Intermittent systemic exposure to lipopolysaccharide-induced inflammation disrupts hippocampal long-term potentiation and impairs cognition in aging male mice.

Age-related cognitive decline, a common component of the brain aging process, is associated with significant impairment in daily functioning and quality of life among geriatric adults. While the complexity of mechanisms underlying cognitive aging are still being elucidated, microbial exposure and the multifactorial inflammatory cascades associated with systemic infections are emerging as potential drivers of neurological senescence. The negative cognitive and neurobiological consequences of a single pathogen-associated inflammatory experience, such as that modeled through treatment with lipopolysaccharide (LPS), are well documented. Yet, the brain aging impacts of repeated, intermittent inflammatory challenges are less well studied. To extend the emerging literature assessing the impact of infection burden on cognitive function among normally aging mice, here, we repeatedly exposed adult mice to intermittent LPS challenges during the aging period. Male 10-month-old C57BL6 mice were systemically administered escalating doses of LPS once every two weeks for 2.5 months. We evaluated cognitive consequences using the non-spatial step-through inhibitory avoidance task, and both spatial working and reference memory versions of the Morris water maze. We also probed several potential mechanisms, including cortical and hippocampal cytokine/chemokine gene expression, as well as hippocampal neuronal function via extracellular field potential recordings. Though there was limited evidence for an ongoing inflammatory state in cortex and hippocampus, we observed impaired learning and memory and a disruption of hippocampal long-term potentiation. These data suggest that a history of intermittent exposure to LPS-induced inflammation is associated with subtle but significantly impaired cognition among normally aging mice. The broader impact of these findings may have important implications for standard of care involving infections in aging individuals or populations at-risk for dementia.

Trace copper levels in the drinking water, but not zinc or aluminum influence CNS Alzheimer-like pathology.

Mounting evidence suggests copper may influence the progression of Alzheimer's disease by reducing clearance of the amyloid beta protein (Abeta) from the brain. Previous experiments show that addition of only 0.12 PPM copper (one-tenth the Environmental Protection Agency Human consumption limits) to distilled water was sufficient to precipitate the accumulation of Abeta in the brains of cholesterol-fed rabbits (1). Here we report that addition of copper to the drinking water of spontaneously hypercholesterolemic Watanabe rabbits, cholesterol-fed beagles and rabbits, PS1/APP transgenic mice produced significantly enhanced brain levels of Abeta. In contrast to the effects of copper, we found that aluminum- or zinc-ion-supplemented distilled water did not have a significant effect on brain Ab accumulation in cholesterol-fed rabbits. We also report that administration of distilled water produced a reduction in the expected accumulation of Ab in three separate animal models. Collectively, these data suggest that water quality may have a significant influence on disease progression and Ab neuropathology in AD.

Down regulation of cerebellar memory related gene-1 following classical conditioning.

We have isolated and characterized the mRNA of a mouse gene named cerebellar memory related gene-1, previously found by microarray analysis to be differentially expressed following classical conditioning of the rabbit nictitating membrane response. Quantitative RT-PCR analysis showed a significant reduction in mRNA expression in cerebellar lobule HVI but not in the hippocampus of rabbits that received classical conditioning compared to control rabbits that received either unpaired stimulus presentations or were simply restrained. The mouse mRNA encodes a protein of 485 amino acids that includes different potential post-translational modification sites and five copies of the WD-repeat suggesting involvement in protein-protein interaction and regulatory function. In-situ hybridization experiments show highly localized expression of the transcript in mouse brain with the highest expression levels located in the cerebellum, hippocampus and cortex. Taken together, our results reveal a novel gene encoding a WD-repeat protein that is down-regulated in cerebellar lobule HVI as a result of learning and memory.

Interactions of prefrontal cortex during eyeblink conditioning as a function of age.

Changes in regional cerebral blood flow (rCBF) in eleven elderly subjects during pairings of tone and air puff were compared to rCBF changes during pairings in young subjects. Although all subjects reported being aware of the relationship between tone and air puff, elderly subjects did not condition as well as young subjects and their rCBF measures were attenuated. Covarying the performance differences between young and old subjects did not change this conclusion suggesting that differences in neural activation during learning are related to binding of CS-US information prior to the impact of the association on performance. Both groups showed learning-specific rCBF changes in cerebellum, inferior right prefrontal cortex and posterior cingulate. However, only in young subjects were there learning-specific changes in rCBF in left temporal cortex, midbrain, caudate, and inferior left prefrontal cortex. Analysis of learning-dependent patterns of functional connectivity of inferior left prefrontal cortex showed only young subjects had a strong left prefrontal functional connectivity with cerebellum, hippocampus, thalamus and temporal cortex. Thus, beyond changes in regional activity, these data also suggest that age may alter the operations of functional networks underlying learning and memory.

