorylation has also been found to be important in the neurotrophin-mediated neuronal survival , . Studies showed that ablation of neuronal CREB during development resulted in a massive neuronal apoptosis, and a full CREB-KO mice showed a significant increase in neuronal cell death in dorsal root ganglion neurons , . Based on these, inhibition of cAMP/CREB induced by anesthetics would lead to the decrease of neurogenesis but increase of neuronal cell death, and further aggravated cognitive dysfunctions.
The aim of the present study is to determine whether anesthesia with sevoflurane combined with N2O in aged rats could induce spatial learning and memory deficit. We also evaluated the cAMP/CREB signaling, neurogenesis levels and cell survival in the hippocampus in an effort to test the hypothesis that general anesthesia by Sev+N2O down-regulates cAMP/CREB pathway, and then suppresses neuronal survival and hippocampal DG neurogenesis, subsequently aggravating learning and memory deficit.
Materials and Methods
The experimental protocol was approved by the Shanghai Medical Experimental Animal Care Commission. Male Sprague Dawley rats were obtained from Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. Aged rats (18 months old) were housed one or two per cage in a climate- and humidity-controlled room in the animal facilities on a 12-h light–dark artificial cycle (lights on at 7:00 AM) with free access to food and water. All experiments were performed during the light phase between 7:00 AM and 7:00 PM.
Animals (n = 40) were randomized into four groups (10 in each group): Sev+N2O-MWM, in which rats received sevoflurane-N2O and behavioral training(Morris Water Maze, MWM); Sev+N2O, in which rats received anesthesia without behavioral training; Con-MWM, in which rats received control gas and behavioral training; and Con, in which rats received control gas without behavioral training. Rats that were randomly assigned to the anesthesia groups received 1.3% sevoflurane (USP, Baxter, Deerfield, IL, USA) in 50% N2O/50% oxygen for 4 h at a flow rate of approximately 3 L/min in a Plexiglas anesthetizing chamber, which was adjusted to maintain minimum alveolar concentration, oxygen, and carbon dioxide at constant levels. Gases within the anesthetic chamber were monitored continuously, and arterial oxygen saturation was measured noninvasively using a pulse oximeter during anesthesia. Control groups received 50% oxygen in their home cage at identical flow rates as anesthetized animals for 4 h, but arterial oxygen saturation was not measured to prevent the introduction of stress as a confounding variable. All anesthetized rats were breathing spontaneously, and the temperature of the anesthetizing chamber was controlled to maintain rat temperature at 37°C ±0.5°C using a heating pad. Anesthesia was terminated by discontinuing the anesthetics. Rats were allowed to recover for 48 h to avoid the confounding influence of residual anesthetic. Forty-eight hours after anesthesia, rats in the groups without behavioral training were sacrificed, and the remaining rats were tested in the Morris water maze from day 1 to day 6.
Morris Water Maze Task
Spatial memory ability was examined in a water maze that consisted of a swimming pool as described by Morris  and adapted for rats. It consisted of a circular tank (160-cm diameter, 50 cm high), filled to a depth of 30 cm with water maintained at 22°C and rendered opaque by the addition of white nontoxic paint. The pool was located in a room uniformly illuminated by a halogen lamp and equipped with various distal cues. Located inside the pool was a removable, circular (12-cm diameter) platform (PF) made of transparent Plexiglas, positioned such that its top surface was 1.0 cm below the surface of the water. The platform, which served as a refuge from the water, was generally located in the center of an arbitrarily defined quadrant of the maze. The quadrant where the PF was located was defined as the target quadrant (E quadrant), and the other three were defined as N, S, W quandrant. The swim paths of the animals were captured by a video camera mounted above the water maze, and the corresponding tracks were recorded and analyzed by a computer tracking system (VideoTrack, Viewpoint).
During the learning procedure, rats were tested during the light phase between 7:00 AM and 7:00 PM. Each rat was given a daily four-trial session (30-min intertrial interval) for six consecutive days. Each trial consisted of releasing the rat into the water facing the outer edge of the pool at one of the quadrants (in a random sequence) and letting the animal escape to the submerged platform. Rats were allowed to swim for a maximum of 60 s in each trial, and the time they spent before reaching the platform (i.e., escape latency) was a measure of acquisition of spatial navigation. If the rat failed to find the platform within 60 s, it was manually guided to the platform, and the escape latency was accepted as 60 s. After climbing onto the platform, the rat was left on it for 15 s and then removed from the pool and returned to its cage beneath a heat lamp to reduce the drop in core temperature. The release point differed in each trial, and different sequences of release points were used from day to day.
Twenty-four hours following the acquisition phase, rats were subjected to a probe trial in which the platform was removed. Starting from the quadrant opposite from the target quadrant where the platform had been located, each rat was allowed to swim for 60 s. Time spent and distances covered in the four round probe zones were measured by the tracking system. Data were collected using the VideoTrack system.
Protein Extraction from Hippocampal Tissue
Five rats in Sev+N2O-MWM group and six rats in Con-MWM group were deeply anesthetized at 15 min after the probe trial, the brains were immediately removed, and both hippocampi from each rat were dissected out. Hippocampi were homogenized by brief sonication in an extract buffer (400 µl) containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 µg/ml aprotinin, 1% Triton X-100, and phosphatase inhibitor cocktail 1 and 2 (Sigma, St. Louis, MO). The homogenate was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was removed and stored at −80°C until use.
For the two groups without Morris water maze training, 48 h after Sev+N2O anesthesia or control, hippocampi from five rats per group were extracted and tissues were processed as already described.
Samples (30 µg of protein) were subjected to SDS-PAGE (10% gels) and then transferred from the gel to nitrocellulose membranes using a tank transfer apparatus with buffer containing 25 mM bicine, 25 mM Bis-Tris, and 20% methanol. Membranes were incubated in blocking buffer (TBS/0.1% Tween 20 with 5% nonfat dried milk) for 2 h at room temperature. To control for protein loading, an antibody against β-actin (Santa Cruz Technologies, Santa Cruz, CA) was used. Anti-CREB antibody (1:1000; Cell Signaling Technologies, Danvers, MA), anti-pCREB antibody (1:1000; Cell Signaling Technologies, Danvers, MA) and anti-Bax (1:1000; Santa Cruz Technologies, CA) were added to the buffer solution, and the membranes were incubated overnight at 4°C. The membrane was then washed for 10 min three times in 1× TBS/0.1% Tween 20, and then incubated in HRP-conjugated anti-rabbit IgG (secondary antibody, 1:3000; Cell Signaling) in blocking buffer for 1 h. Subsequently, membranes were washed with 1× TBS/0.1% Tween 20 several times. Blots were visualized with an ECL detection kit (Pierce, IL) and analyzed using Quantity One 1-D Analysis Software (Bio-Rad, San Francisco, CA).
Cyclic AMP Analysis
Cyclic AMP (cAMP) levels were determined using a cAMP Complete ELISA kit (Enzo Life Sciences, USA) according to the manufacturer’s instructions. The level of cAMP in the sample was determined based on a standard curve and expressed as pmol/mg per each sample.
The remaining rats in each group were deeply anesthetized and perfused transcardially with ice-cold phosphate-buffered solution (PBS) and 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4). The brains were then dissected out, and the meninges were carefully removed and then post-fixed in the same fixative overnight, cryoprotected by first sinking in 10% and then in 30% sucrose (in 0.1 M phosphate buffer) at 4°C. Coronal sections (20 µm) were cut on a microtome.
After blocking of nonspecific epitopes with PBS containing 10% donkey serum and 0.5% Triton-100 for 1 h at room temperature and then in the primary antibody : Phospho-CREB Rabbit mAb (1:100, Cell Signaling), NeuN (1:200, Millipore), five sections (20 µm thickness, 80 µm space) of each rat were incubated for 48 h at 4°C. Sections were then washed in PBS and incubated for 4 h with a 1:1000 dilution of anti-rabbit or anti-mouse IgG secondary antibody (Invitrogen, Carlsbad, CA, USA). Nuclear counterstaining was performed with 4′,6-diamidino-2-phenylindole (DAPI, Sigma) for 30 min at room temperature before three washes with dH2O for 10 min each time and coverslipped. Quantitative analysis was performed using an imaging analysis system (Leica Qwin). Assessment of pCREB staining was performed in CA1 and CA3 area of the dorsal hippocampus, while assessment of NeuN-positive neurons was performed in the CA1 area of the dorsal hippocampus.
Sections rinse in tap water and then in distilled water. Stain in 0.1% cresyl violet solution for 3–10 min. Rinse quickly in distilled water. Then differentiate and dehydrate in alcohol then clearing and finally mount with permanent mounting medium. The Nissl body will be stained purple-blue.
After Nissl staining, neuronal cells in the hippocampal were identified from five sections (20 µm thickness, 80 µm apart) per rat (5 rats in each of Sev+N2O group, Con group, and Sev+N2O-MWM group, and 4 rats in Con-MWM group). In each section, the number of neurons was averaged from three random different vision fields in the hippocampal CA1 in each hemisphere (six vision fields per section) under Leica microscope (400× magnification). Only intact neurons with a clearly defined cell body and nucleus were counted.
