The role of CA3 GABA A receptors on anxiolytic-like behaviors and avoidance memory deficit induced by NMDA receptor antagonists

Abstract

Cognitive functions are influenced by memory and anxiety states. However, a non-linear relation has been shown between these two domains. The important role of the hippocampus in memory and emotional responses may link the pathogenesis of anxiety to memory-related GABAergic and glutamatergic processes in the hippocampus. To investigate the role of GABA_A receptors in relation to blocking N-methyl-D-aspartate (NMDA) receptors in the CA3 region, and balancing the glutamatergic and GABAergic system activities as an approach for the management of related disorders, the elevated plus-maze test–retest paradigm was used to investigate the anxiolytic-like state on the test day and avoidance memory state on the retest day. The data showed that injection of D-AP5, the NMDA receptor antagonist, induced anxiolytic-like behavior and impaired avoidance memory. Injection of GABA_A agonist (muscimol), but not the antagonist (bicuculline), induced avoidance memory impairment. Neither muscimol nor bicuculline altered anxiety-like behaviors. Muscimol pretreatment did not change D-AP5-induced anxiolytic-like behaviors but potentiated avoidance memory impairment. Bicuculline pretreatment blocked D-AP5-induced anxiolytic-like behaviors and contradicted its effect on avoidance memory. Our findings indicate that alteration of the CA3 GABA_A receptor activity can effectively affect the anxiolytic-like behaviors and avoidance memory deficit induced by D-AP5.

Keywords

D-AP5 memory anxiety elevated plus-maze rats

Introduction

The simple amino acids glutamate and γ-aminobutyric acid (GABA) are two main neurotransmitters in the mammalian brain (Wierońska et al., 2011). Moreover, a form of neuronal plasticity is thought to lie behind learning and memory at glutamatergic synapses after the breakthrough of long-term potentiation (LTP; Bliss and Lomo, 1970). GABAergic inhibition has been shown to block this plasticity (Wigstrom and Gustafsson, 1983). Glutamate activates two families of receptors: the ionotropic [including N-methyl-D-Aspartate (NMDA); α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors, responsible for fast neurotransmission] and the metabotropic [G-protein-coupled receptors] receptors (Cortese and Phan, 2005; Swanson et al., 2005). On the other hand, distinct receptor subtypes mediate GABA functions, including the ionotropic GABA_A and GABA_C receptors, and the metabotropic GABA_B receptor. Ionotropic GABA receptor activation leads to the activation of Cl^– channels (Bormann, 2000). Several normal and pathological brain mechanisms such as sleep, memory, epilepsy, and various emotions are regulated by GABA_A receptors (Chapouthier and Venault, 2002; Kalueff and Nutt, 1996; Mihalek et al., 1999). As for the regulation of behavior processes, research shows a close interaction between the brain glutamatergic and GABAergic systems in such a way that the GABA_A receptor presynaptically balances the release of glutamate from some glutamatergic neurons (Long et al., 2009).

Cognitive functions are obviously influenced by memory and anxiety domains. The interaction between memory and anxiety has been described by some behavioral, physiological, pharmacological, and genetic research (Barad, 2005; Quirk and Gehlert, 2003; Wall and Messier, 2000). However, a persistent synergistic rule does not exist, which shows the complexity of the non-linear relation of these behavioral functions. Changes in anxiety and memory (or vice versa) have been observed together in reality. Strengthened or weakened memory formation may modulate emotional states (i.e., fear and aversion; McGaugh, 2004). This, however, does not contradict the idea that under certain conditions, any of these domains may change independently. Similarly, either domain is a multidimensional one, and should not be studied as a single entity (Kalueff, 2007).

Learning and memory, and anxiety are cognitive functions involving glutamatergic and GABAergic systems (Cheetham et al., 1988; Danysz et al., 1988; Kulkarni et al., 1990). Much research has been conducted on glutamate as one of the main excitatory neurotransmitters in the central nervous system (CNS) and because of its role in several cognitive and non-cognitive processes, including neurodevelopment, learning and memory, pathogenesis of anxiety-related disorders, and psychiatric conditions such as schizophrenia and mood disorders (Cortese and Phan, 2005; Daniel and Dohanich, 2001; Walker and Davis, 2002).

