Activation of CREB-BDNF Pathway in Pyramidal Neurons in the Hippocampus Improves the Neurological Outcome of Mice with Ischemic Stroke

Cerebral ischemia is characterized by several pathological reaction evolving over time. Hyperactivation of glutamatergic neurons is the main factor leading to excitotoxicity which potentiates oxidative stress and triggers the mechanisms of neural apoptosis after cerebral ischemia. However, it is unclear whether glutamate in the ventral hippocampal Cornus Ammonis 1 (vCA1) acts a part in neurological deficits, pain perception, anxiety, and depression induced by ischemic stroke. We investigated the effects of chemogenetic inhibition or activation of vCA1 pyramidal neurons which are mainly glutamatergic neurons on sequelae induced by cerebral ischemia. Our results revealed that inhibition of vCA1 pyramidal neurons by chemogenetics alleviated neurological deficits, pain perception, anxiety, and depression caused by cerebral ischemia in mice, but activation of vCA1 pyramidal neurons had limited effects. Moreover, we found that stroke was accompanied by decreased levels of cAMP-response element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in vCA1, which are modulated by glutamate. In this study, overexpression of CREB protein in pyramidal neurons in vCA1 by AAV virus significantly upregulated the content of BDNF and ameliorated the dysfunction induced by ischemic stroke. Our results demonstrated activation of the CREB-BDNF pathway in vCA1 pyramidal neurons significantly improved neurological deficits, pain perception, anxiety, and depression induced by ischemic stroke.


Introduction
Stroke is the leading cause of both disability and mortality in the adult population because of the brain's limited recovery after large strokes [1]. Ischemic stroke accounting for 87% of all stroke occurs as a result of the blockage of brain blood vessels [2]. During ischemic stroke, the acute interruption of blood flow causes deprivation in oxygen and nutrient supplies leading to poor production of ATP in mitochondria which set off a chain reaction, such as the Na + /K + ATPase pumps inactivation, cell swelling and Ca 2+ influx into the cells, and the intracellular accumulation of Ca 2+ and Na + ions induce the intracellular depolarization, which stimulate the magnanimous release of glutamate from the presynaptic terminal eliciting neurotoxicity, with the consequent neuronal necrosis [3]. Subsequently, secondary reactions, such as inflammatory response and excitotoxicity, enlarge the area the neuronal death [3,4].
Reperfusion or reconstruction of blood flow in the ischemic region is the key treatment strategy for stroke-associated ischemic brain injury [5]. But over half of stroke survivors require rehabilitation for residual neurological deficits and perceptual and emotional disorders [2,6]. The damage caused by cerebral ischemia consists of different pathological mechanisms in which excitotoxicity, a disturbed homeostasis of excitatory amino acids leading to stroke progression, plays a vital role [7,8]. Moreover, glutamate, as a representative of excitatory amino acids, is a prominent part as neurotransmitter in the central neural system [8]. Several animal models of cerebral ischemia and in vitro studies have provided unequivocal evidence that glutamate-mediated excitotoxicity is a primary contributor to ischemic neuronal death [8,9].
Under normal physiological conditions, glutamate is involved in synaptic plasticity, sensory, movement, emotional, memory, and other brain functions by acting on synaptic N-methyl-D-aspartate receptors (NMDARs) and other receptors [4]. For example, NMDAR-dependent long-term potentiation (LTP) in the hippocampus participates in the formation of long-term memory [10]. Besides, some studies revealed the involvement of glutamate-related genes in risk for mood disorders [11]. Glutamate is also involved in acupuncture analgesia by acting on ascending excitatory pathways [12]. Moreover, the action of glutamate release could modulate processes in the motor center that control orienting movements [13]. In summary, glutamate acts widely in the central nervous system and is involved in many physiological activities.
Normally, synaptic activity promotes neuronal survival in part by NMDA receptor-mediated CREB, and CREB can upregulate BDNF which is the pro-survival transcription gene, then in turn BDNF release can further promote CREB activation [13]. Previous studies have well established that CREB is a ubiquitous transcription factor that acts pleiotropic roles in the nervous system and controls gene expression programs essential for long-term synaptic plasticity and memory in a variety of organisms [14]. BDNF is a crucial neurotrophic factor adding to the development and endurance of neurons and supports synaptic integrity by modulating the NMDAR [15]. In addition, our previous studies have shown that BDNF in the hippocampus plays an important role in pain perception and related mood disorders [16]. However, during cerebral ischemia, excessive release of glutamate which means overstimulation of the extrasynaptic NMDARs blocks the expression of CREB, thereby disturbing the positive feedback loop between CREB and BDNF, leading to neuronal death [9,17]. CREB has an important role in motor capacity recovery after stroke [18], but the role of CREB, as well as its downstream BDNF, in poststroke perceptual and emotional disorders is less clear.
Previous studies and our results in the past have shown that ventral hippocampal Cornus Ammonis 1 (vCA1) is involved in perceptual and emotional disorder [19]. For instance, vCA1 glutamatergic neurons was involved in spontaneous pain and inflammatory pain [20,21]. "Anxiety cells" have been discovered in vCA1 glutamatergic neurons in mice [22]. Patients with major depression showed a marked reduction in vCA1 volume, and rodents subjected to stress also have decreased spine density in vCA1 pyramidal neurons [23].
But how glutamatergic neurons in vCA1 mediates the disorders such as pain perception, anxiety, and depression induced by stroke remains unclear. We hypothesized that hyperactivation of glutamatergic neurons in vCA1 caused by cerebral ischemia would act on the CREB-BDNF pathway leading to neurological deficits, pain perceptual, anxiety, and depression disorders. Previously, the study of glutamate and related molecules in stroke mainly focuses on glutamate receptors [9,24]. Due to the limitation of technology, there are few literatures on manipulating the excitability of glutamatergic neurons to modulate the release of glutamate neurotransmitter from their terminals. DREADD (designer receptors exclusively activated by designer drugs) is a widely applied chemogenetic technique that reversibly inhibits or activates neurons by expressing inhibitory designer receptors (hM4Di) or activation designer receptor (hM3Dq) in neurons through the specific neuronal promoter with an AAV vector [25]. After intracerebral stereotaxic injection of the chemogenetic virus in mice, intraperitoneal injection of the designer drug (clozapine N-oxide, CNO) could site and neuronal type specifically inhibit or activate the activity of these neurons [26]. In the present study, we applied chemogenetic manipulations on vCA1 pyramidal neurons which are mainly glutamatergic neurons to reversibly regulate its excitability in a mouse model of cerebral ischemia induced by middle cerebral artery occlusion (MCAO). Moreover, overexpress CREB in vCA1 pyramidal neurons by infusing AAV virus and observed its effect on BDNF and the sequelae of MCAO.