Double Labeling with NeuN and Annexin V
Apoptosis in hippocampus was detected using an Annexin V- FITC/neuronal marker NeuN doubled staining according to the protocol by Bian Z et al . Brains were sectioned at 12 um thickness and mounted on cover slides followed by air-drying before labeling with Annexin V-FITC. Five Sections (60 µm space) per animal were stained first for Annexin V-FITC using an Annexin V staining Kit (BD Biosciences, CA) according to the manufacturer’s instructions, then blocked with blocking solution as described above, incubated with primary anti-NeuN antibody (1:100, cell signaling) at RT for 2 h and secondary antibody for 1 h. Nuclear countstaining was performed with Hochest (Invitrogen, CA). Assessment of the doubled NeuN and Annexin V- positive neurons was performed in the CA1 subfield of the hippocampus.
Nestin and DCX Immunohistochemistry
Nestin, a type VI intermediate filament protein, is predominantly expressed in neurogenic and myogenic stem cells, and has been known as a marker of neuroepithelial/progenitor cells. Doublecortin (DXC) is a microtubule-associated protein and has been known as a marker of immature neurons.
In this experiment, five sections (12 µm thickness, 60 µm space) per rat (n = 5 in each of Sev+N2O group, Con group, Sev+N2O-MWM group, and n = 4 in Con-MWM group) were analyzed. The protocol for Nestin and DCX staining was essentially the same as for pCREB staining, expect the primary antibody: anti-Nestin (1:100, Boster, Wuhan, China) and anti-DCX (1:100, Epitomics, Burlingame, CA). Analysis of the Nestin and DCX positive neurons was performed in the DG subfield of the hippocampus.
All analyses were performed using SPSS 18.0 for Windows (SPSS, Inc.). Data were submitted to repeated-measures ANOVA (the escape latencies and the swimming speed) and Tukey honestly significant difference test was used for post hoc testing in the Morris water maze. All the other analyses were conducted using Student unpaired two-tailed t test. A value of p<0.05 was considered significant.
Aged Rats Exhibit Impaired Performance on Spatial Learning and Memory After Sevoflurane–N2O Anesthesia
No rat had an episode of hypoxia (defined as SpO2<90%) during the sevoflurane–N2O exposure, and rats did not exhibit floating behavior in our study.
Spatial acquisition for learning in the water maze is presented in Figure 1, which depicts the escape latency to reach the platform. In the first day of spatial acquisition trials, all rats tended to swim around the edges looking for an escape, but after being placed on the platform at the end of each trial, rats gradually learned that there was an escape platform and would venture into the center of the pool to locate it. By the second day’s session, all rats could rapidly locate the platform and the latency was significant less than the first day. A two-way repeated measure ANOVA on the escape latency revealed a significant effect of days for testing the water maze (p<0.001) and a main effect of group (p = 0.016), but no day × group interaction (p= 0.406; Figure 1A) and no effect of speed (p = 0.699; Figure 1B). In the Sev+N2O-MWM group, the impairment was found on day 5 (p = 0.02) and day 6 (p = 0.024), postanesthesia days 7 and 8, respectively, compared to the control rats, which represented the learning deficit after sevoflurane-N2O anesthesia. These results indicated that both the Con-MWM group and Sev+N2O-MWM group exhibited improvement in spatial learning and memory over time during the acquisition phase, but the latter had a longer latency to reach the target quadrant. The lack of effect of speed suggested that the poorer performance of the Sev+N2O-MWM group did not result from lack of motivation or reduced motor ability.
The probe trial conducted 24 h after the last water maze acquisition session was used to assess the spatial memory based on the percentage of time spent in the target quadrant (E quadrant) where platform had been located as well as the distance covered in the E quadrant (Figure 2). Figure 2A shows that Sev+N2O-MWM rats spent significantly less time in the E quadrant compared with Con-MWM rats (p<0.05), and Figure 2B shows that the distances travelled by Con-MWM rats in the E quadrant were more than those by Sev+N2O-MWM rats (p<0.05). Figure 2C shows that Con-MWM rats spent much more time in the E quadrant than the other three ones (all p<0.01), as revealed by a significant effect of quadrant (p<0.01). But unlike the Con-MWM group, the Sev+N2O-MWM rats spent no more time in the E quadrant than in the W quadrant (p = 0.381). Furthermore, t-test comparison confirmed that animals spent significantly more time in the target quadrant than would be expected by chance (p<0.01).
Figure 2. Probe trial conducted after the last water maze acquisition.
(A) The percentage of time spent in the target quadrant. (B) The percentage of distance traveled in the target quadrant. (C) The percentage of time spent in each quadrant. Results are represented as mean ± SEM. *p<0.05, **p<0.01.doi:10.1371/journal.pone.0079408.g002
The Expression of pCREB was Decreased in the Hippocampus of Aged Rats 48 h after Sevoflurane-N2O Anesthesia
We killed the rats 48 h after anesthesia and measured the pCREB and total CREB levels in the Sev+N2O and Con rats. The Western blot results showed that the immunoreactive bands of pCREB as well as CREB appeared at 43 kDa, and the pCREB levels in the Sev+N2O group were significantly decreased (p = 0.029), but no significant difference in the levels of total CREB was observed (p = 0.923, Figure 3A, 3B). Figure 4A shows the pCREB immunoreactivity (pCREB-ir) distribution in all the subfields of the hippocampus, while Figure 4C shows the pCREB-ir in all the layers of CA1 and CA3 sections. The immunofluorescence revealed that pCREB was exclusively located in neuronal nuclei and there was a significant reduction of pCREB-ir in Sev+N2O rats compared with the Con group. These results implied that sevoflurane-N2O anesthesia would inhibit the activation of CREB 48 h after exposure in aged rats.
Figure 3. Phosphorylated CREB decreased 48-N2O anesthesia as well as 15 min after probe test.
(A) The levels of pCREB in the Sev+N2O group were lower than the Con group, but no differences of CREB levels were detected. (B) Relative levels of pCREB and CREB were quantified (p = 0.029 for pCREB, p = 0.923 for CREB). (C) The pCREB levels in the hippocampus of Sev+N2O-MWM rats were significantly reduced compared to the Con-MWM rats, but CREB expression had no significant difference. (D) Relative levels of pCREB and CREB were quantified (p = 0.01 for pCREB, p = 0.58 for CREB). *p<0.05, **p<0.01.doi:10.1371/journal.pone.0079408.g003
Figure 4. pCREB immunoreactivity (pCREB-ir) distribution in all the subfields of hippocampus and exclusively located in the neuronal nuclei. The pCREB-ir decreased both 48 h after sevoflurane-N2O anesthesia and 15 min after probe test.
(A) There was a derease of pCREB-ir in all the subfields of the hippocampus in Sev+N2O rats compared with the Con group. (B) The pCREB-ir significantly decreased in the Sev+N2O-MWM group compared with Con-MWM group. (C) pCREB-ir both in CA1 and CA3 areas decreased in the exposed rats compared with controls. Magnification: ×200 for (A), ×400 for (B).doi:10.1371/journal.pone.0079408.g004
Expression of pCREB in the Hippocampus of Aged Rats after Morris Water Maze
Both the Sev+N2O-MWM group and the control group were sacrificed 15 min after the probe trial on day7, and we compared the levels of pCREB between groups by Western blot. The results showed that the pCREB levels in the Sev+N2O-MWM rats were significant lower than those in the Con-MWM rats, but no significant difference of total CREB was detected (Figure 3C, 3D).
The immunofluorescence results showed that pCREB was higher in the Con-MWM group than in the Con group (Figure 4A, 4C), which was in accordance with our previous study showing that the retrieval of spatial memory could activate the phosphorylation of CREB . However, the levels of pCREB in the Sev+N2O-MWM rats were significantly lower than those in the Con-MWM rats, suggesting that the decrease of pCREB expression induced by general anesthesia with Sev+N2O was sustained until at least the end of the behavioral procedures (Figure 4B, 4C).
CREB phosphorylation can be achieved by a number of up-stream signaling cascades, among which a pathway is triggered by cAMP accumulation to cause liberation of catalytic subunits of cAMP – dependent protein kinase A (PKA) . Because general anesthesia induced the decrease of pCREB expression, we assessed whether it was related to the cAMP pathway. The ELISA results revealed that the cAMP levels decreased by 65% in hippocampus of Sev+N2O rats compared to the Con rats (p = 0.02, Figure 5A). Similarly, in the Sev+N2O-MWM group, there was a 58% decrease of cAMP levels compared to Con-MWM (p = 0.048, Figure 5B). Thus, the results suggested that general anesthesia with Sev+N2O for 4 hours decreased the cAMP levels and in turn suppressed the cAMP/CREB signaling.
Figure 5. cAMP concentration in the hippocampus of all the groups.
(A) cAMP levels decreased by 65% in hippocampus of Sev+N2O rats compared to the Con rats (p = 0.02). (B) cAMP levels decreased by 58% in Sev+N2O-MWM compared to Con-MWM (p = 0.048).doi:10.1371/journal.pone.0079408.g005
Neuronal Cell Death Increased after Sev+N2O General Anesthesia
To understand whether cAMP/pCREB down-regulation affect the neuronal survival after Sev+N2O anesthesia, we evaluated the neuronal survival by Nissl staining and NeuN staining. Nissl staining is used for the detection of Nissl Body in the cytoplasm of neurons and identifying the basic neuronal structure. Quantitative analysis of Nissl-positive cells in hippocampal CA1 showed a neuronal loss in exposed rats compared with their controls (p<0.05, Figure 6A, B). Otherwise, expression of NeuN, a neuronal marker, also decreased in the Sev+N2O group as well as Sev+N2O-MWM group (Figure 6C). To confirm the neuronal loss is due to the neuronal apoptosis, double labeled of Annexin V-FITC and NeuN was performed.Figure 7B shows that the total number of Annexin V-positive cells in the hippocampal CA1 significantly increased in the rats exposed to anesthetics compared with the controls. However, most of the Annexin V-positive cells also stained positive for the neuronal marker NeuN. As loss of CREB triggered Bax-dependent apoptosis , it is possible that inhibition of CREB signaling by general anesthesia induces the Bax overexpression and contributes to the neuronal apoptosis. To verify it, we tested the Bax protein levels by Western Blot and found that the Bax expression significantly increased in the Sev+N2O group compared with Con group (Figure 7A). However, the Bax expression was only slightly higher in Sev+N2O-MWM rats than in Con-MWM rats (Figure 7A).
