The NR2B subunit has emerged as a potential therapeutic target for a variety of neuropsychiatric and neurological conditions, including Alzheimer’s and Huntingdon’s disease, schizophrenia, and mood and anxiety disorders (Cryan and Dev, 2008
; Danysz and Parsons, 2002
; Gogas, 2006
). The main findings of the present study were that systemic administration of the selective NR2B antagonist, Ro 25-6981, impaired the acquisition of fear memory in mice, and that this effect was modified by ageing.
The potential effects of systemically administered Ro 25-6981 on behaviors associated with NMDAR function, including locomotor exploration, anxiety-like behavior and sensorimotor gating, have not been well characterized in either rats or mice. At the dose range currently tested, Ro 25-6981 treatment had minimal effects on spontaneous locomotor exploration/activity (consonant with data in rats obtained by Kosowski and Liljequist, 2004
) or anxiety-like behavior in the elevated plus-maze. Ro 25-6981 treatment also failed to alter responses on the hot plate assay, suggesting that anti-nociceptive actions were unlikely to account for drug effects on fear conditioning. In addition, we observed no effects of Ro 25-6981 treatment on acoustic startle reactivity or prepulse inhibition of the startle response, which is consistent with a previous study in rats which found that another NR2B selective antagonist, Ro 63-1908, failed to alter acoustic startle reactivity or sensorimotor gating at doses that impaired cognition (Higgins et al., 2003
). The only significant effect currently observed was a decrease in center exploration in a novel open field at the highest dose tested, which may be indicative of an anxiety-like response to the drug (Cryan and Holmes, 2005
) However, as no effect was seen in a second test for anxiety-related behavior, the elevated plus-maze, any effect on anxiety-like behavior does not appear to be robust. Indeed certain other NR2B-selective antagonists, including ifenprodil, have also failed to produce anxiety-related activity in mice (Dere et al., 2003
). This provides an interesting contrast with the anxiolytic-like profile of subunit non-selective NMDAR antagonists (reviewed in Cryan and Dev, 2007
) and gene knockout of the NR2A subunit (Boyce-Rustay and Holmes, 2006
). Nonetheless, further studies will be needed to fully characterize the effects of Ro 25-6981 for potential anxiety-related effects, as well as sensorimotor gating and nociception, for example by using alternate assays or testing intracerebral region-specific injections. Notwithstanding, in the context of the present study, the absence of systemic Ro 25-6981 activity on these behaviors serves to exclude some of the potentially confounding effects on fear learning.
On a standard multi-trial delay cued fear conditioning paradigm, pre-training Ro 25-6981 treatment produced a significant deficit in tone recall measured twenty-four hours later. While a pre-training treatment experimental design does not dissociate drug effects on acquisition versus post-conditioning consolidation, the finding that Ro 25-6981 reduced freezing to the final tone presentation during conditioning is consistent with an impairment of fear acquisition rather than consolidation. Further supporting a selective effect of NR2B antagonism on fear acquisition, pre-training but not pre-recall intra-amygdala injection of the selective antagonist CP101,606 impaired subsequent fear recall in rats (Walker and Davis, 2008
), while another study in rats found that systemic Ro 25-6981 injection prior to fear recall disrupted within-session extinction but also failed to affect fear recall (Dalton et al., 2007
It was notable that Ro 25-6981’s impairing effects on multi-trial conditioning were quite modest. It does not however appear that this is due to a failure to overcome a strong fear response produced by repeated tone × high-shock pairings in a multi-trial paradigm. This is because similarly modest effects were observed in the putatively less fear intensive low-shock multi-trial paradigm and 1-trial cued fear conditioning paradigms; both of which generally produced lower levels of fear than the multi-trial high-shock paradigm. Thus, the magnitude of the fear learning deficit produced by Ro 25-6981 is similar across strong and weak fear learning conditions, and thereby argues against a differential recruitment of NR2B as a function of conditioning strength. The alternative, and currently most parsimonious, interpretation is that selective blockade of NR2B-containing NMDARs does not disrupt fear learning to the same extent as more widespread blockade of NMDARs (at least when NR2B antagonists are delivered systemically). Another possibility is that because NR2B antagonists are more potent at diheteromeric than triheteromeric NR2B-containing NMDARs (Hatton and Paoletti, 2005
; Kash and Winder, 2007
), a proportion of NR2B-containing NMDARs could be relatively resistant to the memory impairing effects of Ro 25-6981.
