In the above studies, we were interested in determining the effect of altered GABAB function on learning and extinction using the trace cued and contextual fear conditioning paradigm. In Experiment 1, the GABAB agonist baclofen and GABAB antagonist phaclofen were administered throughout the entirety of the experiment. Interestingly, the baclofen-treated animals did not exhibit extinction to the CS or to the context as did the saline- and phaclofen-treated groups. These extinction deficits are evidenced by the lack of a significant decrease in freezing to the CS from Day 2 to 4 and by the lack of a significant decrease in freezing to the original context from Day 3 to 5. Further, we did not observe any significant differences between groups 24 hours following training, suggesting that these differences cannot be attributed to alterations in initial learning.
In Experiment 2, we investigated the effects of altered GABAB function in extinction after subjects had already learned the association (drug starting Day 2). Similar to Experiment 1, the baclofen-treated animals in Experiment 2 also exhibited an extinction deficit, but only to the context, as demonstrated by the lack of significantly decreased freezing from Day 3 to 5. Contrary to Experiment 1, the baclofen group did demonstrate significantly greater freezing to the CS presentations compared to the saline group on Days 2 and 4. This difference suggests that enhanced GABAB receptor activity after the initial fear conditioning may create an exaggerated fear response (association) compared to the control group. The baclofen group in Experiment 2 also demonstrated a significant reduction in freezing to the CS from Day 2 to 4, similar to the saline group, thus demonstrating some extinction to the CS. When this result is taken into consideration with the baclofen-induced cued fear extinction deficit from Experiment 1, these data suggest that increased GABAB receptor function during the initial training impairs the ability to extinguish a fear response to the CS. This difference is interesting because it suggests that facilitating GABAB receptor activity during training and not after training may induce an association that is more resistant to extinction. The baclofen treated animals did display equivalent learning of the association 24 hours post-training; however, this conditioned fear did not dissipate after repeated CS presentations analogous to controls. Similar to Experiment 1, the phaclofen-treated animals showed no behavioral differences compared to the saline control group. Future investigations will help further clarify whether the extinction deficits observed in this study are inducible via baclofen administration during training only or whether they require the administration of baclofen during training and throughout testing.
In order to ensure the above change is tied to extinction, we analyzed the data for the first two minutes of each Cued Fear test session, as well as the time between CS presentations, in order to determine if the treatment groups were simply freezing throughout the entire session. Neither the baclofen or phaclofen group demonstrated increased freezing during the first two minutes of the Cued Fear sessions compared to the saline groups, which suggests that the increased freezing is specific to the CS presentations. Furthermore, there were no significant differences between groups in Experiment 2 for freezing between CS presentations, demonstrating that all of the treatment groups were behaving equivalently when the CS was not present. However, in Experiment 1 the baclofen group froze significantly more than the saline group between CS presentations only on Day 4. This result, taken in conjunction with the above data, suggests that the baclofen group in this experiment did not benefit from the extinction training on Day 2. As this difference was only observed during the session when the baclofen group displayed a lack of reduction in freezing to the CS (that is, not during Days 2 or 6), we are confident this behavior is not representative of a general elevated anxiety phenotype due to the administration of baclofen.
For contextual fear, all groups demonstrated equivalent freezing to the context on Day 3 (the first exposure to the original training context after training), demonstrating equivalent learning of the association between the context and US. The phaclofen-treated groups in both experiments had no behavioral differences compared to saline controls. However, the baclofen groups in both experiments demonstrated a contextual fear extinction deficit, as evidenced by a lack of a significant reduction in freezing from Day 3 to Day 5. These results suggest that increased GABAB function impairs the extinction of the hippocampally-mediated contextual fear, regardless of whether or not the drug is present during the initial training. Further, these differences between groups disappear on Day 7, which occurred 48 hours after the reminder trial, suggesting the reinstatement of the fear association between the US and the context in the saline and phaclofen groups without any elevation in the baclofen groups.