Figure 6. More neuronal loss in the CA1 subfield of hippocampus in exposed rats than control rats by Nissl staining and NeuN staing.
(A) Nissl-positve cells in CA1 subfield decreased in the Sev+N2O group and Sev+N2O-MWM group compared with their respective controls. (B) The number of Nissl-postive cells was quantified. The number of neurons in each section was averaged from three random different vision fields in the CA1 area of hippocampus per hemisphere (six vision fields per section) under 40×objective Leica microscope. 5 sections per rat were analyzed (n = 5 in each of Sev+N2O, Con and Sev+N2O-MWM group, and n = 4 in Con-MWM group. p = 0.0286, 0.016, respectively). (C) Expression of the mature neuronal marker NeuN in CA1 subfield decreased in exposed rats compared with controls. Magnification: ×400 for (A), ×200 for (C).doi:10.1371/journal.pone.0079408.g006
Figure 7. Expression of Bax was detected by Western Blot and neuronal apoptosis was evaluated by doubled staining with Annexin V-FITC and NeuN.
(A) Bax expression was significantly increased in the Sev+N2O group compared with Con group, but only slightly increased in the Sev+N2O-MWM compared with Con-MWM group. (B) Doubled staining with Annexin V-FITC and NeuN showed that there were more neurons underwent apoptosis in the CA1 subfield of hippocampus in Sev+N2O and Sev+N2O-MWM rats. Magnification: ×200 for (B).doi:10.1371/journal.pone.0079408.g007
Neurogenesis in the Hippocampal DG
As training on hippocampus-dependent tasks, such as the spatial Morris water maze, increases hippocampal neurogenesis . Decreasing hippocampal neurogenesis has been demonstrated to impair long-term spatial memory . We examined the possibility that the spatial learning and memory impairments in aged rats after general anesthesia with Sev+N2O might be related to a deficiency in DG neurogenesis due to the downregulation of cAMP/pCREB. We analyzed the differences in expression of various neuronspecific markers related to different neuronal stages, Nestin used as a marker of neuroepithelial/progenitor cells and DCX as immature neuronal marker. The immunofluorescence staining results showed that less neurons expressed Nestin in the Sev+N2O group as well as Sev+N2O-MWM group (Figure 8A). Moreover, we found less DCX-positive cells in the Sev+N2O-MWM group compared with Con-MWM group (Figure 8B). These results indicated that neuronal progenitor proliferation and differentiation was inhibited.
Figure 8. Expression of Nestin and DCX in DG area of hippocampus was detected by Immunofluorescence.
(A) Nestin-postive cells were less in the exposed rats than controls. (B) DCX-positive cells decreased in the Sev+N2O-MWM group compared with Con-MWM group. Magnification: ×200 for (A) and (B).doi:10.1371/journal.pone.0079408.g008
With the coming of the aging society, more and more surgical procedures currently performed are in elderly patients. Patients with an age≥60 years have an increased incidence of POCD, which is associated with an increased mortality , , . The aged brain might be more susceptible to anesthetic-mediated changes, as the aged brain is different from the younger in several respects, including size, distribution and type of neurotransmitters, metabolic function, and capacity for plasticity . As sevoflurane and N2O are commonly used anesthetic agents in clinical practice, the current study focused on the behavioral and biochemical effects to aged rats by general anesthesia with sevoflurane and N2O. We first found that 18-month-old rats developed cognitive deficits after exposure to 1.3% sevoflurane combined with 50% N2O for 4 h without surgery. It was unlikely that this impairment was caused by residual anesthetic because we did not perform the MWM training until 48 h after anesthesia. Culley et al showed that aged rats subjected to ISO+N2O anesthesia exhibited poor performance on a spatial memory task for at least 2 weeks after general anesthesia, and the induced impairment may have been worse than ISO alone. Moreover, a 4-h exposure of aged rats to N2O alone has been reported to cause learning impairment up to 2 weeks . Recent studies also showed that exposure to sevoflurane increased β-amyloid protein levels, which could induce further apoptosis and contribute to or cause POCD , , , , . Sevoflurane at doses from 0.5% to 2.6% administered either during or immediately after a learning task has been shown to inhibit memory retention , , , , . Although the inhalation anesthesia–POCD model has been well documented, it is debated. Rammes et al  reported ISO enhanced long-term potentiation (LTP) in CA1 hippocampal neurons and improved hippocampus-dependent cognitive performance, while a recent study demonstrated that 4 h of sevoflurane exposure did not impair acquisition learning or retention memory and might even improve learning in young adult or aged rat . Different methodologies, including different anesthetic, dosage, rat strain, duration of exposure, outcome measurements, and anesthetic carrier gas may have contributed to these contradictory results. In the current study, we used 1.3% sevoflurane and 50% N2O as the anesthetics and 50% O2 for the carrier gas, which could be one of the factors that contributed to the differences.
The precise mechanism of molecular biology of POCD is still not very clear so far. Our current study tried to demonstrate the effects of anesthetics (i.e., sevoflurane, N2O) on biochemical changes associated with cognitive dysfunction. Current hypotheses attribute the neurocognitive deficits produced by anesthetics to neurotoxic effects, endogenous neurodegenerative, neuroinflammatory mechanisms, or age-sensitive suppression of neuronal stem cell proliferation and differentiation , , . Moreover, brain-derived neurotrophic factor has been implicated in the neurotoxicity of N2O, midazolam, and ISO . As mentioned previously, pCREB promotes the transcription of immediate-early gene mRNA, which is then translated into proteins. These proteins are necessary for the maintenance of LTP and long-term memory, while spatial learning has been shown to increase CREB phosphorylation in the dorsal hippocampus , , , , , . In the current study, we measured the pCREB and CREB levels in the hippocampus 48 h after exposure but before water maze test, as well as 15 min after the probe trial. The results found that pCREB levels in the Sev+N2O and Sev+N2O-MWM group were significantly lower than their respective controls. Our results suggested that sevoflurane-N2O anesthesia decreased pCREB levels and lasted until at least the probe trial finished, which would affect the capacity of pCREB’s maintenance of LTP and long-term memory. However, we found the decrease of pCREB could be due to the phosphorylation of preexisting CREB, because there was no difference in total CREB levels among groups.
To identity the upstream regulators of CREB-signaling, studies have demonstrated that NMDA-receptor activation, BDNF (brain derived neurotrophic factor) signaling or growth factor signaling can trigger intracellular signaling cascades that phosphorylate CREB at Ser133, which is a rate-limiting step in the CREB-signaling , . Among various signaling pathways, the most thoroughly pathway is to stimulate adenylyl cyclase and accumulate second messenger cAMP to activate PKA and lead to the release of the catalytical subunit of PKA which then shuttles to the nucleus and phosphorylates CREB . In the current study, we found the cAMP levels decreased in the hippocampus of Sev+N2O group and Sev+N2O-MWM group compared to their respective control group. The above evidence indicated that general anesthesia with Sev+N2O down-regulated the cAMP/CREB signaling and further affected its downstream targets.
New neurons are produced each day through the process of neurogenesis in the hippocampus, while physical training can modify the process by increasing the number of new cells that mature into functional neurons in the adult brain and improve the cognitive ability including learning and memory . In the past years, researchers have found that rats exposure to isoflurane on P7 showed decreased neuronal progenitor proliferation with deficits in fear conditioning and spatial reference task, and P14 rats exposed to isoflurane showed a decreased neurogenesis in hippocampus and impaired cognitive function compared with controls . It has previously been noted that CREB signaling is essential for survival and morphological development of newborn neurons, and for maintenance of expression of proteins involved in neuronal development and the control of neurogenic transcriptional programs, while activation of cAMP signaling promotes the proliferation and morphological maturation of newborn cells , . The in vivo function of CREB-signaling in adult neurogenesis has been examined using pharmacological, genetic and retrovirus-mediated gene transfer . Thus, examination of DG neurogenesis may provide some information about how anesthetics cause learning and memory deficits via downregulation of CREB signaling. We found that the down-expression of pCREB in the hippocampus of rats exposed to Sev+N2O anesthesia disrupted the DG neurogenesis and exacerbated the cognitive dysfunction. This was underscored, at least in part, by our observation that the number of Nestin-positve cells and Dcx-positive cells was less in the exposed rats compared to the controls, which indicated the decrease of neuronal progenitor proliferation, differentiation and maturation.