Experimental Animals
Male C56BL/6 J mice aged 8-10 weeks were obtained from Vital River Laboratories (Beijing, China). All animals were housed under pathogen-free circumstances, in a 12-h light/12-h dark cycle with a maximum of five mice per cage, and with ad libitum access of food and water. All behavioral tests were performed in a blind manner. All experiments with mice were performed in accordance with the guidelines set by National Institute of Health Guide for the Care and Use of Laboratory Animals, approved by the institutional Animal Care and Use Committee of Beijing Neurosurgical Institute (Permit Number: 201904004) and reported following the ARRIVE guidelines [27].
The total number of mice used was 245, but due to the mortality of the MCAO model, 167 mice were finally used for data analysis. The groups are as follows.
For the damage caused by cerebral ischemia (Fig. 1, 2 and 7), mice were assigned to eight groups: (1) sham MCAO

Middle Cerebral Artery Occlusion (MCAO) Model
The MCAO model detailed steps referred to Koizumi's method [28]. Briefly, mice were anaesthetized with isoflurane (RWD, China) and make a midline neck incision. The external common carotid artery and right common carotid artery (CCA) were permanently ligated using silk suture. A second loose collar suture was tied around the CCA and then clip the right internal common carotid artery. An arteriotomy was performed between the two sutures around the CCA. A silicone coated filament (RWD, China) was introduced into the arteriotomy and advanced into the middle cerebral artery. After 45 min, withdrew the filament and tightened the loose collar suture around CCA. For sham MCAO (SMCAO), all procedures are identical except that the filament is not inserted.

