Associative and motor learning in 12-month-old transgenic APP + PS1 mice

Abstract

Doubly transgenic 12-month-old amyloid precursor protein and presenilins 1 (APP + PS1) mice (n = 14) and littermate control mice (n = 17) were tested on eyeblink classical conditioning—a task impaired in humans with Alzheimer's disease (AD). Mice were also tested on a motor learning task (rotorod) and on sensory tasks (prepulse inhibition [PPI] and acoustic startle). Transgenic mice had impaired motor performance on rotorod. Overall, APP + PS1 mice performed similarly to controls on both 500 ms delay and 500 ms trace eyeblink conditioning as well as on prepulse inhibition (PPI) and acoustic startle. However, within the transgenic group, cortical amyloid burden correlated significantly with decreased trace eyeblink conditioning. Moreover, cortical amyloid burden and hippocampal microglia activation correlated significantly with decreased PPI. These data suggest that only those transgenic mice with the most severe amyloid pathology exhibited deficits in hippocampus-dependent tasks. Transgenic mouse models of amyloid deposition differ from Alzheimer patients not only by the absence of major neuronal loss, but also by the general absence of severe impairments in eyeblink conditioning, except for mice with the greatest amyloid pathology.

Introduction

Impaired learning and memory ability are the cognitive hallmarks of Alzheimer's disease (AD)–a progressive dementia characterized by extensive loss of neurons and the presence of amyloid-containing senile plaques as well as neurofibrillary tangles [7], [8], [43]. Many scientists maintain that the initiation of the cascade of events resulting in AD begins with genetic triggering of excess production of β-amyloid (Aβ) [45]. One major neuropathological event, which has been linked to neurodegeneration in AD, is the deposition of fibrillar β-amyloid [46]. Several studies have shown that neuritic plaques and microgliosis, are correlated with cognitive decline in patients with AD [5], [17], [38], [41].

In familial AD, mutations in the gene encoding the amyloid precursor protein (APP) and presenilins 1 and 2 (PS1 and PS2) have been linked to development of AD in humans. Recently, several transgenic mouse lines expressing minigene copies of the human mutations have been developed [18], [26]. Mice that are doubly transgenic for both APP and PS1 mutations (APP + PS1) show a rapid progressive development of compact plaques within the hippocampus and other cortical areas beginning at about 3 months of age, where the number of compact plaques reaches a plateau at 12 months of age [21], [25]. In addition to the deposition of amyloid plaques, which consists of similar isoforms of Aβ present in AD patients [36], other AD-related pathologies such as microgliosis and oxidative stress occur in transgenic AD mice carrying an APP mutation [19], [21], [42], [51]. However, despite the rapid increase in compact plaques until the age of 12 months, normal performance on the Morris water maze and radial arm water maze was observed at 5–7, 9 and 11.5 months of age in APP + PS1 mice [2], [25]. At 15–17 months of age, a working memory deficit was observed on a modified version of the water maze task, but not on other tasks [2], suggesting that the detection of memory impairment depends upon the particular task employed.

In the current study we examined behavioral performance in 12-month-old APP + PS1 mice on two forms of eyeblink classical conditioning: delay and trace. In delay eyeblink classical conditioning a neutral stimulus such as a tone is called the conditioned stimulus (CS), and it is followed half a second after its onset by blink-eliciting stimulation to the eye muscles called the unconditioned stimulus (US). Repeated presentations of CS and US result in associative learning—the organism blinks to the tone-CS before the onset of the stimulation-US. This learned eyeblink response is called the conditioned response (CR). The cerebellum is the essential brain substrate for delay eyeblink classical conditioning (reviewed in [13]). In trace eyeblink conditioning the CS turns on and then off, and a blank period ensues before the onset of the US. The hippocampus as well as the cerebellum are essential for eyeblink conditioning to occur if the trace period exceeds a critical time interval. For mice this critical interval is 250 ms [53]. In delay eyeblink conditioning, the hippocampus is not essential, but disruption of the septohippocampal cholinergic system impairs learning (e.g., [48]).

A number of studies have demonstrated that delay eyeblink classical conditioning is severely impaired in patients with AD and in adults over the age of 35 with Downs's syndrome and AD neuropathology but not in patients with other neurodegenerative diseases such as Parkinson's or Huntington's disease (for review, see [58]). In the 400 ms delay eyeblink conditioning procedure, 20 patients diagnosed with probable AD were extremely impaired in comparison to 20 age-matched control subjects [59]. This result was replicated [47] and extended to a larger sample [63]. Patients with AD actually performed better in trace than in delay eyeblink conditioning when the trace interval was 500 ms [62]. Subsequently it was demonstrated that in humans, the critical interval for trace conditioning to be hippocampus-dependent is 1000 ms [14]. There are no published reports of AD patients’ performance at this long trace interval.

The brain circuitry forming the substrate for delay eyeblink conditioning is similar in all mammals that have been tested [13], making this paradigm useful for evaluating behavioral validity of mouse models of AD. In trace eyeblink conditioning the brain circuitry is similar in all mammals, but the critical interval making the paradigm hippocampus-dependent varies from 250 ms in mice [53] to 500 ms in rabbits [40] to 1000 ms in humans [14].

We also tested prepulse inhibition (PPI), acoustic startle, and rotorod to assess sensory and motor capacity in these transgenic mice. PPI is a model of sensorimotor gating that is modulated by hippocampal function in rats [29]. PPI is impaired in patients with schizophrenia, possibly due to a nicotinic cholinergic deficit within the hippocampus [1]. Acoustic startle has been tested in both humans and rodents and shows similar parametric characteristics across different species [49]. The hippocampus is an early site of cell loss and neuropathology in AD. Because eyeblink classical conditioning, PPI, and possibly acoustic startle are influenced by hippocampal function, these tasks are promising tools to use in modelling behavioral deficits in the APP + PS1 mouse model of AD. We further asked whether performance on these tasks correlates with amyloid pathology and microglial activation within the cortex and hippocampus of the APP + PS1 mice.