Researchers have shown that CREB DNA binding activity and phosphorylation of CREB are necessary for nerve growth factor (NGF)-dependent survival of sympathetic neurons. Bonni et al indicated that CREB may also have a function in the regulation of neuronal survival in the developing central nervous system. Furthermore, experiments using genetic transfer have demonstrated that CREB is a key executor of neurotrophin-mediated cell survival and loss of CREB triggers Bax-dependent apoptosis in vivo . These findings demonstrated a critical role for CREB and CREB-dependent gene expression in supporting neuronal survival. In our work, we found an increase of neuronal loss in the exposed rats compared with controls. Annexin V-FITC by immunofluorescence and Bax expression by Westren blot revealed the neuronal loss was due to the trigger of neuronal apoptosis. Importantly, double staining of Annexin V and the neuronal marker NeuN indicated that most of the apoptotic cells in these structures were indeed neurons. In conjunction with the role of CREB in DG neurogenesis, these findings partly support our prediction that downregulation of cAMP/CREB induced by general anesthesia leads to a decrease of the neurogenesis in hippocampus as well as an increase of neuronal apoptosis, both of which play critical roles in the deficit of learning and memory after Sev+N2O exposure.
In summary, we found that general anesthesia with 1.3% sevoflurane and 50% N2O impaired hippocampus-dependent learning and memory in aged rats. To explore the mechanism of neurotoxicity induced by Sev+N2O, we found the downregulation of cAMP/CREB signaling which was implicated in the learning and memory, long term potentiation, and neuroprection. Furthermore, we observed the decrease of DG neurogenesis and neuronal survival in the hippocampus, both of which highly depended on the normal activation of CREB signaling. Thus, the pathway of cAMP/CREB-neurogensis/neuronal apoptosis contributed to the neurotoxicity and impairment of learning and memory induced by Sev+N2O. The study provided some theoretical basis for the further study of neurotrophin factors, which could prevent POCD in elderly patients and decrease the postoperative morbidity and mortality.
Impaired Spatial Learning and Memory after Sevoflurane–Nitrous Oxide Anesthesia in Aged Rats Is Associated with Down-Regulated cAMP/CREB Signaling
Wan-Xia Xiong , Guo-Xia Zhou , Bei Wang, Zhang-Gang Xue, Lu Wang, Hui-Chuan Sun, Sheng-Jin Ge
Autism spectrum disorder is a neurodevelopmental syndrome diagnosed primarily by persistent deficits in social interactions and communication, unusual sensory reactivity, motor stereotypies, repetitive behaviors, and restricted interests. No FDA-approved medical treatments exist for the diagnostic symptoms of autism. Here we interrogate multiple pharmacological targets in two distinct mouse models that incorporate well-replicated autism-relevant behavioral phenotypes. Compounds that modify inhibitory or excitatory neurotransmission were selected to address hypotheses based on previously published biological abnormalities in each model. Shank3B is a genetic model of a mutation found in autism and Phelan-McDermid syndrome, in which deficits in excitatory neurotransmission and synaptic plasticity have been reported. BTBR is an inbred strain model of forms of idiopathic autism in which reduced inhibitory neurotransmission and excessive mTOR signaling have been reported. The GABA-A receptor agonist gaboxadol significantly reduced repetitive self-grooming in three independent cohorts of BTBR. The TrkB receptor agonist 7,8-DHF improved spatial learning in Shank3B mice, and reversed aspects of social deficits in BTBR. CX546, a positive allosteric modulator of the glutamatergic AMPA receptor, and d-cycloserine, a partial agonist of the glycine site on the glutamatergic NMDA receptor, did not rescue aberrant behaviors in Shank3B mice. The mTOR inhibitor rapamycin did not ameliorate social deficits or repetitive behavior in BTBR mice. Comparison of positive and negative pharmacological outcomes, on multiple phenotypes, evaluated for replicability across independent cohorts, enhances the translational value of mouse models of autism for therapeutic discovery. GABA agonists present opportunities for personalized interventions to treat components of autism spectrum disorder. Autism Res 2019. © 2019 The Authors. Autism Research published by International Society for Autism
Research published by Wiley Periodicals, Inc.
Many of the risk genes for autism impair synapses, the connections between nerve cells in the brain. A
drug that reverses the synaptic effects of a mutation could offer a precision therapy. Combining pharmacological and
behavioral therapies could reduce symptoms and improve the quality of life for people with autism. Here we report reductions
in repetitive behavior by a GABA-A receptor agonist, gaboxadol, and improvements in social and cognitive behaviors
by a TrkB receptor agonist, in mouse models of autism.
The invention relates to a neuroprotective composition derived from mesenchymal stem cells , especially a neuro protective composition derived from the primary culture of dental pulp mesenchymal stem cells . The invention also relates to a process for preparing the neuroprotective com position , as well as the medical use of the neuroprotective composition in the treatment of Parkinson ' s disease .
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Adaptive fear responses to external threats rely upon efficient relay of computations underlying contextual encoding to subcortical circuits. Brain-wide analysis of highly coactivated ensembles following contextual fear discrimination identified the dorsolateral septum (DLS) as a relay of the dentate gyrus–CA3 circuit. Retrograde monosynaptic tracing and electrophysiological whole-cell recordings demonstrated that DLS somatostatin-expressing interneurons (SST-INs) receive direct CA3 inputs. Longitudinal in vivo calcium imaging of DLS SST-INs in awake, behaving mice identified a stable population of footshock-responsive SST-INs during contextual conditioning whose activity tracked and predicted non-freezing epochs during subsequent recall in the training context but not in a similar, neutral context or open field. Optogenetic attenuation or stimulation of DLS SST-INs bidirectionally modulated conditioned fear responses and recruited proximal and distal subcortical targets. Together, these observations suggest a role for a potentially hard-wired DLS SST-IN subpopulation as arbiters of mobility that calibrate context-appropriate behavioral fear responses
Neurobiology of Disease, Volume 65, May 2014, Pages 55-68
Null mutations in the gene encoding the major myelin protein of the central nervous system, proteolipid protein 1 (PLP1), cause an X-linked form of spastic paraplegia (SPG2) associated with axonal degeneration. While motor symptoms are the best known manifestations of this condition, its somatosensory disturbances have been described but poorly characterized. We carried out a longitudinal study in an animal model of SPG2 — mice carrying a deletion of the Plp1 gene (Plp-null mice). Plp-null mice exhibited severe early-onset thermal hyperalgesia, in the absence of thermal allodynia. We first performed an electrophysiological testing which showed an early decrease in peripheral and spinal conduction velocities in Plp null mice. Such as the abnormal sensitive behaviors, this slowing of nerve conduction was observed before the development of myelin abnormalities at the spinal level, from 3 months of age, and without major morphological defects in the sciatic nerve. To understand the link between a decrease in nerve velocity and an increased response to thermal stimuli before the appearance of myelin abnormalities, we focused our attention on the dorsal horn of the spinal cord, the site of integration of somatosensory information. Immunohistochemical studies revealed an early-onset activation of astrocytes and microglia that worsened with age, associated later in age with perturbation of the expression of the sensory neuropeptides calcitonin-gene-related peptide and galanin. Taken together, these results represent complementary data supporting the hypothesis that Plp-null mice suffer from ganglionopathy associated with late onset central demyelination but with few peripheral nerve alterations, induced by the glial-cell-mediated sensitization of the spinal cord. The mechanism suggested here could underlie pain experiments in other leukodystrophies as well as in other non-genetic demyelinating diseases such as multiple sclerosis.
Mice with a deletion of the major central myelin protein exhibit hypersensitivity to noxious thermal stimuli: involvement of central sensitization
Bérengère Petita,, Fabrice Giraudetb, Céline Béchona, Laurent Bardinf, Paul Avanb, Odile Boespflug-Tanguyd, Mélina Bégou
Neuron, Volume 83, Issue 1, p189–201, 2 July 2014
- The DG and CA3 contain memory traces for fear inducing contexts
- Over time, contextual memories generalize and become less localized in CA3
- Optical inhibition of context-specific DG or CA3 cells inhibits memory expression
- Ablating neurogenesis impairs memory traces in CA3, but not in the DG
SummaryMemory traces are believed to be ensembles of cells used to store memories. To visualize memory traces, we created a transgenic line that allows for the comparison between cells activated during encoding and expression of a memory. Mice re-exposed to a fear-inducing context froze more and had a greater percentage of reactivated cells in the dentate gyrus (DG) and CA3 than mice exposed to a novel context. Over time, these differences disappeared, in keeping with the observation that memories become generalized. Optogenetically silencing DG or CA3 cells that were recruited during encoding of a fear-inducing context prevented expression of the corresponding memory. Mice with reduced neurogenesis displayed less contextual memory and less reactivation in CA3 but, surprisingly, normal reactivation in the DG. These studies suggest that distinct memory traces are located in the DG and in CA3 but that the strength of the memory is related to reactivation in CA3.
Christine A. Denny, Mazen A. Kheirbek, Eva L. Alba, Kenji F. Tanaka, Rebecca A. Brachman, Kimberly B. Laughman, Nicole K. Tomm, Gergely F. Turi, Attila Losonczy, René Henemail
We hypothesize that the trisomy 21 (Down syndrome) is the additive and interactive outcome of the triple copy of different regions of HSA21. Because of the small number of patients with partial trisomy 21, we addressed the question in the Mouse in which three chromosomal regions located on MMU10, MMU17 and MMU16 carries almost all the HSA21 homologs. Male mice from four segmental trisomic strains covering the D21S17-ETS2 (syntenic to MMU16) were examined with an exhaustive battery of cognitive tests, motor tasks and MRI and compared with TS65Dn that encompasses D21S17-ETS2. None of the four strains gather all the impairments (measured by the effect size) of TS65Dn strain. The 152F7 strain was close to TS65Dn for motor behavior and reference memory and the three other strains 230E8, 141G6 and 285E6 for working memory. Episodic memory was impaired only in strain 285E6. The hippocampus and cerebellum reduced sizes that were seen in all the strains indicate that trisomy 21 is not only a hippocampus syndrome but that it results from abnormal interactions between the two structures.