Neurological Severity Scoring (NSS)
An expanded six-point scale was used by an individual blinded to experimental group as previously described [28,29]: 0, normal; 1, mild turning behavior with or without inconsistent rotation when picked up by the tail; 2, mild consistent turning, > 50% attempts to curl to the contralateral side; 3, consistent strong and immediate circling, mouse holds a rotation position for more than 1-2 s; 4, severe curling progressing into barreling, loss of walking or righting reflex; and 5, comatose or moribund.

5-Triphenyltetrazolium Chloride (TTC) Staining
After 24 h of reperfusion, mice were dissected to perform TTC staining under 1% sodium pentobarbitone (100 mg/kg, i.p.). Brain was extracted and placed within a brain matrix, and serial sections were made at 1-mm thickness. Sections were incubated in TTC for 10 min at 37 °C and fixed in 4% paraformaldehyde until imaging. The infarct volume was quantified by ImageJ software (National Institutes of Health, USA) [30].

Mechanical Pain Measurement
Mechanical pain threshold was measured when mouse stayed calm and awake using a Dynamic Plantar Aesthesiometer (Ugo Basile, Italy) by applying increasing pressure to the left paw until the mouse withdrew the paw. A maximal cut-off value is 5 g to prevent tissue damage. The paw withdrawal threshold (PWT) was tested four times at an interval of 10 min for each time point [21].

Open Field Test (OF)
OF (50 × 50 × 40 cm) was performed as previously described [16,21]. The mouse is placed into the center of the field, and its activity was videotaped for 5 min and calculated by the SMRAT software (v 3.0, Harvard Apparatus).

Elevated Plus Maze Test (EPM)
The EPM was placed 40 cm above the floor and consisted of two open arms (30 × 5 cm) and two closed arms (30 × 5 × 15 cm). Mice were placed in the center of the maze facing the same closed arm, videotaped for 5 min and calculated by the SMRAT software [16,21].

Tail Suspension Test (TST)
The TST was performed as previously described [31]. Briefly, tails of mice are suspended using adhesive tape to a horizontal for 6 min. Latency to immobility, defined as latency to the first immobility, and total immobility time, defined as the period during which each mouse was not struggling throughout the test, were manually measured.

Forced Swim Test (FST)
The FST was as previously described [32]. The test assesses the tendency to give up attempting to escape from an inescapable cylinder (15.0 × 25 cm) filled with water (24-26 °C).  The open field of four groups (grouping as above) on 15 days after operation. N = 8 ~ 9/group. *p < 0.05, two-way ANOVA with Bonferroni's test. (E) The elevated plus maze of four groups (grouping as above) on 16 days after operation. N = 8 ~ 9/group. *p < 0.05, **p < 0.01, and ***p < 0.001; two-way ANOVA with Bonferroni's test. (F) The tail suspension test of four groups (grouping as above) on 17 days after operation. N = 8 ~ 9/group. *p < 0.05, two-way ANOVA with Bonferroni's test. (G) The forced swim test of four groups (grouping as above) on 21 days after operation. N = 8 ~ 9/group. **p < 0.01 and ***p < 0.001; two-way ANOVA with Bonferroni's test Similar to TST experiment, latency to the first immobility and total immobility time were manually observed.

Chemogegetics
After expression of chemogenetic virus, the MCAOinduced cerebral ischemia model was established. Since clozapine N-oxide (CNO, Tocris) started to take effect half an hour after the injection, lasted for about 8 h, began to metabolize, and the metabolism was completed until 24 h [26], mice in our study were injected intraperitoneally (1.0 mg/kg dissolved in normal saline) with CNO [21,33] at 4 days after MCAO or SMCAO operation once a day for 4 consecutive days (i.e., 4-7 days postoperatively). To verify the chemogenetics, 2 h before perfusion, mice were injected with CNO. Then c-Fos immunofluorescence staining was performed to verify the chemogenetic system.

