The studies reported here were designed to test the hypothesis that excess neural activity in the hippocampus of aged rats contributes to memory impairment. We first overexpressed an inhibitory neuropeptide (NPY 13–36) targeting the CA3 region of the hippocampus of aged rats using methods shown to attenuate aberrant excitability in limbic seizure models. We also tested two widely used antiepileptic agents administered systemically. In each case, aged rats with established memory impairment showed improved behavioral performance in spatial memory tasks. The converging evidence for benefits of these treatments indicates that excess excitation in the hippocampus may have a key role underlying age-related impairment in hippocampal-dependent memory processes as proposed for this model (Wilson et al, 2006
Aged rats in the current research came from a study population with well-characterized individual differences in memory impairment. Importantly, previous studies have shown a high degree of test-retest reliability for the presence and severity of those individual differences in behavioral assessments; aged rats that fall outside the range of normative young performance in the standardized assessment show a similar behavioral profile in subsequent tests of hippocampal-dependent memory (Colombo et al, 1997
; Robitsek et al, 2008
). Such evidence increases confidence that the assignment of subjects based on the standardized background assessment ensured a valid test of treatment efficacy in experiments using between group comparisons. In addition, the data obtained in the experiments using within-subject designs support the reliability of individual differences because impairment was routinely evident under vehicle treatment in those studies, as discussed further below.
The hypothesis examined here is also grounded in much background research in this model showing that behavioral status in the aged rat study population is strongly correlated with neurobiology in the underlying circuitry of the hippocampal formation. Although aged rats in this study population maintain similar number of neurons and largely preserved synaptic connections in the medial temporal lobe system (Geinisman et al, 2004
; Rapp and Gallagher, 1996
; Rapp et al, 2002
; Smith et al, 2000
), functional alterations are closely tied to age-related cognitive decline. The striking increase in neural activity in the CA3 region led to a proposal for the basis of cognitive impairment, which links that excess activity to loss of encoding that normally depends on the CA3/dentate gyrus subdivisions of the hippocampal network (Wilson et al, 2006
). As the majority of synaptic input to the CA3 pyramidal neurons comes from the excitatory recurrent CA3 collaterals, elevated activity would be expected to promote pattern completion, a computational function attributed to the CA3 autoassociative network that favors retrieval of previously stored representations (Guzowski et al, 2004
; Treves and Rolls, 1994
). In addition, the joint occurrence of elevated activity and reduced plasticity is well established in studies of neural networks, including the hippocampus; synaptic plasticity most readily occurs in an optimal range of activity, and thresholds for synaptic modification can rise in conditions of excess activity (see Abraham et al, 2001
for example, and Abraham, 2008
for recent review). Recording data from hippocampal neurons in this study population are in agreement with this concept linking excess activity to a failure to encode new information in the CA3 network (Wilson et al, 2005
). By that view, experimental treatments that lower activity would be expected to lower the threshold for synaptic modification to allow the encoding of new information.
As an initial test of this idea, we locally expressed an inhibitory neuropeptide, NPY13–36, which has been shown elsewhere to be effective in modulating neuronal excitability under both normal and epileptic conditions (Foti et al, 2007
; McQuiston and Colmers, 1996
; Vezzani et al, 1999
). NPY13–36 preferentially activates the Y2 receptor subtype which has a predominant role in mediating the NPY-induced inhibition of glutamate release in the CA1 and CA3 of the hippocampus (El Bahh et al, 2005
; Silva et al, 2005
). We engaged the inhibitory property of Y2 receptor activation by constitutively expressing NPY 13–36 via an AAV vector delivered into the CA3; this resulted in significantly improved hippocampal-dependent memory in the aged rats. In situ
histochemistry for the vector-produced mRNA confirmed that expression of NPY13–36 was restricted to the hippocampus and centered primarily on the CA3, likely providing only local control of inhibition in that area. Because no elevated activity is observed in the CA1 subregion of the hippocampus in our aging model (Wilson et al, 2005
), and CA3 connections (Shaffer collaterals) onto CA1 neurons have reduced efficacy in the aged brain (Lister and Barnes, 2009
), boosting inhibition in the adjacent CA1 area would not be expected to have beneficial effects on network function. These data therefore support the view that controlling excess neural activity in the CA3 of aged rats improves hippocampal network function and hippocampal-dependent memory performance, and serves as a proof of principal for further exploration into more clinically accessible methods of modulating excess neural activity.
Subsequent tests centered on the same rationale of targeting excess excitation by administering two widely used antiepileptic agents, VPA and LEV, which each showed the ability to improve spatial memory in aged rats. In the first of those studies, VPA was assessed in a spatial memory task when aged memory-impaired rats learned new escape platform locations in each day's session in the water maze. Rats implanted with osmotic mini-pumps to administer 100
mg/kg had substantial savings after a 6
h delay, escaping with similar proficiency relative to the end of the previous session's training trials. Those rats differed from the control vehicle group and a group receiving a lower dose of VPA, which showed little savings for previous training after the delay. In a subsequent study using a within-subject design, VPA doses of 100 and 200
mg/kg significantly improved memory performance on the radial maze after a 1-h delay. The improvement obtained with effective doses of VPA reduced errors to levels typically seen in young rats tested on this task (Chappell et al, 1998
). Together those experiments show a benefit from VPA treatment across spatial memory tasks with different motivational and performance demands. It is important to note that blood levels of VPA in rats at the doses effective for neurocognitive aging (10
μg/ml; see Materials and methods section for details) are well below those needed for antiepileptic efficacy to control seizure activity (225
μg/ml and above; Tulloch et al, 1982
). Indeed, high dosing might be less effective for cognitive improvement in aging by producing more pronounced and widespread effects on brain function.