Anxiety, stress, and trauma-related disorders are large-scale health concerns in many countries (Greenberg et al., 1999). As the drugs that affect the GABAergic and serotonergic systems, benzodiazepines and selective serotonin reuptake inhibitors, respectively, are extensively prescribed (Liberzon et al., 2003; Rayen et al., 2011). However, with the knowledge that drugs that alter glutamatergic function can enhance existing treatments, the glutamate role in anxiety disorders is more relevant. Studies have shown that modification of glutamate transmission can potentially cause an anxiolytic action in paradigms such as fear-potentiated startle (Walker and Davis, 2002), punished responding (Klodzinska and Chojnacka-Wojcik, 2000), and the elevated plus-maze (EPM; Ho et al., 2005). The competence of glutamatergic drugs in anxiety-related disorder treatments has also been shown in human clinical drug trials, and magnetic resonance imaging (MRI) data have also contributed an additional link between anxiety and the glutamate system (Rauch et al., 2003; Yamasue et al., 2003). Based on the proposed models, fear-related learning and reactivity is related to an elevated transmission of glutamate and/or extra glutamate release within the limbic system (Walker and Davis, 2002). Thus, future investigation on the mechanism and clinical efficacy of glutamatergic agents in anxiety disorders is warranted (Cortese and Phan, 2005). On the other hand, the GABAergic system rapidly emerged as a target for production of anxiety and mood disorder medications (Drugan et al., 1986). Consequently, recognizing the possible neural basis of anxiety relating to GABA is of great importance (Andrews and File, 1993; Muller et al., 1997).

The recognized role of the hippocampus in memory, and the role of memory in anxiety and depression, may link their pathogenesis to memory-related glutamatergic and GABAergic processes in the hippocampus (Nasehi et al., 2015; Naseri et al., 2014; Yousefi et al., 2013). The hippocampus, with its high density of glutamate and GABA receptors, has CA1, CA2, CA3, and CA4 as its four main subregions, and is involved in the encoding, consolidation, and retrieval of memory, irrespective of the type. The hippocampus is a heterogeneous structure, with the dorsal hippocampus being involved in working memory (Daniel and Dohanich, 2001), spatial learning (Moser et al., 1993), and contextual memory retrieval (Holt and Maren, 1999), and the ventral hippocampus being involved in emotionality through its connections with the amygdala (Kjelstrup et al., 2002). Receiving patterned information from the dentate gyrus or from the entorhinal cortex, the CA3 network has the capacity to acquire and store unique associations for a short period of time (Rolls and Treves, 1996). CA1 and CA3 are engaged in encoding higher-order representations of the stimuli or objects enclosed in contexts and places (Daumas et al., 2005; Ji and Maren, 2008). It has also been proposed that CA3 provides a channel between the dorsal hippocampus and the amygdala for the transmission of contextual information (Maren and Holt, 2004), thereby involving it in emotional responses.

CNS arousal and neuronal excitability is regulated by the mutual homeostasis between glutamate and GABA, and the excitatory action of glutamate is counterbalanced by GABA, which avoids unnecessary neuronal hyperexcitability that occurs in seizure disorders and pathologic anxiety (Lydiard, 2003). The interaction is central to the adaptation to stress and the avoidance of the development of anxiety disorders (Lydiard, 2003) inasmuch as it has been proposed that balancing the activities of the glutamatergic and GABAergic systems may be considered an approach for the management of anxiety disorders (Sajdyk and Shekhar, 1997). To investigate this further, the present research aimed to study the interactions between the CA3 glutamatergic system and GABA_A receptor on the anxiety level and emotional memory of rats performing in an EPM test. We used the EPM test–retest paradigm to evaluate the effects of drugs on learning and memory, and anxiety simultaneously (Bertoglio et al., 2006; Chegini et al., 2014; Wall and Messier, 2000), since some learning and memory animal models do not have the ability to reveal the drugs’ effect on anxiety and memory thoroughly. As a common test of animal anxiety (Lister, 1987), the EPM model can also be used to measure the effect of emotional states on memory in a repeated-measures protocol (Carobrez and Bertoglio, 2005). During exploration on the test day, the animals obtain information about safe and dangerous areas of the maze (Galvis-Alonso et al., 2010). Based on the observed experience-dependent behavioral changes, the test–retest model gives a measure of acquisition and memory retention (Serafim et al., 2013).