Immunostaining
Mice were anesthetized with 1% sodium pentobarbitone and transcardially perfused with normal saline followed by 4% PFA and postfixed, then dehydrated in sucrose solutions. Twenty-micrometer sections were sliced coronally. vCA1 slices were blocked with a buffer containing 5% goat serum and 0.3% Triton X-100 for 2 h at 24 °C, then incubated with primary antibodies (rabbit anti-CREB,

Statistical Analysis
Data were expressed as means ± SD (standard deviation). The normal distribution of data was evaluated using the Kolmogorov-Smirnov test. All statistical analyses were performed using the GraphPad Prism 8 software. Differences between two or multiple groups were calculated using unpaired Student's t test, or ANOVA followed by Bonferroni post hoc analyses, respectively. p values lower than 0.05 indicated significant differences. All detailed statistical descriptions are presented in the tables in the Supplementary Information.

MCAO-Induced Cerebral Ischemia Causes Neurological Deficits and Perceptual and Emotional Disorders
To investigate the damage caused by cerebral ischemia, MCAO, the most used ischemic model, was built. After 24 h of ischemia reperfusion, TTC staining was performed and revealed a significant cerebral infarction in the MCAO group compared to the sham MCAO (SMCAO) group (t = 6.59, p < 0.001, Fig. 1A and B).
Then, a series of behavioral experiments was conducted to investigate the dysfunction induced by MCAO. To assess the neurological deficits of mice after MCAO, neurological severity score (NSS) was used and a significant increase of NSS in MCAO group than that in SMCAO group on 1, 3, 7, and 14 days after operation was observed (Fig. 1C, Supplementary  Table 1). Similarly, paw withdrawal threshold in the MCAO group was significantly lower than that in the SMCAO group on 3, 7, and 14 days after operation, which indicated mechanical allodynia (Fig. 1D, Supplementary Table 1).
Emotional disorder is a serious sequelae of patients encountering stroke [36]. We observed increased anxietylike behaviors in mice with cerebral ischemia (15 days after MCAO) indicated by less distance traveled, time spent and entries into the central area of open field (Fig. 1E, Supplementary Table 1), as well as less time spent and fewer entries into the open arms of elevated plus maze (Fig. 1F, Supplementary Table 1).
No statistically significant effects of cerebral ischemia (15 days after MCAO) on locomotion were observed, indicated by similar total distance in open field (Fig. 1E, Supplementary Table 1) and similar total entries in elevated plus maze (Fig. 1F, Supplementary Table 1) between the MCAO and SMCAO groups.
In addition to detecting anxiety, depression was also examined by tail suspension test (TST) (17 days after MCAO) and forced swim test (FST) (21 days after MCAO). Both TST and FST of the MCAO group spent less time struggling behavior until the first immobility (latency to immobility) and stayed more immobile time (immobility time) compare with SMCAO ( Fig. 1G and  H, Supplementary Table 1).
These data indicate that MCAO-induced cerebral ischemia increases neurological severity score, mechanical allodynia, anxiety, and depression behaviors in mice.

MCAO-Induced Cerebral Ischemia Activates Glutamatergic Neurons in vCA1
Since cerebral ischemia causes numerous insults, including excitotoxicity [17], we examined the excitability of pyramidal neurons on 3 days after MCAO or SMCAO within the vCA1, which plays an important role in perception and emotion. We detected a sixfold increase in the number of c-Fos which is immediate early genes (t = 9.25, p < 0.001, Fig. 2A and B, Supplementary Fig. 1), indicating neuronal excitation in the vCA1, and 88.2 ± 7.7% of c-Fos-positive neurons colabeled with EAAT3 (Fig. 2B), a marker of glutamatergic neurons. These findings indicate strong activation of vCA1 glutamatergic neurons after MCAO.