Down syndrome (DS) is a common genetic cause of intellectual disability yet no pro-cognitive drug therapies are approved for human use. Mechanistic studies in a mouse model of DS (Ts65Dn mice) demonstrate that impaired cognitive function is due to excessive neuronal inhibitory tone. These deficits are normalized by chronic, short-term low doses of GABAA receptor (GABAAR) antagonists in adult animals, but none of the compounds investigated are approved for human use. We explored the therapeutic potential of flumazenil (FLUM), a GABAAR antagonist working at the benzodiazepine binding site that has FDA approval. Long-term memory was assessed by the Novel Object Recognition (NOR) testing in Ts65Dn mice after acute or short-term chronic treatment with FLUM. Short-term, low, chronic dose regimens of FLUM elicit long-lasting (>1 week) normalization of cognitive function in both young and aged mice. FLUM at low dosages produces long lasting cognitive improvements and has the potential of fulfilling an unmet therapeutic need in DS.
S 38093, a novel histamine H3 receptor inverse agonist, was tested in a series of neurochemical and behavioral paradigms designed to evaluate its procognitive and arousal properties.
In intracerebral microdialysis studies performed in rats, S 38093 dose-dependently increased histamine extracellular levels in the prefrontal cortex and facilitated cholinergic transmission in the prefrontal cortex and hippocampus of rats after acute and chronic administration (10 mg/kg i.p.).
Acute oral administration of S 38093 at 0.1 mg/kg significantly improved spatial working memory in rats in the Morris water maze test. The compound also displayed cognition enhancing properties in the two-trial object recognition task in rats, in a natural forgetting paradigm at 0.3 and 1 mg/kg p.o. and in a scopolamine-induced memory deficit situation at 3 mg/kg p.o. The property of S 38093 to promote episodic memory was confirmed in a social recognition test in rats at 0.3 and 1 mg/kg i.p.
Arousal properties of S 38093 were assessed in freely moving rats by using electroencephalographic recordings: at 3 and 10 mg/kg i.p., S 38093 significantly reduced slow wave sleep delta power and induced at the highest dose a delay in sleep latency. S 38093 at 10 mg/kg p.o. also decreased the barbital-induced sleeping time in rats.
Taken together these data indicate that S 38093, a novel H3 inverse agonist, displays cognition enhancing at low doses and arousal properties at higher doses in rodents.
Severity of lithium poisoning depends on the ingested dose, previous treatment duration and renal function. No animal study has investigated neurobehavioral differences in relation to the lithium poisoning pattern observed in humans, while differences in lithium pharmacokinetics have been reported in lithium-pretreated rats mimicking chronic poisonings with enhanced brain accumulation in rats with renal failure. Our objectives were: 1)-to investigate lithium-related effects in overdose on locomotor activity, anxiety-like behavior, spatial recognition memory and anhedonia in the rat; 2)-to model the relationships between lithium-induced effects on locomotion and plasma, erythrocyte, cerebrospinal fluid and brain concentrations previously obtained according to the poisoning pattern. Open-field, elevated plus-maze, Y-maze and sucrose consumption tests were used. In acutely lithium-poisoned rats, we observed horizontal (p < 0.001) and vertical hypolocomotion (p < 0.0001), increased anxiety-like behavior (p < 0.05) and impaired memory (p < 0.01) but no altered hedonic status. Horizontal (p < 0.01) and vertical (p < 0.001) hypolocomotion peaked more markedly 24 h after lithium injection and was more prolonged in acute-on-chronically vs. acutely lithium-poisoned rats. Hypolocomotion in chronically lithium-poisoned rats with impaired renal function did not differ from acutely poisoned rats 24 h after the last injection. Interestingly, hypolocomotion/concentration relationships best fitted a sigmoidal Emax model in acute poisoning and a linear regression model linked to brain lithium in acute-on-chronic poisoning. In conclusion, lithium overdose alters rat behavior and consistently induces hypolocomotion which is more marked and prolonged in repeatedly lithium-treated rats. Our data suggest that differences between poisoning patterns regarding lithium-induced hypolocomotion are better explained by the duration of lithium exposure than by its brain accumulation.
Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of α and β subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4β2-, and α6-containing nAChRs, respectively. By contrast, GABAA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs.
Journal of International Society of Psychoneuroendocrinology
Early-life exposure to calorie-dense food, rich in fat and sugar, contributes to the increasing prevalence of obesity and its associated adverse cognitive and emotional outcomes at adulthood. It is thus critical to determine the impact of such nutritional environment on neurobehavioral development. In animals, maternal high-fat diet (HFD) consumption impairs hippocampal function in adult offspring, but its impact on hippocampal neuronal morphology is unknown. Moreover, the consequences of perinatal HFD exposure on the amygdala, another important structure for emotional and cognitive processes, remain to be established. In rats, we show that adult offspring from dams fed with HFD (45% from fat, throughout gestation and lactation) exhibit atrophy of pyramidal neuron dendrites in both the CA1 of the hippocampus and the basolateral amygdala (BLA). Perinatal HFD exposure also impairs conditioned odor aversion, a task highly dependent on BLA function, without affecting olfactory or malaise processing. Neuronal morphology and behavioral alterations elicited by perinatal HFD are not associated with body weight changes but with higher plasma leptin levels at postnatal day 15 and at adulthood. Taken together, our results suggest that perinatal HFD exposure alters hippocampal and amygdala neuronal morphology which could participate to memory alterations at adulthood.
Learning & Memory
Retrieval of an associative memory can lead to different phenomena. Brief reexposure sessions tend to trigger reconsolidation, whereas more extended ones trigger extinction. In appetitive and fear cued Pavlovian memories, an intermediate “null point” period has been observed where neither process seems to be engaged. Here we investigated whether this phenomenon extends to contextual fear memory. Adult rats were subjected to a contextual fear conditioning paradigm, reexposed to the context 2 d later for 3, 5, 10, 20, or 30 min, with immediate injections of MK-801 or saline following reexposure, and tested on the following day. We observed a significant effect of MK-801 with the 3- and 30-min sessions, impairing reconsolidation and extinction, respectively. However, it did not have significant effects with 5-, 10-, or 20-min sessions, even though freezing decreased from reexposure to test. Further analyses indicated that this is not likely to be due to a variable transition point at the population level. In conclusion, the results show that in contextual fear memories there is a genuine “null point” between the parameters that induce reconsolidation and extinction, as defined by the effects of MK-801, although NMDA receptor-independent decreases in freezing can still occur in these conditions.
One role of BACE 1 (Beta-site amyloid precursor protein cleaving enzyme 1) is to cleave the sequential amyloid precursor protein (APP) into β-Amyloid (Aβ), the accumulation of which is an important participant in the formation of the amyloid plaques and neurofibrillary tangles of Alzheimer’s disease (AD). Our previous study showed BACE 1, the potential functional downstream target of miR-124, to be connected to cell death in AD cell models. Recent studies have shown that autophagy is altered in AD, however, as to whether miR-124 is involved in this alteration is not clear. In this study, 7-month-old APP/PS1 transgenic mice were transfected with miR-124 lentiviral vectors, injected bilaterally into the dentate gyrus (DG) of mice hippocampi. Following 7 days of recovery, both behavior and biochemical pathology tests were implemented. The results demonstrated learning ability improvement and specific AD pathology alleviation. Meanwhile there was down-regulation of Bcl-2 to Bax ratio expression, increase in Beclin-1 and decreases in expression of LC3II, Atg5 and p62/SQSTMl. In view of this, we hypothesis that miR-124 conducts its neuroprotective effect through BACE 1 by regulation of autophagic pathways.
PLoS ONE 12(9): e0184811.
Traumatic brain injury (TBI) results in white matter injury (WMI) that is associated with neurological deficits. Neuroinflammation originating from microglial activation may participate in WMI and associated disorders. To date, there is little information on the time courses of these events after mild TBI. Therefore we investigated (i) neuroinflammation, (ii) WMI and (iii) behavioral disorders between 6 hours and 3 months after mild TBI. For that purpose, we used experimental mild TBI in mice induced by a controlled cortical impact. (i) For neuroinflammation, IL-1b protein as well as microglial phenotypes, by gene expression for 12 microglial activation markers on isolated CD11b+ cells from brains, were studied after TBI. IL-1b protein was increased at 6 hours and 1 day. TBI induced a mixed population of microglial phenotypes with both pro-inflammatory, anti-inflammatory and immunomodulatory markers from 6 hours to 3 days post-injury. At 7 days, microglial activation was completely resolved. (ii) Three myelin proteins were assessed after TBI on ipsi- and contralateral corpus callosum, as this structure is enriched in white matter. TBI led to an increase in 2',3'-cyclic-nucleotide 3'-phosphodiesterase, a marker of immature and mature oligodendrocyte, at 2 days post-injury; a bilateral demyelination, evaluated by myelin basic protein, from 7 days to 3 months post-injury; and an increase in myelin oligodendrocyte glycoprotein at 6 hours and 3 days post-injury. Transmission electron microscopy study revealed various myelin sheath abnormalities within the corpus callosum at 3 months post-TBI. (iii) TBI led to sensorimotor deficits at 3 days post-TBI, and late cognitive flexibility disorder evidenced by the reversal learning task of the Barnes maze 3 months after injury. These data give an overall invaluable overview of time course of neuroinflammation that could be involved in demyelination and late cognitive disorder over a time-scale of 3 months in a model of mild TBI. This model could help to validate a pharmacological strategy to prevent post-traumatic WMI and behavioral disorders following mild TBI.