Materials and methods

Animals

A total of 167 male Wistar rats weighing 220–270 g and aged seven to eight weeks at the time of the experiment were tested. The animals were maintained in an air-conditioned room (21±2°C) under a 12-hour light–dark cycle (lights on at 7:00am). Groups comprising six to eight animals were kept in standard polycarbonate cages (40 cm×28 cm×18 cm), and had access to food and water ad libitum. The animals were handled two days before the test for five minutes. The experimental protocol was in accordance to the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).

Drug treatment

Ketamine and xylazine were used for anesthesia, and were purchased from a local approved vendor. NMDA receptor antagonist, D-AP5 ((2R)-amino-5-phosphonovaleric acid; (2R)-amino-5-phosphonopentanoate), muscimol (GABA_A receptor agonist), and bicuculline (GABA_A receptor antagonist) were all purchased from Tocris (Bristol, UK). The drug doses were selected based on pilot and previous studies (Kangarlu-Haghighia et al., 2015; Rezvanfard et al., 2009).

Stereotaxic surgery and drug infusion

The rats were anesthetized intraperitoneally (i.p.) using ketamine hydrochloride 10% (50 mg/kg) plus xylazine 2% (4 mg/kg) in a mixture of 2:1, which was injected at a volume of 2 mL/kg. The rats were then positioned in a stereotaxic frame. Two stainless steel guide cannulae (length 11.0 mm, outer diameter 0.6 mm) were implanted bilaterally, aiming at the ventral CA3 area, following the coordinates (AP −4.5, ML ±5.2, DV −7.6; Figures 1A and 1B) from the rat brain atlas by Paxinos and Watson (2007), and fixed to the skull with instant glue and acrylic resin. The cannulae tips were 1.3 mm above the drug injection site. A stylet was introduced inside each guide cannula to prevent occlusion. At least one week after surgery, the rats received a bilateral infusion into the ventral CA3 area with dental needles (outer diameter 0.3 mm), introduced through the guide cannulae, until their tips were 1.3 mm below the cannula end. Using a micro-syringe, 0.5 µL/side of either the vehicle or drug was manually injected for one minute. To connect the upper end of the dental needle and the micro-syringe, a polyethylene catheter was used. The displacement of an air bubble inside the polyethylene was used to monitor the drug flow. Needles were removed 30 seconds after the end of drug infusion (Stern et al., 2010).

Behavioral study

EPM apparatus

The device consisted of two opposite open arms (50 cm×10 cm) surrounded by a 1 cm high Plexiglas ledge, and two enclosed arms (50 cm×10 cm×40 cm), set up 50 cm above the floor, and was made of wood. The junction area of the four arms (the central platform) was 10 cm×10 cm (Chegini et al., 2014).

Experimental protocol

Seven rats were used in each experimental group. After recovery, the rats were subjected to the following experimental procedures. The stylet was removed, the microinjection cannulae were inserted into the guide cannulae, and manual injections were made bilaterally (0.5 µL/side) with either the vehicle or one of the GABAergic drugs: the GABA_A agonist, muscimol (0.125, 0.25, 0.5 µg/rat), or the GABA_A antagonists, bicuculline (0.05, 0.1, 0.2 µg/rat). All drugs were dissolved in sterile 0.9% saline, except for bicuculline, which was dissolved in a drop of glacial acetic and then diluted with saline just before the experiments. Five minutes after the completion of the injection, the rats were placed at the center of the EPM apparatus. The response of each animal was recorded for five minutes, and the video recordings were scored later. After a 24-hour interval, each animal’s response on the EPM was recorded again. In the rats receiving two drugs (Figures 3 and 4, panels 3 and 4), the injections were made 10 and 5 minutes before the first test day. Rats in the control groups were injected with saline or vehicle.