Chemogenetic Inhibition of vCA1 Glutamatergic Neurons Improves Neurological Deficits and Perceptual and Emotional Disorders Induced by MCAO
Since cerebral ischemia leads to massive activation of vCA1 glutamatergic neurons, chemogenetics was used to inhibit these neurons [25]. We expressed inhibitory chemogenetic viruses (pAAV 9 -CaMKIIα-hM4D (Gi)-mCherry, referred to as AAV-Gi below) or control virus (pAAV 9 -CaMKIIα-mCherry, referred to as AAV-mCherry below) [33] in vCA1 pyramidal neurons in mice (Fig. 3A-C). 91.4 ± 7.4% of NeuN-positive neurons which is the neuronal marker were infected with AAV-Gi (mCherry) (Fig. 3D). Also, the virus, in which AAV-Gi expression is under the regulation of the pyramidal neuronal-specific CaMKIIα promoter was mostly colocalized with EAAT3 (Fig. 3E), which means the glutamatergic neurons in vCA1 were transfected by the inhibitory chemogenetic virus (mCherry). The Gi-coupled receptor activated exclusively by clozapine N-oxide (CNO) [33], a synthetic ligand and inhibited local glutamatergic activities was verified by c-Fos. The inhibitory effect was verified by a decrease of c-Fos immunofluorescence in vCA1 slice of MCAO mice after CNO (Fig. 3G) than that after saline (Fig. 3F) intraperitoneal injection (t = 5.27, p < 0.001, Supplementary Fig. 2), which colocalized with mCherry from the AAV-Gi virus (Fig. 3F).
Studies have shown that 5-14 days after stroke occurs is the critical period of post-stroke rehabilitation [37]. In our design, 3 weeks after AAV-Gi or AAV-mCherry virus injection, MCAO or SMCAO operation was carried out, and then CNO intraperitoneal injection was performed once daily from 4 to 7 days (Fig. 4A). We found that vCA1 inhibition significantly attenuated MCAO-induced neurological severity score on 14 days after MCAO (Fig. 4B, Supplementary Table 2) and mechanical allodynia was similarly attenuated on 7 and 14 days after MCAO (Fig. 4C, Supplementary Table 2) between the AAV-Gi + MCAO group and AAV-mCherry + MCAO group.
Open field and elevated plus maze were also used to assess the effect of chemogenetically inhibiting vCA1 glutamatergic neurons on anxiety on 15 and 16 days after MCAO, respectively (Fig. 4A). We observed vCA1 inhibition significantly attenuated MCAO-induced anxiety-like behaviors in mice indicated by elevated distance traveled, time spent, and entries into the central area of open field (Fig. 4D, Supplementary Table 2), as well as increased time spent and entries into the open arms of elevated plus maze (Fig. 4E, Supplementary Table 2).
No statistically significant effects of MCAO or chemogenetics on locomotion were observed (15 and 16 days after MCAO), indicated by similar total distance in open field (Fig. 4D, Supplementary Table 2) and similar total entries in elevated plus maze (Fig. 4E, Supplementary Table 2).
Meanwhile, tail suspension test and forced swim test were used to assess the effect of chemogenetically inhibiting vCA1 glutamatergic neurons on depression caused by cerebral ischemia (17 and 21 days after MCAO) (Fig. 4A). The results showed that both tail suspension test and forced swim test mice of the MCAO group after vCA1 inhibition spent longer time struggling behavior until the first immobility (latency to immobility) and stayed less immobile time (immobility time) between AAV-Gi + MCAO group and AAV-mCherry + MCAO group ( Fig. 4F and G, Supplementary Table 2).
These data indicate that chemogenetically inhibiting glutamatergic neurons in vCA1 at early stage (4-7 days) after MCAO attenuates neurological severity score, mechanical allodynia, anxiety and depression behaviors induced by cerebral ischemia in mice.

Chemogenetic Activation of vCA1 Glutamatergic Neurons Maintains Neurological Deficits and Perceptual and Emotional Disorders Induced by MCAO
On the other hand, to explore whether further activation of vCA1 glutamatergic neurons aggravates the dysfunction caused by MCAO, similar to chemogenetic inhibition, we also chemogenetically activated vCA1 glutamatergic neurons ( Fig. 5A-C). The excitatory effect was verified by the increase of c-Fos immunofluorescence in vCA1 slice of MCAO mice after CNO (Fig. 5E) than that after saline (Fig. 5D) intraperitoneal injection (t = 3.62, p < 0.01, Supplementary Fig. 3), which colocalized with mCherry from the AAV-Gq virus (Fig. 5D and E).
Open field and elevated plus maze were also used to assess the effect of chemogenetic activation of vCA1 glutamatergic neurons on anxiety on 15 and 16 days after MCAO (Fig. 6A). We observed vCA1 activation had no effect on anxiety-like behaviors between the AAV-Gq + MCAO group and AAV-mCherry + MCAO group indicated by similar distance traveled, time spent, and entries into the central area of open field (Fig. 6D, Supplementary Table 3), as well as identical time spent and entries into the open arms of elevated plus maze (Fig. 6E, Supplementary Table 3).
No statistically significant effects of MCAO or chemogenetics on locomotion were observed (15 and 16 days after MCAO), indicated by similar total distance in open field (Fig. 6D, Supplementary Table 3) and similar total entries in elevated plus maze (Fig. 6E, Supplementary Table 3).
Moreover, tail suspension test and forced swim test were also used to assess the effect of chemogenetic activation of vCA1 glutamatergic neurons on depression caused by MCAO (17 and 21 days after MCAO) (Fig. 6A). The results showed that both tail suspension test and forced swim test in the MCAO group (AAV-Gq + MCAO vs AAV-mCherry + MCAO) spent similar time struggling behavior until the first immobility (latency to immobility) and stayed identical immobile time (immobility time) ( Fig. 6F and G, Supplementary Table 3).
These results showed that chemogenetically activating glutamatergic neurons in vCA1 at early stage (4-7 days) after MCAO maintained neurological severity score, mechanical allodynia, anxiety, and depression behaviors induced by MCAO in mice.