Sleep restriction (SR) impairs short term memory (STM) that might be related to different processes. Neuropeptide S (NPS), an endogenous neuropeptide that improves short term memory, activates arousal and decreases anxiety is likely to counteract the SR-induced impairment of STM. The objective of the present study was to find common cerebral pathways in sleep restriction and NPS action in order to ultimately antagonize SR effect on memory.
The STM was assessed using a spontaneous spatial alternation task in a T-maze. C57-Bl/6 J male mice were distributed in 4 groups according to treatment (0.1 nmol of NPS or vehicle intracerebroventricular injection) and to 20 h-SR. Immediately after behavioural testing, regional c-fos immunohistochemistry was performed and used as a neural activation marker for spatial short term memory (prefrontal cortex, dorsal hippocampus) and emotional reactivity (basolateral amygdala and ventral hippocampus). Anxiety-like behaviour was assessed using elevated-plus maze task.
Results showed that SR impaired short term memory performance and decreased neuronal activation in cingular cortex.NPS injection overcame SR-induced STM deficits and increased neuronal activation in infralimbic cortex. SR spared anxiety-like behavior in the elevated-plus maze. Neural activation in basolateral nucleus of amygdala and ventral hippocampus were not changed after SR.In conclusion, the present study shows that NPS overcomes SR-induced STM deficits by increasing prefrontal cortex activation independently of anxiety-like behaviour.
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is pathologically characterized by the deposition of β-amyloid (βA) peptides in senile plaques and neurofibrillary tangles in the brain. Flavonoids have recently been used to prevent and treat a variety of neurodegenerative diseases, but little is known about bioflavonoids. In this study, we evaluate whether a biflavonoid fraction (BF) exerts neuroprotective effects on an aged triple transgenic mouse mode of AD (3xTg-AD). Then, 21–24-month-old 3xTg AD mice were i.p. injected with 25 mg/kg of a BF from Garcinia madruno composed of morelloflavone (65%), volkensiflavone (12%), GB 2a (11%), fukugiside (6%) and amentoflavone (0.4%) every 48 h for 3 months. The BF treatment reduced βA deposition in different regions of the brain (the hippocampus, entorhinal cortex and amygdala), reduced βA1-40 and βA1-42 levels, BACE1-mediated cleavage of APP (CTFβ), tau pathology, astrogliosis and microgliosis in the brains of aged 3xTg-AD mice. Although the BF treatment weakly improved learning, animals treated with BF spent more time in the open arms of the elevated plus maze test and displayed greater risk assessment behavior than the control groups. In summary, the BF reverses histopathological hallmarks and reduces emotional disorders in the 3xTg-AD mouse model, suggesting that the biflavonoids from G. madruno represent a potential natural therapeutic option for AD if its bioavailability is improved.
During development, experience continuously interacts with genetic information to shape and optimize neuronal circuits and behaviour. Therefore, environmental conditions have a powerful impact on the brain. To date, accumulating evidence shows that raising animals in a so-called “enriched environment” elicits remarkable effects on the brain across molecular, anatomical, and functional levels when compared to animals raised in a “standard cage” environment. In our article, we provide a brief review of the field and illustrate the different results of “enriched” versus standard cage-raised rodents with examples from visual system plasticity. We also briefly discuss parallel studies of enrichment effects in humans. Collectively, these data highlight that results should always be considered in the context of the animals’ environment.
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Neurobiology of Learning and Memory
The Hamlet test is a novel complex environment for testing topographic memory in mice.
Exploration and memory differ in different mouse strains but not gender.
The hippocampus-subiculum-parahippocampal gyrus axis and dopaminergic structures are activated.
Training increased hippocampal neurogenesis (cell proliferation and neuronal maturation).
Topographical disorientation was measured in the Hamlet test using a pharmacological model of Alzheimer's disease.
The Hamlet test is an innovative device providing a complex environment for testing topographic memory in mice. Animals were trained in groups for weeks in a small village with a central agora, streets expanding from it towards five functionalized houses, where they can drink, eat, hide, run, interact with a stranger mouse. Memory was tested by depriving mice from water or food and analyzing their ability to locate the Drink/Eat house. Exploration and memory were analysed in different strains, gender, and after different training periods and delays. After 2 weeks training, differences in exploration patterns were observed between strains, but not gender. Neuroanatomical structures activated by training, identified using FosB/ΔFosB immunolabelling, showed an involvement of the hippocampus-subiculum-parahippocampal gyrus axis and dopaminergic structures. Training increased hippocampal neurogenesis (cell proliferation and neuronal maturation) and modified the amnesic efficacy of muscarinic or nicotinic cholinergic antagonists. Moreover, topographical disorientation in Alzheimer's disease was addressed using intracerebroventricular injection of amyloid β25-35 peptide in trained mice. When retested after 7 days, Aβ25-35-treated mice showed memory impairment. The Hamlet test specifically allows analysis of topographical memory in mice, based on complex environment. It offers an innovative tool for various ethological or pharmacological research needs. For instance, it allows to examine topographical disorientation, a warning sign in Alzheimer's disease.
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Journal of Neuroscience Methods
Zebrafish larvae have a high potential as model system to replace rodents, especially in screening and drug discovery applications. However, an experimental setup to deliver mild electrical stimuli with simultaneous high throughput behavioural tracking has not yet been described.
A new tool was designed, making the delivery of electrical stimuli in a 96-well plate format possible. Using custom made electrode clips that can be slid over the walls of a square 96-well plate, 80 larvae could be tested simultaneously and behavioural responses recorded.
As proof of principle, two applications were tested: 1) The behavioural response after a single stimulus and the effect of buprenorphine on this response. 2) Habituation of locomotor activity to multiple stimuli and the involvement of the NMDA receptor. Reduced locomotor activity was observed after a single 5 V stimulus, however not with lower intensity stimuli. Pre-treatment with the analgesic buprenorphine prevented this response. Specificity of buprenorphine was confirmed using the antagonist naloxone. Habituation of locomotor activity was seen in response to multiple stimuli, depending on the inter stimulus interval. Treatment with the NMDA receptor antagonist memantine disrupted behavioural habituation.
Comparison with existing methods
The equipment and setup described here are the first of its kind using a 96-well plate format, thereby increasing the potential throughput in screening applications using zebrafish larvae.
The combination of the described electrode clips for stimulus delivery and behavioural tracking allows for the use of zebrafish larvae in a new array of medium to high throughput applications.
electrical stimulation; buprenorphine; naloxone; memantine; NMDA; zebrafish larvae
Phospholipid alterations in the brain are associated with progressive neurodegeneration and cognitive impairment after acute and chronic injuries. Various types of treatments have been evaluated for their abilities to block the progression of the impairment, but effective treatments targeting long-term post-stroke alterations are not available. In this study, we analyzed changes in the central and peripheral phospholipid profiles in ischemic rats and determined whether a protective monoterpene, Linalool, could modify them. We used an in vitro model of glutamate (125 μM) excitotoxicity and an in vivo global ischemia model in Wistar rats. Linalool (0.1 μM) protected neurons and astrocytes by reducing LDH release and restoring ATP levels. Linalool was administered orally at a dose of 25 mg/kg every 24 h for a month, behavioral tests were performed, and a lipidomic analysis was conducted using mass spectrometry. Animals treated with Linalool displayed faster neurological recovery than untreated ischemic animals, accompanied by better motor and cognitive performances. These results were confirmed by the significant reduction in astrogliosis, microgliosis and COX-2 marker, involving a decrease of 24:0 free fatty acid in the hippocampus. The altered profiles of phospholipids composed of mono and polyunsaturated fatty acids (PC 36:1; 42:1 (24:0/18:1)/LPC 22:6)/LPE 22:6) in the ischemic hippocampus and the upregulation of PI 36:2 and other LCFA (long chain fatty acids) in the serum of ischemic rats were prevented by the monoterpene. Based on these data, alterations in the central and peripheral phospholipid profiles after long-term was attenuated by oral Linalool, promoting a phospholipid homeostasis, related to the recovery of brain function.
Ischemia; Phospholipids; Neurodegeneration hallmarks; Linalool; Functional recovery
Current Protocols in Mouse Biology
Here we provide instructions to measure topographic memory in mice using the Hamlet test, a complex environment. The apparatus mimics a small hamlet with a central agora and five houses, which are functionalized since mice can drink, eat pellets, hide within a small maze, run in an activity wheel, or interact with a stranger mouse behind a grid. The houses are interconnected through a network of streets in a five‐arm star shape, and a video tracking system takes information from the activity in each house or follows a single mouse by trajectometry. Training the mice in the Hamlet, in groups for several hours per day over several days or weeks, allows consolidation of topographic memory (i.e., route learning involving both allocentric and egocentric strategies). Analysis of topographic memory can be performed a posteriori in a probe test by depriving mice of water or food and measuring their ability to efficiently reach the “eat” or “drink” house. Control groups include mice tested in non‐deprived condition and mice naïve to the Hamlet and tested in deprived or non‐deprived conditions. The present article details the apparatus, procedures, and protocols that can be used to reliably habituate mice in this complex environment and measure topographic memory.