General conditions and data collection

The tests were undertaken in a low illumination (40 lx) condition space, during the diurnal phase, between 9:00am and 3:00pm. Each EPM session was five minutes long, and was recorded by a video camera while a monitor was installed in an adjacent room. The apparatus was cleaned with wet tissue paper (10% ethanol solution) after each test to avoid urine impregnation. The following behavioral measures were scored: the number of open- (OAE) and enclosed-arms entries (EAE) with the four paws, and the time spent in the open arms (OAT). The resulting data were used to calculate the percentage of time spent in the open arms (Carobrez and Bertoglio, 2005; Rodgers et al., 1997). The percentage of OAE and OAT as the standard anxiety indexes was calculated as follows: (a) %OAT (the ratio of time spent in the open arms to total time spent in any arm×100); (b) %OAE (the ratio of entries into the open arms to total entries×100); (c) EAE (total closed-arm entries were measured as a relative pure index of locomotor activity; Zarrindast et al., 2008).

Histology

After completion of all the experiments, an i.p. injection of a lethal dose of pentobarbital (100 mg/kg) was administered. The site of implantations was marked by an injection of 50% Indian ink solution (0.5 μL/site). The brains were removed and fixed in 10% neutral buffered formalin for at least 48 hours. Slices (50 µm thick) were obtained and mounted on glass microscope slides for localization according to the diagrams from Paxinos and Watson’s (2007) rat brain atlas. If the drug infusion was outside the CA3 region, the data for the rat were excluded from the analysis. In total, 37/167 were excluded from the analyses because of the misplacement of the guide cannulae and/or injection, on either one side or both sides.

Statistical analysis

Repeated measures or two-way analysis of variance (ANOVA) was used for data analysis. Following significant ANOVA results, independent and dependent t-tests were performed. SPSS for Windows v16 (SPSS, Inc., Chicago, IL) was used for data processing and analysis, and the level of statistical significance was p<0.05.

Results

Experiment 1: effects of the pretest intra-CA3 microinjection of muscimol and bicuculline on the open-arms exploratory behaviors

Repeated-measures analysis showed that the intra-CA3 injection of muscimol altered %OAT (intra-groups: F(3, 28)=2.73, p>0.05; inter-groups: F(1, 28)=0.73, p>0.05; inter–intra group interaction: F(3, 28)=5.76, p<0.01; Figure 2, panels 1A and 2A), and %OAE (intra-groups: F(3, 28)=5.06, p<0.01; inter-groups: F(1, 28)=1.35, p>0.05; inter–intra group interaction: F(3, 28)=2.73, p>0.05; Figure 2, panels 1B and 2B), but not EAE (intra-groups: F(3, 28)=0.17, p>0.05; inter-groups: F(1, 28)=57.97, p<0.001; inter–intra group interaction: F(3, 28)=0.06, p>0.05; Figure 2, panels 1C and 2C). Dependent and independent t-test analyses between the test and retest days showed that on the retest day, %OAT and %OAE increased at a dose of 0.5 µg/rat, suggesting that muscimol induced avoidance memory impairment.

Furthermore, according to the repeated-measures analysis, the intra-CA3 injection of bicuculline changed the %OAE (intra-groups: F(4, 35)=3.47, p<0.05; inter-groups: F(1, 35)=18.99, p<0.001; inter–intra group interaction: F(4, 35)=5.02, p<0.01; Figure 2, panels 3B and 4B), but not the %OAT (intra-groups: F(4, 35)=1.36, p>0.05; inter-groups: F(1, 35)=9.37, p<0.01; inter–intra group interaction: F(4, 35)=2.70, p>0.05; Figure 2, panels 3A and 4A), or the EAE (intra-groups: F(4, 35)=5.58, p<0.01; inter-groups: F(1, 35)=59.14, p<0.001; inter–intra group interaction: F(4, 35)=3.44, p<0.05; Figure 2, panels 3C and 4C). The data showed that the difference between the vehicle group and the saline group was not significant. Dependent and independent t-test analyses showed that at a dose of 0.2 µg/rat, the %OAT did not change significantly, EAE decreased on the test day, and %OAE increased on the retest day, indicating that the highest dose of bicuculline attenuated locomotor activity on the test day, and at lower doses (0.05 and 0.1 µg/rat) remained subthreshold for all parameters.