Cerebral Ischemia Decreases the CREB and BDNF Protein Levels
To further explore why glutamatergic neurons within vCA1 are involved in dysfunction after MCAO, we studied the CREB protein and its downstream signaling pathways, and found that the expression of CREB in right vCA1, as well as the downstream molecule BDNF, decreased on 3 days after MCAO (for CREB/GAPDH, t = 7.19, p < 0.001; for BDNF/ GAPDH, t = 3.55, p < 0.01, Fig. 7A and B).
It was found that the protein content of CREB and BDNF in vCA1 was significantly increased in both SMCAO and MCAO models after AAV-CREB virus injection ( Fig. 7C and D, Supplementary Table 4), indicating that the virus was effective.
Similarly, after overexpression of CREB on pyramidal neurons in vCA1, we examined its effect on sequelae induced by MCAO (Fig. 8A). The details are as follows: overexpression of CREB in vCA1 pyramidal neurons did not affect the neurological severity score (Fig. 8B) and paw withdrawal threshold (Fig. 8C) Table 5).
Likewise, open field and elevated plus maze were also used to assess the effect of overexpression of CREB in vCA1 pyramidal neurons on anxiety on 15 and 16 days after MCAO, respectively (Fig. 8A). We observed overexpression of CREB in vCA1 pyramidal neurons significantly attenu-  Table 5).
No statistically significant effects of MCAO or CREB overexpression on locomotion were observed (15 and 16 days after MCAO), indicated by similar total distance in open field (Fig. 8D, Supplementary Table 5) and similar total entries in elevated plus maze (Fig. 8E, Supplementary  Table 5).
Meanwhile, tail suspension test and forced swim test were used to assess the effect of overexpression of CREB in vCA1 pyramidal neurons on depression caused by MCAO (17 and 21 days after MCAO) (Fig. 8A). The results showed that both tail suspension test and forced swim test mice of the MCAO group spent longer time struggling behavior until the first immobility (latency to immobility) and stayed less immobile time (immobility time) after MCAO (AAV-CREB + MCAO vs. AAV-mCherry + MCAO) ( Fig. 8F and G, Supplementary Table 5).
These data indicate that overexpression of CREB in vCA1 pyramidal neurons attenuates neurological severity score, mechanical allodynia, anxiety, and depression behaviors induced by cerebral ischemia in mice.