Progress in Neuro-Psychopharmacology and Biological Psychiatry
Emerging evidence have posited that dysregulated microglia impair clearance and containment of amyloid‐β (Aβ) species in the brain, resulting in aberrant buildup of Aβ and onset of Alzheimer's disease (AD). Hematopoietic cell kinase (Hck) is one of the key regulators of phagocytosis among the Src family tyrosine kinases (SFKs) in myeloid cells, and its expression is found to be significantly altered in AD brains. However, the role of Hck signaling in AD pathogenesis is unknown. We employed pharmacological inhibition and genetic ablation of Hck in BV2 microglial cells and J20 mouse model of AD, respectively, to evaluate the impact of Hck deficiency on Aβ‐stimulated microglial phagocytosis, Aβ clearance, and resultant AD‐like neuropathology. Our in vitro data reveal that pharmacological inhibition of SFKs/Hck in BV2 cells and genetic ablation of their downstream kinase, spleen tyrosine kinase (Syk), in primary microglia significantly attenuate Aβ oligomers‐stimulated microglial phagocytosis. Whereas in Hck‐deficient J20 mice, we observed exacerbated Aβ plaque burden, reduced microglial coverage, containment, and phagocytosis of Aβ plaques, and induced iNOS expression in plaque‐associated microglial clusters. These multifactorial changes in microglial activities led to attenuated PSD95 levels in hippocampal DG and CA3 regions, but did not alter the postsynaptic dendritic spine morphology at the CA1 region nor cognitive function of the mice. Hck inhibition thus accelerates early stage AD‐like neuropathology by dysregulating microglial function and inducing neuroinflammation. Our data implicate that Hck pathway plays a prominent role in regulating microglial neuroprotective function during the early stage of AD development.
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European Journal of Neuroscience
Early non‐motor symptoms such as mood disorders and cognitive deficits are increasingly recognised in Parkinson's disease (PD). They may precede the characteristic motor symptomatology caused by dopamine (DA) neuronal loss in the substantia nigra pars compacta (SNc). It is well known that striatal cholinergic interneurons (ChIs) are emerging as key regulators of PD motor symptom, however, their involvement in the cognitive and affective alterations occurring in the premotor phase of PD is poorly understood. We used optogenetic photoinhibition of striatal ChIs in mice with mild nigrostriatal 6‐hydroxydopamine (6‐OHDA) lesions and assessed their role in anxiety‐like behaviour in the elevated plus maze, social memory recognition of a congener and visuospatial object recognition. In transgenic mice specifically expressing halorhodopsin (eNpHR) in cholinergic neurons, striatal ChIs photoinhibition reduced the anxiety‐like behaviour and reversed social and spatial short‐term memory impairment induced by moderate DA depletion (e.g., 50% loss of tyrosine hydroxylase TH‐positive neurons in the SNc). Systemic injection of telenzepine (0.3 mg/kg), a preferential M1 muscarinic cholinergic receptors antagonist, improved anxiety‐like behaviour, social memory recognition but not spatial memory deficits. Our results suggest that dysfunction of the striatal cholinergic system may play a role in the short‐term cognitive and emotional deficits of partially DA‐depleted mice. Blocking cholinergic activity with M1 muscarinic receptor antagonists may represent a possible therapeutic target, although not exclusive, to modulate these early non‐motor deficits.
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Neurobiology of Disease
This study aimed to determine the effects of diet‐induced paternal obesity on cognitive function in mice offspring.
Male mice (F0) were randomized to receive either a control diet (10 kcal% fat) or a high‐fat diet (HFD; 60 kcal% fat) for 10 weeks before being mated with normal females to generate F1 offspring. Male F1 offspring were mated with normal females to generate F2 offspring. Behavioral tests were used to assess cognitive functions in F1 and F2 offspring. Reduced representation bisulfite sequencing was used to the explore mechanisms of epigenetic inheritance.
HFD‐induced paternal obesity resulted in cognitive impairments in F1 offspring, potentially due, at least in part, to increased methylation of the BDNF gene promoter, which was inherited from F0 spermatozoa. BDNF/tyrosine receptor kinase B signaling was associated with cognitive impairments in HFD‐fed F1 offspring. However, there were no significant changes in F2 offspring.
The findings provide evidence of intergenerational effects of paternal obesity on cognitive function in offspring occurring via epigenetic spermatozoan modifications.
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International Journal of Fisheries and Aquatic Studies
Due to industrialization and urbanization many pollutants are being introduced directly and indirectly into aquatic ecosystem. Behavioural bioassay have been widely used in toxicity assessment. Bioassay based on behavior is faster, more sensitive and ecologically more relevant as assessing growth and reproduction need longer bioassay. Behavioural bioassay is more promising alternatives than lethality evaluating bioassay which are currently used for the risk assessment of toxicant. Behavioural changes provide early warning signals about the health of exposed population which other standard tests do not take in to consideration. These endpoints may be 10–100 times more sensitive than those derived from acute or chronic tests because chemicals can induce rapid behavioural responses in organisms even at very low concentrations. Behaviour is an organism-level effect defined as the action, reaction, or functioning of a system under a set of specific circumstances. We rationalize that a greater understanding of behavioural responses in effect to chemical stress may increase. Therefore in current scenario there is a need of developing newer and effective methods to study the behavioural responses. Behavioural changes in a fish form an efficient index to measure any alterations in the environmental conditions.
Link to the publication: https://www.researchgate.net/profile/Madhu_Sharma6/publication/330090387_Behavioural_responses_in_effect_to_chemical_stress_in_fish_A_review_Madhu_Sharma/links/5c2cd5b892851c22a35547bc/Behavioural-responses-in-effect-to-chemical-stress-in-fish-A-review-Madhu-Sharma.pdf
The Journal of neuroscience
Memory reconsolidation is hypothesised to be a mechanism by which memories can be updated with new information. Such updating has previously been shown to weaken memory expression or change the nature of the memory. Here we demonstrate that retrieval-induced memory destabilization also allows that memory to be strengthened by additional learning. We show that for rodent contextual fear memories, this retrieval-conditioning effect is observed only when conditioning occurs within a specific temporal window opened by retrieval. Moreover, it necessitates hippocampal protein degradation at the proteasome and engages hippocampal Zif268 protein expression, both of which are established mechanisms of memory destabilization-reconsolidation. We also demonstrate a conceptually analogous pattern of results in human visual paired-associate learning. Retrieval-relearning strengthens memory performance, again only when relearning occurs within the temporal window of memory reconsolidation. These findings link retrieval-mediated learning in humans to the reconsolidation literature, and have potential implications both for the understanding of endogenous memory gains and strategies to boost weakly-learned memories.
The mineralocorticoid receptor (MR) in mammals mediates the effects of aldosterone in regulating fluid balance and potassium homeostasis. While MR signalling is essential for survival in mammals, there is no evidence that MR has any physiological role in ray-finned fish. Teleosts lack aldosterone and emerging evidence suggest that cortisol mediates ion and fluid regulation by activating glucocorticoid receptor (GR) signalling. Consequently, a physiological role for MR signalling, despite its conserved and ancient origin, is still lacking. We tested the hypothesis that a key physiological role for MR signalling in fish is the regulation of stress axis activation and function. Using either MR or GR knockout zebrafish, our results reveal distinct and complementary role for these receptors in stress axis function. GR−/− mutants were hypercortisolemic and failed to elicit a cortisol stress response, while MR−/− mutants showed a delayed, but sustained cortisol response post-stressor. Both these receptors are involved in stress-related behaviour, as the loss of either receptors abolished the glucocorticoid-mediated larval hyperactivity to a light stimulus. Overall, the results underscore a key physiological role for MR signalling in ray-finned fishes, and we propose that the regulation of the highly conserved stress axis as the original function of this receptor.
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Dopamine D1 receptor (D1R) signalling is involved in contextual fear conditioning. The D1R antagonist SCH23390 impairs the acquisition of contextual fear when administered systemically or infused locally into the dorsal hippocampus or basolateral amygdala.
We determined if state dependency may account for the impairment in contextual fear conditioning caused by systemic SCH23390 administration. We also examined if the dorsomedial prefrontal cortex (dmPFC), nucleus accumbens (NAc), and ventral hippocampus (VH) are involved in mediating the effect of systemic SCH23390 treatment on contextual fear conditioning.
In experiment 1, SCH23390 (0.1 mg/kg) or vehicle was given before contextual fear conditioning and/or retrieval. In experiment 2, SCH23390 (2.5 μg/0.5 uL) or vehicle was infused locally into dmPFC, NAc, or VH before contextual fear conditioning, and retrieval was tested drug-free. Freezing was quantified as a measure of contextual fear.
In experiment 1, SCH23390 given before conditioning or before both conditioning and retrieval decreased freezing at retrieval, whereas SCH23390 given only before retrieval had no effect. In experiment 2, SCH23390 infused into dmPFC before conditioning decreased freezing at retrieval, while infusion of SCH23390 into NAc or VH had no effect.
The results of experiment 1 confirm those of previous studies indicating that D1Rs are required for the acquisition but not retrieval of contextual fear and rule out state dependency as an explanation for these findings. Moreover, the results of experiment 2 provide evidence that dmPFC is also part of the neural circuitry through which D1R signalling regulates contextual fear conditioning.