The amygdala has been demonstrated to play a role in cued fear association (Goosens & Maren, 2001
; Phillips & LeDoux, 1992
; Vazdarjanova & McGaugh, 1998
), and would be a likely site for changes impacting extinction. Additionally, previous investigations of GABAA
systems in extinction have argued that cortical GABAergic neurons projecting to the amygdala mediate some aspects of extinction (Akirav & Maroun, 2007
; Akirav, Raizel, & Maroun, 2006
; Makkar, Zhang, & Cranney, 2010
). The observed cued fear extinction deficit in Experiment 1 may be related to altered receptivity to GABA in the amygdala. In Experiment 1, there was a significant increase in the total protein levels of the GABAB2
subunit in the amygdala of the baclofen group, the only group that failed to display cued fear extinction. The possibility exists that an increase in the GABAB2
subunit may have altered network connectivity in the amygdala and may be related to the lack of cued fear extinction seen in Experiment 1. Since the baclofen group in Experiment 2 did show a significant decease in freezing from Day 2 to Day 4, the lack of altered GABAB2
protein levels in the amygdala in this experiment may further support the hypothesis that the GABAB2
subunit is linked with cued fear extinction.
The phaclofen-treated group from Experiment 1 also demonstrated a non-significant increase in total GABAB2 protein levels (see ), but there were no behavioral differences observed. It is possible, therefore, that the increases in total protein level observed (significant for baclofen, non-significant for phaclofen) in this experiment are in response to alterations in GABAB tone, and are not necessarily linked to the behavioral differences. Equally interesting is that this increase in total protein was not observed in either treatment group in Experiment 2. It is possible that altering GABAB signaling during the training session induced a different effect as compared to altering GABAB tone during other sessions. Specifically, these data suggest that alterations in GABAB signaling coupled with the delivery of the US appears to produce different alterations in total GABAB receptor protein levels than when the drug treatments are administered after training. The extent to which this difference directly impacts brain function and behavior is unclear and certainly merits further investigation.
Data from the tissue analyses cannot account for all of the behavioral differences mentioned above. None of the protein markers we examined in the hippocampus indicated a difference that may be related to the contextual fear extinction deficit seen in both baclofen groups, suggesting the administration of baclofen may have altered other transmitter systems, or other GABAergic targets (see below). However, it is clear that increasing GABAB
function via baclofen does inhibit the extinction of a hippocampally-mediated association. Research indicates that the dorsal hippocampus mediates contextual fear conditioning (Esclassan et al., 2009
; Kim & Fanselow, 1992
; Quinn et al., 2005
), so it is possible that analyzing whole hippocampus GABAergic protein levels hindered the discovery of a significant target related to the observed contextual fear extinction deficits. Alternatively, it is also possible that the total protein changes in the amygdala could contribute to these observed behavioral changes. For instance, it has been established that amygdala activity is capable of mediating performance of hippocampal-dependent behavioral tasks (Galliot et al., 2010
; McIntyre, Marriott, & Gold, 2003
; Vafaei et al., 2007
). Considering the extent to which amygdala function contributes to hippocampal-dependent processes, the protein changes in the present study may have relevance to the behavioral data. The increase in total GABAB2
protein levels found in the amygdala could have had a direct effect on the hippocampally-mediated contextual fear extinction deficit seen in Experiment 1. However, the lack of significant changes in total protein levels of the targets we examined in the amygdala or in the hippocampus of any of the treatment groups in Experiment 2 make a direct connection impossible. Thus, while it is possible for the amygdala to be contributing to the contextual fear extinction deficit in Experiment 1, this explanation does not sufficiently address the contextual fear extinction deficit observed in Experiment 2.
The extinction deficits associated with the administration of baclofen found in these studies are surprising considering the extensive literature that suggests extinction is largely facilitated by inhibitory mechanisms (see Makkar, Zhang, & Cranney, 2010
). Possibly because the bulk of the data are derived from research that used compounds that target GABAA
-driven alterations of extinction may just need to be characterized further. Alternatively, it is possible that altering the GABAB
system may alter existing circuits through autoinhibition. By preventing the presynaptic release of GABA via enhancing the function of autoreceptors, baclofen administration could result in the lack of inhibition on the circuits responsible for maintaining the fear associations, thus the associations stay intact.