Quantitative distribution of protein kinase C alpha, beta, gamma, and epsilon mRNAs in the hippocampus of control and nictitating membrane conditioned rabbits.

We used oligonucleotide in situ hybridization and film autoradiography to quantitate the distributions of protein kinase C (PKC) alpha, beta, gamma, and epsilon mRNAs in subregions of rabbit hippocampus. Levels of each of the hippocampal PKC isozyme mRNAs and patterns of their regional distributions were remarkably invariant between individuals. Within stratum pyramidale, the highest levels of PKC alpha mRNA were in the CA2 region, while PKC beta mRNA was maximally expressed in CA1, and PKC epsilon mRNA in CA3; PKC gamma mRNA was abundantly expressed throughout Ammon's horn. Previous experiments employing quantitative autoradiography for [3H]PDBU (Olds et al., Science, 245 (1989) 866-869) revealed an increase in membrane-bound PKC in the CA1 region of rabbit hippocampus up to 3 days following classical conditioning of the nictitating membrane response. We report here that there were no differences in levels of PKC alpha, beta, gamma, or epsilon mRNA between conditioned and control rabbits in any hippocampal region one day after training. These data are consistent with the hypothesis that PKC is post-translationally activated and translocated to the membrane during memory storage.

Stimulation of the spinal trigeminal nucleus supports classical conditioning of the rabbit's nictitating membrane response.

Acquisition, extinction, and differential conditioning of the rabbit's nictitating membrane response to a tone conditioned stimulus were supported by electrical stimulation of the spinal trigeminal nucleus. Stimulation of the accessory abducens nucleus, the abducens nucleus, and the reticular formation at the level of the spinal trigeminal nucleus supported lower, transient levels of conditioning. The results are discussed in terms of stimulation of sensory inputs to the brainstem and cerebellum.

Acquisition of conditioned associations in Hermissenda: additive effects of contiguity and the forward interstimulus interval.

Conditioned suppression of photokinesis by the marine mollusc Hermissenda was examined in 3 experiments. In each experiment, groups of animals received light (the conditioned stimulus, CS) that was paired with high-speed orbital rotation (the unconditioned stimulus, UCS), light and rotation explicitly unpaired, or no exposure to these stimuli. Twenty-four hours after training, all animals were tested for suppression of photokinesis in the presence of the light. To establish the effectiveness of our conditioning procedure, in Experiment 1 individual groups of animals received either 50, 100, or 150 CS-UCS pairings. Fifty pairings resulted in a marginal suppression of photokinesis, whereas 100 and 150 pairings produced strong suppression. In Experiment 2, the delay between CS onset and UCS onset was varied between 1 and 10 s. The 10-s interstimulus interval (ISI) did not support conditioning, whereas 1-s and 2-s ISIs were effective. As predicted by the current understanding of Hermissenda's neural network, in Experiment 3 it was found that CS-UCS pairings in which the CS preceded the onset of the UCS and terminated with the offset of the UCS evoked stronger conditioned suppression than either a CS that preceded the UCS and terminated with its onset or a CS that was paired in simultaneous compound with the UCS. This result indicates that CS-UCS contiguity as well as the forward ISI act additively to establish the CS-UCS association. In none of the 3 experiments were any differences observed between groups that were untreated and that received the CS and UCS unpaired. In total, these experiments suggest strong similarities in the temporal characteristics of associative learning in Hermissenda and vertebrate species.

Conditioning-specific modification of the rabbit's unconditioned nictitating membrane response.

Robust classical conditioning modifies responding to the unconditioned stimulus (US) in the absence of the conditioned stimulus (CS), a phenomenon the researchers called conditioning-specific reflex modification. Unconditioned responses (URs) to periorbital stimulation varying in intensity and duration were assessed before and after 1, 3, or 6 days of paired, explicitly unpaired, or no presentations of tone and electrical stimulation. After 3 days of pairings, conditioned responding (CRs) reached 94%, and there was an increase in latency to the peak of URs. The peak latency increase was replicated in a second experiment where rabbits reached asymptotic conditioning during 6 days of pairings. There was also a conditioning-specific increase in the amplitude of URs. There were no UR changes as a function of low level of CRs following 1 day of pairings. Data suggest that there are learning-specific changes in pathways mediating the US/UR, as well as in those mediating the CS/CR.