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The protective effect of resveratrol (RSV), a stimulator of the silent mating
type information regulation 2 homolog (SIRT1), on the neurotoxicity of rat brain induced
by chronic fluorosis was investigated. Thirty-two Sprague-Dawley (SD) rats were
divided randomly into four groups: (i) a control group; (ii) a RSV treatment group; (iii) a
fluoride-exposed group (50 ppm F- in drinking water); and (iv) a fluoride plus RSV group.
The experimental period was 7 months. The protein levels of SIRT1 in the cortex and the
hippocampus of the rat brains were determined by Western blotting, the SOD activity
and the MDA content in the brains by biochemical methods, and the 8-OHdG content by
ELISA. The spatial learning ability and the memory of the rats was examined by the
Morris Water Maze test. The results showed chronic fluorosis caused changes to the
brains of the rats with a reduced SIRT1 protein, elevated MDA and 8-OHdG contents,
inhibited SOD activity, and, in addition, decreased learning and memory. Interestingly,
RSV pretreatment attenuated the reduced SIRT1, the raised level of oxidative stress, and
the lowered ability for learning and memory resulting from the chronic fluorosis. The
results indicate that RSV may have a neuroprotective effect on fluoride toxicity.
Pharmacological targeting of memory reconsolidation is a promising therapeutic strategy for the treatment of fear memory-related disorders. However, the success of reconsolidation-based approaches depends upon the effective destabilisation of the fear memory by memory reactivation. Here, we show that the nootropic nefiracetam stimulates tone fear memory destabilisation to facilitate reconsolidation disruption by the glucocorticoid receptor antagonist mifepristone. Moreover, the enhancing effect of nefiracetam was dependent upon dopamine D1 receptor activation, although direct D1 receptor agonism was not sufficient to facilitate destabilisation. Finally, while the combined treatment with nefiracetam and mifepristone did not confer fearreducing effects under conditions of extinction learning, there was some evidence that mifepristone reduces fear expression irrespective of memory reactivation parameters. Therefore, the use of combination pharmacological treatment to stimulate memory destabilisation and impair reconsolidation has potential therapeutic benefits, without risking a maladaptive increase of fear.
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- • Intranasal linalool reduces infarct volume and recovers neurological function after ischemia.
- • Linalool intranasal induces anti-inflammatory microglia.
- • Nrf-2 microglial redistribution under anti-inflammatory context by linalool.
Behavioural Brain Research
• Sleep deprivation worsened the memory impairment of rotenone treated zebrafish.
- • DA metabolic process was significantly increases after sleep deprivation.
- • The expression of dopamine D2 receptors were decreased in SD + Rotenone group.
Olfactory perceptual learning is defined as an improvement in the discrimination of perceptually close odorants after passive exposure to these odorants. In mice, simple olfactory perceptual learning involving the discrimination of two odorants depends on an increased number of adult-born neurons in the olfactory bulb, which refines the bulbar output. However, the olfactory environment is complex, raising the question of the adjustment of the bulbar network to multiple discrimination challenges. Perceptual learning of 1 to 6 pairs of similar odorants led to discrimination of all learned odor pairs. Increasing complexity did not increase adult-born neuron survival but enhanced the number of adult-born neurons responding to learned odorants and their spine density. Moreover, only complex learning induced morphological changes in neurons of the granule cell layer born during the first day of life (P0). Selective optogenetic inactivation of either population confirmed functional involvement of adult-born neurons regardless of the enrichment complexity, while preexisting neurons were required for complex discrimination only.
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Introduction Bexarotene, a retinoid X receptor agonist, improves cognition in murine models of Alzheimer’s disease (AD). This study evaluated the effects of bexarotene on pathological and electrophysiological changes in very old triple transgenic AD mice (3xTg-AD mice).
Methods 24-month-old 3xTg-AD mice were treated with bexarotene (100 mg/kg/day for 30 days). The Morris water maze was used to evaluate spatial memory; immunofluorescence and confocal microscopy were used to evaluate pathological changes; and in vivo electrophysiological recordings were used to evaluate basal transmission and plasticity in the commissural CA3-CA1 pathway.
Results In addition to cognitive improvement, bexarotene-treated 3xTg-AD mice were found to have 1) reductions of astrogliosis and reactive microglia both in cortex and hippocampus; 2) increased ApoE expression restricted to CA1; 3) increased number of cells co-labeled with ApoE and NeuN; 4) recovery of NeuN expression, suggesting neuronal protection; and, 5) recovery of basal synaptic transmission and synaptic plasticity.
Link to the publication :
British Journal of pharmacology
Background and Purpose
Excessive GABAergic inhibition contributes to cognitive dysfunctions in Down syndrome (DS). Selective negative allosteric modulators (NAMs) of α5‐containing GABAA receptors such as α5IA restore learning and memory deficits in Ts65Dn mice modelling DS. This study aimed at testing long‐lasting effects of α5IA on in vivo long‐term potentiation (LTP) and behavior in Ts65Dn mice.
We performed in vivo long‐term potentiation (LTP) recordings for six consecutive days in freely moving Ts65Dn mice and their wild‐type littermates, treated with vehicle or α5IA. In parallel, Ts65Dn mice were subjected to various learning and memory tests (Y‐maze, Morris water maze or the novel object recognition) up to seven days following one single injection of α5IA or vehicle.
We found that LTP could not be evoked in vivo in Ts65Dn mice at the hippocampal CA3‐CA1 synapse. However, this deficit was sustainably reversed for at least six consecutive days following a single injection of α5IA. This long‐lasting effect of α5IA was also unveiled when assessing working and long‐term memory deficits in Ts65Dn mice.
Conclusion and Implications
We show for the first time in vivo LTP deficits in Ts65Dn mice. These deficits are restored for at least six days following acute treatment with α5IA and might be the substrate for the long‐lasting pharmacological effects of α5IA demonstrated here on spatial working and long‐term recognition and spatial memory tasks. Altogether, these results highlight the interest of NAMs of α5‐containing GABAA receptors for treating cognitive deficits associated with DS.
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Microglia of the developing brain have unique functional properties but how their activation states are regulated is poorly understood. Inflammatory activation of microglia in the still-developing brain of preterm-born infants is associated with permanent neurological sequelae in 9 million infants every year. Investigating the regulators of microglial activation in the developing brain across models of neuroinflammation-mediated injury (mouse, zebrafish) and primary human and mouse microglia we found using analysis of genes and proteins that a reduction in Wnt/β-catenin signalling is necessary and sufficient to drive a microglial phenotype causing hypomyelination. We validated in a cohort of preterm-born infants that genomic variation in the Wnt pathway is associated with the levels of connectivity found in their brains. Using a Wnt agonist delivered by a blood–brain barrier penetrant microglia-specific targeting nanocarrier we prevented in our animal model the pro-inflammatory microglial activation, white matter injury and behavioural deficits. Collectively, these data validate that the Wnt pathway regulates microglial activation, is critical in the evolution of an important form of human brain injury and is a viable therapeutic target.
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Journal of Neurotrauma
Traumatic brain injury (TBI) is a chronic pathology, inducing long term deficits that remains understudied in preclinical studies. In this context, exploration, anxiety-like behavior, cognitive flexibility and motor coordination were assessed until 5 and 10 months after an experimental TBI in the adult mouse, using two cohorts. In order to differentiate age, surgery, and remote gray and white matter lesions, three groups, unoperated, sham-operated, and TBI, were studied. TBI induced delayed motor coordination deficits at the pole test, 4.5 months after injury, that could be explained by gray and white matter damages in ipsilateral nigrostriatal structures (striatum, internal capsule) that were spreading to new structures between cohorts, at 5 versus 10 months after the injury. Furthermore, TBI induced an enhanced exploratory behavior during stressful situations (active phase during actimetry test, object exploration in an open field), risk-taking behaviors in the elevated plus maze 5 months after injury, and a cognitive inflexibility in the Barnes maze that persists until 9 months after the injury. These behavioral modifications could be related to the white and gray matter lesions observed in ipsi- and contra-lateral limbic structures (amygdala, hilus/CA4, hypothalamus, external capsule, corpus callosum, cingular cortex) that were spreading to new structures between cohorts, at 5 months versus 10 months after the injury. The present study corroborates clinical findings on TBI, and provides a relevant rodent chronic model which could help validating pharmacological strategies against the chronic consequences of TBI.
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The partial pressure of CO2 in the oceans has increased rapidly over the past century, driving ocean acidification and raising concern for the stability of marine ecosystems1,2,3. Coral reef fishes are predicted to be especially susceptible to end-of-century ocean acidification on the basis of several high-profile papers4,5 that have reported profound behavioural and sensory impairments—for example, complete attraction to the chemical cues of predators under conditions of ocean acidification. Here, we comprehensively and transparently show that—in contrast to previous studies—end-of-century ocean acidification levels have negligible effects on important behaviours of coral reef fishes, such as the avoidance of chemical cues from predators, fish activity levels and behavioural lateralization (left–right turning preference). Using data simulations, we additionally show that the large effect sizes and small within-group variances that have been reported in several previous studies are highly improbable. Together, our findings indicate that the reported effects of ocean acidification on the behaviour of coral reef fishes are not reproducible, suggesting that behavioural perturbations will not be a major consequence for coral reef fishes in high CO2 oceans.
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