It is possible the baclofen-induced extinction deficits in Experiment 1 may be influenced by altered auditory processing based on data from the acoustic startle experiments. However, we believe this is unlikely based on equivalent performance in the fear conditioning experiments between groups 24 hours post-training. If baclofen impaired detection of the auditory CS, we would expect differences in the initial cued fear session (e.g. decreased freezing compared to the saline controls). Given the data of equivalent performance between groups it seems likely the baclofen group detected the auditory CS consistent with controls. Further, the differences in acoustic startle were not observed in Experiment 2. A closer evaluation of the acoustic startle data between the two experiments suggests that the saline controls in Experiment 1 displayed a much larger startle response than controls in Experiment 2. This is in contrast to consistent startle amplitudes for both drug treatment groups in Experiment 1 and 2, suggesting the significant difference may be related to an elevated response in a subset of controls. These data merit additional investigations and could be evaluated using both acoustic and tactile startle approaches. Similarly, we believe the differences in the tail flick nociception task observed only in Experiment 2 are not directly related to the above extinction deficits. If a true nociceptive reduction were induced by baclofen, we would expect differences in freezing (decreased compared to the saline controls) in the initial cued and contextual fear sessions on Days 2 and 3 as reported elsewhere with drugs associated with analgesia (see Abbot, Melzack, & Leber, 1982
). No such reduction was observed in the above experiments, and data from other investigations have demonstrated that systemic administration of baclofen at the dosage used in the current study does not inhibit nociception (De Luca & Massotti, 1990
; Smith et al., 1994
). As the difference in tail flick was only significant in one experiment and we have not observed a similar difference in preliminary studies with baclofen and phaclofen (unpublished results) we do not believe the extinction deficits are related to altered nociception. These data do merit additional investigation to determine why the administration of baclofen induced changes in the above tests; however, given equivalent learning between all groups it seems unlikely that the extinction deficits observed could be mediated by these differences.
The administration of phaclofen did not alter performance in either experiment, but it did produce a significant decrease in total protein levels of the GABAB1b subunit compared to the saline group in the amygdala tissue from Experiment 2. This result suggests that although phaclofen used at the above concentration may not be altering behavior in CCF, the drug did induce a physiological effect at the cellular level. The decrease in total protein may be an initial response to the drug, but these changes do not appear to specifically impact the fear conditioning as examined in these studies. It is possible that the alteration of total GABAB1 protein levels may impact performance in other tasks and/or that a higher dosage of phaclofen may be sufficient to alter performance in trace CCF. The novel finding that altering GABAB tone impairs extinction merits substantial additional investigations, some of which include examining alternative doses of phaclofen and baclofen, determining if phaclofen is able to reverse the baclofen-induced extinction deficits, as well as extending these investigations into additional learning and memory tasks.
Future experiments are also needed to help elucidate the protein correlates to these behavioral differences. It is possible that other GABAergic targets (such as inward-rectifying potassium (GIRK) channels, GAD65/67, or GABA transporters) were altered. Studies have demonstrated that the GABAB
receptors do not respond in a typical manner seen with other G-protein coupled receptors. For instance, Wetherington and Lambert (2002)
demonstrated that GIRK-associated postsynaptic GABAB
receptors rapidly desensitized to agonist treatment, whereas GIRK-associated presynaptic GABAB
receptors failed to desensitize even after 24 hours of agonist treatment. Therefore, it is possible that presynaptic GABAB
receptors are less sensitive to prolonged ligand treatment. Other research has demonstrated that GABAB
receptors do not internalize in response to agonist treatment (Fairfax et al., 2004
; Mutneja et al., 2005
; Perroy et al., 2003
), but that agonist treatment does produce a decrease in cell surface-expressed receptors (Fairfax et al., 2004
). Further, it would be interesting to determine if the total protein level differences observed translate to functional or even membrane-expressed differences in GABAB
receptors. Additional studies are also needed to help determine if altering GABAB
receptor function using GABAB
antagonists can affect fear learning and extinction.
Overall, the data presented in the above studies suggest a significant role of GABAB receptor function in fear learning and extinction. In particular, enhanced GABAB function during training appears to influence the ability to extinguish the fear response to a conditioned stimulus, and may be mediated by the GABAB2 subunit in the amygdala. Regardless of when the drug was administered, baclofen impaired the extinction of the fear association to the original context. It appears that the GABAB receptor may play an alternative role in learning and extinction compared to the GABAA receptor. Generally, GABAA agonists tend to impair learning, but enhance extinction. The current study found that using a GABAB agonist did not impair learning, but that it did prevent extinction. Future experiments are needed to determine if the administration of phaclofen is capable of rescuing the extinction deficit induced by baclofen, as well as if a higher dose of phaclofen alone may be able to affect a change in behavior, and further characterize GABAergic targets that may mediate these extinction deficits.