The lateral amygdala is the most likely site of the fear impairing memory impairing effects of systemically administered Ro 25-6981. The lateral amygdala is the principle brain region mediating cued fear acquisition (Fanselow and Poulos, 2005
; LeDoux, 2000
; Maren and Quirk, 2004
), and NR2B is expressed on the majority of thalamo-amygdala dendritic spines (Radley et al., 2007
). Antagonism of NR2B-containing NMDARs via ifenprodil or genetic disruption of NR2B tyrosine phosphorylation disrupts synaptic plasticity in the basolateral amygdala (Bauer et al., 2002
; Li et al., 1995
; Nakazawa et al., 2006
; Weisskopf and LeDoux, 1999
). Furthermore, intra-amygdala injection of the NR2B antagonists ifenprodil or CP101,606 is sufficient to impair fear conditioning in rats while, in contrast, pre-training siRNA knockdown or selective pharmacological blockade of NR2B with Ro 25-6981 in the anterior cingulate impairs the acquisition of context (but not cued) fear memory and intra-hippocampal infusion of Ro 25-6981 impairs trace but not delay cued fear conditioning (Blair et al., 2005
; Rodrigues et al., 2001
; Sotres-Bayon et al., 2007
; Valenzuela-Harrington et al., 2007
; Walker and Davis, 2008
; Zhao et al., 2005
). However, given the modest fear memory impairing effects of Ro 25-6981 in the current study, it is noteworthy that the aforementioned memory impairing effects of pre-training intra-amygdala CP101,606 which, as with Ro 25-6981, is more 2B-selective than ifenprodil (Kash and Winder, 2007
), were only evident within a narrow dose range (Walker and Davis, 2008
Nonetheless, present data provide novel evidence that a NR2B-mediated component of fear learning may be compromised with ageing. The specific finding was that twelve month old C57BL/6Tac mice showed good basal fear, similar to that of three month old C57BL/6Tac counterparts but, unlike the younger mice, were resistant to the fear memory impairing effects of Ro 25-6981. We chose to examine the memory impairing effects of Ro 25-6981 at twelve months because rodents are not yet considered ‘aged’ and likely to exhibit global cognitive deficits (Barnes et al., 1997
; Clayton et al., 2002
; Magnusson et al., 2007
). Rather twelve months perhaps more closely approximate to ‘middle age’ in humans when more subtle age-related changes begin to manifest. It should be made clear that for reasons of availability C57BL/6Tac mice were used for this ageing experiment rather than the C57BL/6J line used in the other experiments. However, vehicle-treated mice from the two lines showed very similar levels of fear (i.e., forty-eight percent freezing for C57BL/6Tac, forty-nine percent freezing for C57BL/6J). In addition, the three month old mice in this experiment were also C57BL/6Tac and showed a clear fear recall deficit at the highest dose of Ro 25-6981, replicating the effect of this dose in C57BL/6J tested across conditioning paradigms. Thus, the loss of the drug’s efficacy in the twelve month old C57BL/6Tac mice appears to be a genuine effect of ageing rather than an idiosyncrasy of this line of C57BL/6 mice. Nonetheless, a final point to bear in mind is that our data do not demonstrate that ageing is only associated with a loss of the fear impairing effects of Ro 25-6981 and cannot not exclude the possibility that other positive (and as yet to determined) effects of Ro 25-6981 are not also diminished in older mice.
Magnusson and colleagues’ work demonstrating that loss of NR2B at the protein expression level does not manifest until fifteen months in the frontal cortex of C57BL/6Nia mice and even later in hippocampus (Magnusson, 2000
; Magnusson et al., 2002
; Magnusson et al., 2007
; Ontl et al., 2004
). Consistent with their data, we found no evidence of reduced NR2B protein levels in the amygdala, dorsal hippocampus or medial prefrontal cortex of the twelve month old C57BL/6Tac mice. This suggests that the loss of sensitivity to Ro 25-6981 reflects functional alterations preceding the loss of protein itself, perhaps involving improper synaptic targeting or coupling to downstream signaling mechanisms (Malenka and Bear, 2004
). Another, and not necessarily exclusive, possibility is that the ratio of NR2B to NR2A, at the protein and/or functional level, changes with ageing having the effect of reducing the importance of NR2B relative to NR2A for fear memory formation. These mechanisms await elucidation but could provide insight into how NMDAR-mediated learning processes are dynamically regulated with ageing.
In summary, the present study provides further evidence that selective blockade of NR2B-containing NMDARs is sufficient to impair the acquisition of conditioned fear behavior. This effect was demonstrated using various conditioning procedures, but was overall quite modest. The same dose range of Ro 25-6981 had minimal effects on locomotor exploration, anxiety-like behavior, nociception, or sensorimotor gating. Ro 25-6981’s learning impairing effects were also absent in twelve month old mice. These data further support a role for NR2B-containing NMDARs in fear learning and suggest that this role may diminish with ageing. Further studies along these lines could ultimately have implications for understanding the contribution of NMDAR to the pathophysiology and treatment not only of fear-related neuropsychiatric conditions such as post-traumatic stress disorder, but also other disease states in which NMDARs are implicated including age-related cognitive dysfunction and Alzheimer’s disease.