Temporal patterns of the rabbit's nictitating membrane response to compound and component stimuli under mixed CS-US intervals.

The acquisition of the rabbit's nictitating membrane response to a tone and light compound and to its components was examined when compound presentations were reinforced at one conditioned stimulus-unconditioned stimulus (CS-US) interval and individual component presentations were reinforced at another CS-US interval. Examination of the time course of the CRs revealed that (a) despite the mixture of CS-US intervals, conditioned response (CR) timing remained accurate, that is, CRs reached their peaks at the alternative points of US delivery; (b) the momentary magnitude of the CR to the compound was predominantly an additive function of the CR magnitude to the individual components; but (c) there was modest evidence of differentiation between the compound as a unit and the individual components. The results are discussed in terms of their implications for the study of the neural substrates of temporal and sensory integration as they modulate CR acquisition.

Conditioning-specific reflex modification of the rabbit (Oryctolagus cuniculus) nictitating membrane response: generality and nature of the phenomenon.

Conditioning-specific reflex modification (CRM) occurs when classical conditioning modifies responding to an unconditioned stimulus in the absence of a conditioned stimulus. This form of reflex modification suggests that learning modifies the unconditioned reflex pathway. Rabbit (Oryctolagus cuniculus) nictitating membrane responses to 5 intensities and 3 durations of airpuff (AP) or periorbital electrical stimulation (ES) were monitored before and after conditioning. AP tests detected strong CRM after conditioning with ES and modest levels of CRM after conditioning with AP. After conditioning with AP, ES tests failed to detect CRM. After conditioning with a stronger AP, CRM was again detected by AP tests. CRM is a general phenomenon but is more readily detected after training with a relatively aversive stimulus; thus, it may be a function of level of arousal.

The effects of scopolamine, lorazepam, and glycopyrrolate on classical conditioning of the human eyeblink response.

Human eyeblink conditioning, a relatively simple form of learning and memory, has previously been shown to be impaired by the central and peripheral anticholinergic scopolamine. The present study compared the behavioral effects of scopolamine with the benzodiazepine lorazepam and a peripherally active anticholinergic, glycopyrrolate. Thirty-six healthy normal volunteers (mean age: 23.7 years) were studied with 12 assigned double-blind to each of three drug conditions (0.5 mg scopolamine IV, 2 mg lorazepam PO, or 0.2 mg glycopyrrolate IV). Subjects underwent classical conditioning of the eyeblink response in which the conditioned stimulus was an 80 dB binaural tone, and the unconditioned stimulus was a 2 psi airpuff to the right eye. Ten trials of unpaired stimulus presentations were followed by 60 paired trials and finally by an extinction period of five tone-alone presentations. An eyeblink response that occurred during the tone but before the airpuff was scored as a conditioned response (CR). Subjects treated with lorazepam (43% mean CRs) and scopolamine (51% mean CRs) exhibited a significantly lower asymptotic level of conditioning than those treated with glycopyrrolate (85% mean CRs; P < 0.01). However, during extinction, lorazepam-treated subjects (35% CRs) showed a lower overall level of responding to the tone than either scopolamine (60% CRs) or glycopyrrolate (62% CRs) treated subjects (P < 0.05). It seems unlikely that these differences could be accounted for by drug-induced alterations in motor responses because there were no significant differences between the three drug conditions in the frequency, latency, or amplitude of unconditioned responses to the airpuff. Overall, our data indicate that scopolamine and lorazepam impair eyeblink conditioning and suggest that some of the effects of benzodiazepines and anticholinergics on learning and memory can be differentiated using this paradigm.

Pairing-specific long-term depression prevented by blockade of PKC or intracellular Ca2+.

Long-term depression was established in cerebellar Purkinje cells using 20 pairings of a brief, high frequency train of parallel fiber stimulation with a subsequent 100 ms depolarizing current injection. Effects were assessed on the peak amplitude of Purkinje cell excitatory post synaptic potentials (EPSPs) elicited by single parallel fiber test pulses. Intradendritically recorded Purkinje cell EPSPs exhibited long-term (>20 min) reduction in peak amplitude following paired stimulation but not if pretreated with the protein kinase C inhibitor calphostin C or iontophoretically injected with the calcium chelator EGTA. The similarity of the stimulation protocol and behavioral conditioning paradigms suggests pairing-specific long-term synaptic depression is a valuable model for understanding the cellular mechanisms underlying cerebellar cortical contributions to learning.