Experiment 2: effect of the pretest intra-CA3 injection of D-AP5 on the open-arms exploratory behaviors

Repeated-measures analyses showed that D-AP5 altered %OAT (Intra-groups: F(3, 28)=11.57, p<0.001; inter-groups: F(1, 28)=35.28, p<0.001; inter–intra group interaction: F(3, 28)=0.27, p>0.05; Figure 3, panels 1A and 2A) and %OAE (intra-groups: F(3, 28)=20.15, p<0.001; inter-groups: F(1, 28)=46.68, p<0.001; inter–intra group interaction: F(3, 28)=1.35, p>0.05; Figure 3, panels 1B and 2B), while it did not alter the EAE (intra-groups: F(3, 28)=0.07, p>0.05; inter-groups: F(1, 28)=49.82, p<0.001; inter–intra group interaction: F(3, 28)=0.83, p>0.05; Figure 3, panels 1C and 2C). Dependent and independent t-test analyses showed that D-AP5 at its highest dose (0.5 µg/rat) increased %OAT and %OAE, but not EAE on test and retest days, indicating that D-AP5 induced anxiolytic-like behaviors and impaired avoidance memory.

Experiment 3: effects of the pretest intra-CA3 microinjection of muscimol on the D-AP5-induced open-arms exploratory behaviors

Two-way ANOVA and post hoc analyses showed that intra-CA3 injection of the subthreshold dose of muscimol (0.125 µg/rat) did not alter %OAT (intra-groups: F(3, 64)=11.43, p<0.001; inter-groups: F(1, 64)=2.03, p>0.05; inter–intra group interaction: F(3, 64)=2.06, p>0.05; Figure 3, panels 1A and 3A), %OAE (intra-groups: F(3, 64)=6.85, p<0.001; inter-groups: F(1, 64)=1.55, p>0.05; inter–intra group interaction: F(3, 64)=0.99, p>0.05; Figure 3, panels 1B and 3B), or EAE (intra-groups: F(3, 64)=0.50, p>0.05; inter-groups: F(1, 64)=1.30, p>0.05; inter–intra group interaction: F(3, 64)=0.80, p>0.05; Figure 3, panels 1C and 3C) already induced by D-AP5 on the test day, indicating that muscimol did not alter the D-AP5-induced anxiolytic-like behaviors.

Moreover, two-way ANOVA revealed that the subthreshold dose of muscimol (0.125 µg/rat) altered %OAT (intra-groups: F(3, 64)=14.35, p<0.001; inter-groups: F(1, 64)=8.11, p<0.01; inter–intra group interaction: F(3, 64)=2.15, p>0.05; Figure 3, panels 2A and 4A), %OAE (intra-groups: F(3, 64)=21.89, p<0.001; inter-groups: F(1, 64)=9.37, p<0.01; inter–intra group interaction: F(3, 64)=3.21, p>0.05; Figure 3, panels 2B and 4B), and EAE (intra-groups: F(3, 64)=1.93, p>0.05; inter-groups: F(1, 64)=5.49, p<0.05; inter–intra group interaction: F(3, 64)=1.10, p>0.05; Figure 3, panels 2C and 4C). Post hoc analyses showed that compared to the effects already induced by D-AP5, %OAT increased at a dose of 0.25 µg/rat, %OAE increased at doses of 0.125 and 0.25 µg/rat, and EAE decreased at a dose of 0.25 µg/rat on the retest day. The data revealed that the intra-CA3 injection of muscimol potentiated D-AP5-induced avoidance memory impairment.

Effects of the pretest intra-CA3 microinjection of bicuculline on the D-AP5-induced open-arms exploratory behaviors

Two-way ANOVA showed that intra-CA3 injection of the subthreshold dose of bicuculline (0.05 µg/rat) altered the %OAT (intra-groups: F(3, 64)=1.40, p>0.05; inter-groups: F(1, 64)=15.16, p<0.001; inter–intra group interaction: F(3, 64)=4.05, p<0.05; Figure 4, panels 1A and 3A), but not the %OAE (intra-groups: F(3, 64)=8.19, p<0.001; inter-groups: F(1, 64)=7.64, p<0.01; inter–intra group interaction: F(3, 64)=0.34, p>0.05; Figure 4, panels 1B and 3B), or EAE (intra-groups: F(3, 64)=0.79, p>0.05; inter-groups: F(1, 64)=0.88, p>0.05; inter–intra group interaction: F(3, 64)=0.13, p>0.05; Figure 4, panels 1C and 3C). Independent t-test analyses revealed that %OAT reversed on the test day at D-AP5 doses of 0.25 and 0.5 µg/rat compared with the effect already induced by D-AP5, indicating that bicuculline blocked D-AP5-induced anxiolytic-like behaviors.