Discussion
Worldwide, stroke ranked as the second leading cause of death and the primary factor to disability and incapacity [37]. Over the past decades, the decline in stroke mortality has been accompanied by a rise in stroke incidence at younger ages, leading to an increased survival among stroke patients [38]. These survivors suffer from many complications, with an estimated incidence ranging between 24.2 and 95% [39], such as movement, perceptual, emotional, and cognitive impairments, which place an enormous burden on society and families [40,41].
The damage caused by stroke is mainly through excitotoxicity, inflammation, oxidative stress, and calcium overload. Glutamate is the major excitatory neurotransmitter in the central nerve system [9]. The balance between excitatory glutamatergic pyramidal neurons and inhibitory GABAergic interneurons undergoes significant changes post-stroke [7]. Besides, overexposure of neurons to glutamate causes elevated of ions into cells, potentiates oxidative stress and triggers the mechanisms of neural apoptosis by stimulating microglial activation [7]. Moreover, some findings reveal that the removal of glutamatergic afferent neurons attenuates neuronal death in rats subjected to cerebral ischemia [17]. Therefore, inhibition of glutamate release allows for the termination of ischemic strokeinduced excitotoxicity at its most upstream initiation point which is crucial for cerebral ischemic prognosis. However, no feasible treatment candidates, which inhibiting ischemic glutamate release by blocking reverse uptake, have passed the clinical testing.
In order to reversibly regulate the excitability of neurons post-stroke, we adopted chemogenetics (also calls designer receptor exclusively activated by designer drug, DREADD), which is only modulated by CNO to reversibly control neuronal excitability [25,33]. Meanwhile, we found the excitability of glutamatergic neurons in vCA1 in early stroke was increased, and its inhibition by chemogenetics could alleviate the neurological deficits, mechanical allodynia, anxiety and depression induced by cerebral ischemia. These results suggest that the excitatory glutamatergic neurons within vCA1 play a vital role in the recovery from post-stroke.
Glutamate binds to several receptors, including NMDARs [9]. Under normal physiological conditions, glutamate is involved in sensory, movement, emotional, memory and cognitive brain functions by acting on synaptic NMDARs [4]. Synaptic activity promotes neuronal survival in part by NMDAR-mediated CREB, which stimulates the BDNF release, and in turn, BDNF activation of the Trk receptor can further promote CREB activation [4,9]. But, under pathological conditions, such as stroke, excessive release of glutamate transmitters that act on extrasynaptic NMDARs decreases CREB, thereby, shut-off the positive feedback loop between CREB and BDNF [17,42], which is consistent with our results (Fig. 7A).
Our results showed that, CREB is mainly expressed in excitatory neurons (Fig. 7). The previous research indicated that CREB and its mediated transcription, such as BDNF, were required for NMDAR-dependent neuronal survival during development and can render neurons resistant to excitotoxicity and apoptosis [17,43]. Meanwhile, NMDAR-mediated survival signaling involves CREB as a major player [43,44]. Therefore, we hypothesized that the excessive release of glutamate caused by cerebral ischemia would act on the CREB-BDNF pathway. Whereas in our results, overexpression of CREB on pyramidal neurons by using AAV virus significantly ameliorates cerebral ischemia-induced neurological deficits, mechanical allodynia, anxiety, and depression.
The primary goal of cerebral ischemic stroke therapy is to restore blood flow as quickly as possible by recanalizing the occluded vessel [43,44]. But the time window of interventions is limited to the first hours after stroke [7,45]. Therefore, complementary or alternative therapies are urgently needed to reduce stroke-related mortality and disability beyond this short period. In rodents, the critical period of post-stroke rehabilitation is around the 5-14 days after ischemia [37]. Therefore, we chose to intervene at 4 to 7 days after MCAO.
With the inactivated AAV virus already in clinical treatment, and CNO known as a metabolite of widely prescribed medication has been administered to humans [25]. All these provide a good prospect for the potential application of chemogenetics in clinical treat.
In the present study, we used adult mice aged 6-8 weeks, which does not mimic the current situation in which stroke patients in the clinic are mostly elderly. Older patients were more likely to have hypertension, atrial fibrillation, coronary artery disease, but those rarely occur in adults [46]. Due to the different underlying diseases in adult and aged mice, this may lead to different pathogenesis and treatment of stroke at different ages. Therefore, the use of adult mice is a limitation of this study. In order to be more consistent with the clinical situation, we will consider studying stroke in elderly mice.
In general, the data presented herein demonstrate that cerebral ischemia leads to excessive activation of glutamatergic neurons in hippocampal vCA1. Chemogenetically inhibiting glutamatergic neurons in vCA1 at early stage (4-7 days) after MCAO attenuates neurological deficits, pain perception, anxiety, and depression behaviors induced by cerebral ischemia in mice, whereas chemogenetic activation of vCA1 glutamatergic neurons had limited effects. Moreover, cerebral ischemia leads to a decrease in CREB and downstream BDNF levels in vCA1; however, overexpression of CREB increases BDNF levels and significantly alleviates neurological deficits, mechanical allodynia, anxiety, and depression induced by cerebral ischemia.