Classical conditioning increases membrane-bound protein kinase C in rabbit cerebellum.

We examined membrane-bound protein kinase C (PKC) in the cerebellum of rabbits given paired presentations of a tone conditioned stimulus (CS) that co-terminated with a periocular electrical stimulation unconditioned stimulus (US) or unpaired presentations of the CS and US or restraint in the experimental context. PKC activation was measured by quantitative film autoradiography of [3H]phorbol 12,13-dibutyrate ([3H]PBt2) binding in the molecular and granule cells layers of lobule HVI, anterior vermis and Crus I, and in the dentate/interpositus nuclei. There was a statistically significant increase in [3H]PBt2 binding within the molecular layer of lobule HVI in rabbits given paired training relative to controls. The results indicate PKC activation in lobule HVI may be important in acquisition of conditioned eyeblink responses.

Parameters and sites of brainstem stimulation capable of eliciting the rabbit nictitating membrane response.

One hundred and thirty-five different combinations of pulse amplitude, pulse width, pulse train duration, and pulse frequency were delivered to either pars oralis of the spinal trigeminal nucleus, the accessory abducens nucleus, the abducens nucleus, or the reticular formation at the level of the spinal trigeminal nucleus. The percentage of occurrence, amplitude, and latency of the rabbit nictitating membrane response were shown to be lawfully related to the parameters of stimulation. Moreover, the electrode sites were ordered from reticular formation to abducens to accessory abducens to pars oralis in terms of the increasing efficacy with which stimulation elicited the nictitating membrane response.

Rabbit cerebellar slice analysis of long-term depression and its role in classical conditioning.

Cerebellar long-term depression (LTD) has been proposed as a mechanism underlying classical conditioning of the rabbit nictitating membrane/eyelid response (NMR). However, LTD has only been obtained reliably when (1) cerebellar slices are bathed in GABA antagonists which abolish disynaptic inhibitory post synaptic potentials, and (2) the temporal sequence of stimulation used in slice or intact preparations is the opposite of that used in classical conditioning. Based on intradendritic Purkinje cell recordings obtained from rabbit cerebellar slices, we report that stimulation of climbing fibers and then parallel fibers in the presence of the GABA antagonist, bicuculline, produced significant depression of parallel fiber excitatory post synaptic potential (epsp) amplitude that continued to increase for at least 20 min after stimulation. However, application of the same stimulation protocol without GABA antagonists produced a brief depression of parallel fiber epsps that disappeared within minutes. Activation of parallel fibers and then climbing fibers in an order opposite to the LTD-producing sequence (i.e. a classical conditioning-like order) produced a brief depression that dissipated quickly. Stimulation of parallel fibers alone produced a small, slowly developing potentiation, but stimulation of parallel fibers during depolarization-induced local dendritic calcium spikes produced significant depression almost immediately which then declined slowly to more modest levels. Finally, stimulation of parallel fibers at frequencies used in in vivo parallel fiber-climbing fiber stimulation experiments (e.g. 100 Hz) produced an immediate and profound long-lasting epsp depression. The depression occurred, however, whether parallel and climbing fibers were stimulated separately (unpaired) or in a classical conditioning-like protocol (paired) where parallel fiber stimulation coterminated with climbing fiber stimulation (10 Hz).(ABSTRACT TRUNCATED AT 250 WORDS)

High-resolution fluorescent labeling of living cerebellar slices.

In the present study, we extend previous research on staining of living brain slices with fluorescent phospholipids. This new procedure allows high-resolution staining of specific cell types, in particular, Purkinje cells, in the cerebellar slice while not affecting the intrinsic electrical activity of the tissue. Four different nitrobenzoxadiole (NBD)-phospholipids were incorporated into living cerebellar slices via loading from small unilamellar vesicles (SUVs), composed of a carrier and the fluorescent lipid. The labeled acidic phospholipid, NBD-phosphatidic acid (NBD-PA), produced the highest resolution images with exquisite labeling of the dendritic fields. The label was incorporated predominantly into the Purkinje cell body (excluding the nucleus), with more diffuse staining in other cell types, including stellate, basket and granule cells. The labeled lipid concentration and composition of the carrier lipid were significant in determining the specificity of labeling. Labeling, which was optimal after a 1 h incubation, was present throughout the depth of the slice. This procedure provides a promising approach to fluorescent labeling that will allow simultaneous monitoring of changes in cellular morphology and electrophysiology of living brain slices.