Moreover, two-way ANOVA analyses showed that the subthreshold dose of bicuculline (0.05 µg/rat) also altered %OAT (intra-groups: F(3, 64)=5.96, p<0.001; inter-groups: F(1, 64)=12.44, p<0.001; inter–intra group interaction: F(3, 64)=11.94, p<0.001; Figure 4, panels 2A and 4A) and %OAE (intra-groups: F(3, 64)=15, p<0.001; inter-groups: F(1, 64)=6.96, p<0.05; inter–intra group interaction: F(3, 64)=10.49, p<0.001; Figure 4, panels 2B and 4B), but not EAE (intra-groups: F(3, 64)=0.85, p>0.05; inter-groups: F(1, 64)=0.42, p>0.05; inter–intra group interaction: F(3, 64)=1.23, p>0.05; Figure 4, panels 2C and 4C). Independent t-test analyses showed that at the mentioned dose, bicuculline reversed %OAT and %OAE effects already induced by D-AP5 (0.5 µg/rat) on the retest day, but did not alter EAE. The data revealed that the intra-CA3 injection of bicuculline contradicted the D-AP5-induced effect on avoidance memory.

Discussion

In line with previous studies (Cortese and Phan, 2005; Daniel and Dohanich, 2001), our results on the effects of CA3 NMDA receptor blockade on anxiety and memory showed that the intra-CA3 administration of D-AP5 as an NMDA receptor antagonist induced anxiolytic-like behaviors and amnesia. This confirms the data from MRI studies, which have also provided an additional link between the anxiety and memory with the glutamatergic system (Rauch et al., 2003; Yamasue et al., 2003).

Glutamate has a prominent role in many psychiatric conditions such as anxiety and memory loss (Cortese and Phan, 2005; Fonnum et al., 1995). It has been suggested that anxiety arises from increased glutamatergic neurotransmission in the brain (Assie et al., 1993), and the glutamate system notably regulates fear and anxiety responses in the limbic/paralimbic brain sites (Cortese and Phan, 2005). In addition, NMDA receptors in the hippocampus are involved in the regulation of anxiety, and their blockade can produce anxiolytic-like effects (Rezvanfard et al., 2009). Selective targeted suppression of glutamate hyperexcitability in the appropriate fear/anxiety circuits can guide us to more efficacious and well-tolerated anxiolytics (Swanson et al., 2005).

Some studies related to the test–retest paradigm in the EPM have shown the one-trial tolerance phenomenon, which is a result of avoidance learning, and previous exposure of the animal to the EPM apparatus causes a decrease in %OAT (Bertoglio and Carobrez, 2000; File et al., 1990). This causes a qualitative shift from unconditioned fear to an acquired phobic state (Holmes and Rodgers, 1998; Rodgers et al., 1996; Treit et al., 1993).

Our findings revealed that GABA_A receptor activity manipulation within CA3 by the injection of GABA_A receptor agonist (muscimol) or antagonist (bicuculline) did not alter anxiety-like behaviors, but produced avoidance memory impairment at the highest dose of muscimol. Since it has been proposed that the %OAT is more sensitive to drug effects than the number of entries (Pellow et al., 1985), the bicuculline effect on avoidance memory was considered to be insignificant.

In line with the results of our study, a recent report showed that application of muscimol into the ventral hippocampus leaves the open-arm movement measures unaffected (Zhang et al., 2014). However, some previous studies have claimed that that it influences locomotion and coordination (Corbett et al., 1991). Hence, we suggest the addition of locomotion (activity boxes) and motor coordination (rotarod) tests to future similar investigations.

Some reports have shown that muscimol microinjection into the lateral septum (Drugan et al., 1986) and basal amygdala nuclei (Muller et al., 1997) can produce anxiolytic effects. Peripheral administration of muscimol and bicuculline has also been reported to cause anxiolytic and anxiogenic effects (Dalvi and Rodgers, 1996; Sanders and Shekhar, 1995). In agreement with our results, studies by Nagahara and McGaugh (1992) on muscimol application in medial septal also reported memory impairment. The fact that microinjection of muscimol in CA3 did not result in anxiolytic effects may show that the previous observed effects were caused by areas other than CA3.