Similar to VPA, LEV was also found to improve memory performance on the radial maze at doses substantially lower than those used in seizure models. Significant improvement was observed at 5 and 10
mg/kg, but not at a higher dose of 20
mg/kg. Typical antiepileptic doses of LEV in rodent seizure models are in a range of 50–150
mg/kg (eg, Ji-qun et al, 2005
; Stratton et al, 2003
). In addition, the two antiepileptic compounds VPA and LEV exhibited benefit in combination at doses that were otherwise ineffective. Comparing the dose response for the individual drugs with the combinations provided evidence for drug synergy rather than merely additive effects. As an extension of the data obtained in the radial maze, improved retention over a 24
h delay was confirmed with LEV at 10
mg/kg in an independent set of memory-impaired aged rats trained in a novel water maze environment. Finally, as expected under the view that benefit is because of dampening excess activity, we found no improvement in memory performance when young adult rats were given LEV.
Although the route by which VPA and LEV exert antiepileptic efficacy is not known with certainty, and those mechanisms may not directly apply to the doses used in the current studies, these two compounds share little apparent mechanistic overlap. The efficacy of VPA as an antiepileptic potentially involves a variety of mechanisms, including increased γ-amino butyric acid (GABA)-ergic transmission, increased uptake of excitatory amino acids, and blockade of voltage-gated sodium channels (Perucca, 2002
). At the low doses found to be effective in the current research, the most likely mechanisms involve control of excitatory transmission by reducing extracellular accumulation of glutamate and increased GABAergic transmission. An upregulation of hippocampal glutamate transport occurs in rats treated with VPA (Hassel et al, 2001
). We confirmed this finding at doses used in our aged rats and further observed that low doses of VPA administered systemically increase the hippocampal expression of GLT1b, an isoform of the glutamate transporter (M Gallagher, unpublished data). In contrast to the localization of GLT1a in astrocytes, GLT1b is localized to neurons in association with the post-synaptic density (González-González et al, 2008
). Thus, VPA may permit the regulation of excitatory transmission at synapses that drive neuron activity in addition to boosting inhibition. LEV is thought to mediate its effects via a synaptic vesicle protein Sv2a, which renders primed synaptic vesicles competent for activity-dependent (calcium-mediated) release (Chang and Südhof, 2009
). At concentrations of up to 10
μM, LEV does not show binding affinity for a variety of known receptors, such as those associated with benzodiazepines, GABA, glycine, and NMDA (N
-methyl--aspartate). In vitro
and in vivo
recordings of epileptiform activity from the hippocampus have shown that LEV inhibits burst firing. In vivo
data also show that LEV opposes a pilocarpine-induced increase of the orthodromically activated population spike in the CA3 region of hippocampus (Klitgaard et al, 2003
). Synergistic activity has also been noted for use of VPA and LEV in combination in studies of seizure models (Kaminski et al, 2009
Current data suggest that modifying excitatory–inhibitory balance through different routes can ameliorate behavioral memory impairment in aging. The benefit observed with these different treatments suggests that neural activity and encoding properties in the hippocampal network are ameliorated by targeting excess activity. Studies to directly monitor such effects, using electrophysiological recordings in awake behaving rats, are beyond the scope of the current investigation, but will be informative as to whether such restoration is a final common pathway for behaviorally effective treatments in this model.
The functional significance of aberrant activity in the hippocampus may not be limited to animal models of aging but may also have direct implications for memory decline experienced during aging in humans. A number of investigations using functional neuroimaging in human aging have reported increased activation in the medial temporal lobe during memory encoding. This finding has now been observed in patients with mild cognitive impairment (MCI; Celone et al, 2006
; Dickerson et al, 2004
; Dickerson et al, 2005
; Hämäläinen et al, 2007
), in older individuals with genetic or familial risk for developing Alzheimer's disease (Bassett et al, 2006
; Bookheimer et al, 2000
), and in aged subjects showing poor performance in memory but not sufficiently impaired to meet a diagnosis of MCI (Miller et al, 2008a
). The original view of such increased activation was that it served a beneficial function by recruiting additional resources to provide ‘compensation' for a failing neural network. A possible detrimental role of excess activation in humans, however, has been suggested by findings in follow up with MCI patients (Dickerson et al, 2004
; Miller et al, 2008b
). Specifically, Miller and colleagues (2008b)
found that higher activation in the hippocampus, but not in signals imaged elsewhere in the brain, predicted greater subsequent cognitive decline 4–8 years after initial evaluation. Consequently, those authors suggested that elevated hippocampal activation might serve as a therapeutic target for early intervention in cognitive decline. An assessment of whether reduction in elevated hippocampal activation in humans leads to worse memory performance, as suggested by a compensation view, or improves performance, consistent with the animal data obtained here, would provide a needed experimental test of the alternative interpretations now existing in the clinical literature. If the condition in humans' parallels the excess activity observed in studies of aged animals with memory impairment, such a test would be feasible using FDA approved compounds (VPA and LEV) that are effective in aged animals, albeit at lower doses than those used for other clinical indications.
Not much more than a decade ago it was widely believed that loss of neurons in the brain was the cause of memory impairment associated with aging. Now it is generally accepted that memory decline is predominantly a functional disorder in aged brains that are largely structurally intact. This view gives hope to the prospect for corrective therapies that will aid in the restoration of function in the aged brain. Many recent approaches to this problem have focused on boosting mechanisms underlying neural plasticity in young adults (Josselyn and Nguyen, 2005
; Rose et al, 2005
for reviews). The current research supports a novel approach based on a disorder specific to the condition of neurocogntive impairment in the aged brain. As such, the findings may point to novel targets for therapies in this indication.