Learning-specific differences in Purkinje-cell dendrites of lobule HVI (Lobulus simplex): intracellular recording in a rabbit cerebellar slice.

The cerebellum has been directly implicated in the associative learning exemplified by classical conditioning of the rabbit nictitating membrane response. Lesions of cerebellar lobule HVI (Lobulus simplex) can disrupt or even abolish classical conditioning. Electrical stimulation of inputs that converge at cerebellar lobule HVI and the deep cerebellar nuclei has been shown to support classical conditioning of the responses elicited by such stimulation. Here we report that intracellular recording in a slice of rabbit cerebellar lobule HVI revealed a conditioning-specific increase in the excitability of Purkinje-cell dendrites without significant changes in dendritic membrane potential or input resistance. The finding of a conditioning-specific change in lobule HVI lends support to the theory that associative learning involves local dendritic modification and provides further evidence for the involvement of the cerebellar cortex in classical conditioning of the rabbit's nictitating membrane response.

GABA-induced responses in Purkinje cell dendrites of the rabbit cerebellar slice.

Pressure applications of GABA localized to Purkinje cell somas in a rabbit cerebellar slice produced uniphasic hyperpolarizing responses, whereas applications of GABA that were directed at the Purkinje cell dendrites produced complex, triphasic responses with hyperpolarizing and depolarizing components. Both somatic and dendritic application of GABA elicited fast hyperpolarization (GABAhf), but dendritic application also elicited a slower depolarization (GABAd) and a later, long-lasting hyperpolarization (GABAhl). All three types of responses were accompanied by increased conductance. Use of either GABA antagonist, bicuculline or picrotoxin, eliminated the GABAhf and GABAd responses but left the GABAhl response intact. Pressure delivery of the GABA agonist, baclofen, to the dendrites but not the soma elicited a GABAhl response. Application of baclofen paired with membrane depolarization sufficient to elicit local, calcium-dependent dendritic spiking produced a persistent reduction in the GABAhl response, whereas alternating presentations of baclofen and membrane depolarization or presentations of baclofen alone could not. The fact that GABA and baclofen inhibited Purkinje cell activity in the rabbit cerebellar slice and that picrotoxin and bicuculline eliminated some, but not all of the components of the GABA response suggests the presence of both GABAA and GABAB receptors. The ability of baclofen to inhibit Purkinje cells if it was applied to the dendrites but not if applied to the soma suggests that GABAB receptors are located predominantly on Purkinje cell dendrites. The pairing-specific change in the baclofen response suggests the existence of GABAB-mediated modifiability of Purkinje cell dendrites.(ABSTRACT TRUNCATED AT 250 WORDS)

Conditioning the unconditioned response: modification of the rabbit's (Oryctolagus cuniculus) unconditioned nictitating membrane response.

Conditioning-specific reflex modification (CRM) occurs when classical conditioning modifies responding to an unconditioned stimulus (US) in the absence of a conditioned stimulus (CS). Three experiments monitored rabbit nictitating (Oryctolagus cuniculus) membrane unconditioned responses to 5 intensities and 4 durations of periorbital electrical stimulation before and after CS or US manipulation. CRM occurred after 12 days of CS-US pairings but not following unpaired CS/US presentations or restraint. CRM survived CS-alone and CS/US-unpaired extinction of the conditioned response (CR) but not presentations of the US alone, although CRs remained intact. Thus, CRs could be weakened without eliminating CRM and CRM could be weakened without eliminating CRs. Data indicate CRM is a reliable, associative effect that is more than a generalized CR and may not be explained by habituation, stimulus generalization, contextual conditioning, or bidirectional conditioning.

Dietary cholesterol increases ventricular volume and narrows cerebrovascular diameter in a rabbit model of Alzheimer's disease.

Using structural magnetic resonance imaging in a clinical scanner at 3.0T, we describe results showing that following 12weeks on a diet of 2% cholesterol, rabbits experience a significant increase in the volume of the third ventricle compared to rabbits on a diet of 0% cholesterol. Using time-of-flight magnetic resonance angiography, we find cholesterol-fed rabbits also experience a decrease in the diameter of a number of cerebral blood vessels including the basilar, posterior communicating, and internal carotid arteries. Taken together, these data confirm that, despite the inability of dietary cholesterol to cross the blood-brain barrier, it does significantly enlarge ventricular volume and decrease cerebrovascular diameter in the rabbit - effects that are also seen in patients with Alzheimer's disease.