As the CNS’s fast inhibitory responses are mediated by GABA receptors, they are expressed on virtually every neuron (Lydiard, 2003). A high degree of diversity and specificity has been recognized among GABA-releasing cells (Freund and Buzsaki, 1996; Klausberger and Somogyi, 2008), which control the activity of local networks and constitute the output of some brain regions and nuclei (Mody and Pearce, 2004). Hippocampal pyramidal neurons express various GABA_A receptor subtypes. A high level of α1, α2, and α5 subunit expression along with β1–3 and γ2 subunits has been reported (Dalvi and Rodgers, 1996; Hinderer, 1990), which indicates the expression of at least three main GABA_A receptor subtypes in these cells. The previous data indicate that hippocampal synaptic transmission regulated by α5-GABA_A receptors plays an important part in certain forms of learning (Collinson et al., 2002).

In the present study, investigating the impacts of CA3 GABA_A receptor activity on D-AP5-induced effects on memory and anxiety showed that intra-CA3 administration of sub-threshold dose of muscimol and D-AP5 did not alter the anxiety level, but they did cause avoidance memory impairment, indicating a possible additive effect between the drugs. We also found that the sub-threshold doses of bicuculline reversed the anxiolytic-like and avoidance memory effects of D-AP5.

Regulation of behavioral processes by the close relationship between brain glutamatergic and GABAergic systems has already been reported. An example is the glutamate release control by GABA_A receptors (Long et al., 2009) in which GABA_A receptor stimulation creates a protective action against the neuronal injury resulting from NMDA receptor activation (Ohkuma et al., 1994). Another example of the close cross-talk between the NMDA and GABA_A receptors in the hippocampus is the potent inhibition of GAT-4 (GABA transaminase) by zinc, leading to the proposal that the release of zinc with glutamate from glutamatergic nerve endings (e.g., CA3 region of the hippocampus) is involved in rising adjacent GABA levels, which in turn regulates extra glutamate-mediated excitation of neurons (Cohen-Kfir et al., 2005). Hence, the regulation of the behavioral processes in the hippocampus might be regulated by contrary and interdependent functions of glutamatergic and GABAergic systems.

A previous report indicated that muscimol potentiated the anxiolytic effect of Mk-801 in CA1 (Naseri et al., 2014). However, it did not have the same effect in the CA3 region. Differences between the GABA_A receptor subtypes (Korpi et al., 2002) and networks in the mentioned areas might be considered as contributing factors.

Inhibitory postsynaptic potentials are potentiated by the drugs that target the GABA_A receptor (e.g., barbiturates and benzodiazepines). As a result, they can be used for disorders in which a superabundance of excitatory (primarily glutamate-mediated) neurotransmission underlies the pathology (e.g., anxiety). Diverge action mechanisms and pharmacokinetic properties of NMDA channel blockers underlie the paradoxical observation of drug effects working through the same molecular target and production of anesthesia on the one hand and memory improvement on the other (Foster and Kemp, 2006).

A speculative explanation for the observed results is that D-AP5 action on presynaptic NMDA receptors of glutamatergic nerve terminals may increase the glutamate release. In addition, activation of presynaptic GABA_A receptors on glutaminergic nerve terminals has been shown to facilitate glutamate release in postsynaptic CA3 pyramidal neurons, and muscimol amplified the frequency of spontaneous excitatory postsynaptic potentials triggered action potentials (Jang et al., 2006). Hence, the presynaptic action of muscimol would potentiate the D-AP5 effect, and the application of bicuculline will restore the observed effects of D-AP5 on anxiety and memory.

Considering the fan-like distribution of the CA3-to-CA1 axons (Andersen et al., 2000), microinjections in CA3 can affect a wide range of CA1 neurons. The extent to which the manipulation can facilitate/interrupt the performance of the neuronal ensembles needs more detailed analysis.

Based on the presented results, it seems that GABA_A receptor agonist, muscimol, modulates the excitability of neurons by increasing the effectiveness of NMDA receptor antagonist, an effect that was blocked by the application of GABA_A receptor antagonist, bicuculline.

Conclusions

Considering the important balance between the activity of NMDA and GABA systems, application of the methods to keep this balance may be considered as an approach for the management of anxiety disorders while preserving memory function.

Footnotes

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

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