Christopher J. Davis

Effects of extracellular matrix-degrading proteases matrix metalloproteinases 3 and 9 on spatial learning and synaptic plasticity

Rats learning the Morris water maze exhibit hippocampal changes in synaptic morphology and physiology that manifest as altered synaptic efficacy. Learning requires structural changes in the synapse, and multiple cell adhesion molecules appear to participate. The activity of these cell adhesion molecules is, in large part, dependent on their interaction with the extracellular matrix (ECM). Given that matrix metalloproteinases (MMPs) are responsible for transient alterations in the ECM, we predicted that MMP function is critical for hippocampal‐dependent learning. In support of this, it was observed that hippocampal MMP‐3 and ‐9 increased transiently during water maze acquisition as assessed by western blotting and mRNA analysis. The ability of the NMDA receptor channel blocker MK801 to attenuate these changes indicated that the transient MMP changes were in large part dependent upon NMDA receptor activation. Furthermore, inhibition of MMP activity with MMP‐3 and ‐9 antisense oligonucleotides and/or MMP inhibitor FN‐439 altered long‐term potentiation and prevented acquisition in the Morris water maze. The learning‐dependent MMP alterations were shown to modify the stability of the actin‐binding protein cortactin, which plays an essential role in regulating the dendritic cytoskeleton and synaptic efficiency. Together these results indicate that changes in MMP function are critical to synaptic plasticity and hippocampal‐dependent learning.

REM sleep deprivation-induced deficits in the latency-to-peak induction and maintenance of long-term potentiation within the CA1 region of the hippocampus

Sleep loss adversely affects certain types of cognitive processing, particularly associative memory. Given that long-term potentiation (LTP) represents a putative cellular basis of learning and memory consolidation, the influence of sleep deprivation on LTP was examined. Rats were REM sleep deprived for 24, 48, or 72 h using the inverted flowerpot method in temperature-regulated chambers. Hippocampal slices taken from sleep-deprived rats were compared with home cage and pedestal control animals at 5, 15 and 60 min post-tetanization. The results indicated that at 5 min post-tetanization there were no differences in field potentials in any of the sleep-deprived or control groups, suggesting comparable levels of induction. However, analysis of latency-to-peak slope indicated that members of the 48 and 72 h sleep-deprived groups required approximately twice as long to achieve maximum slope as the 24 h group, home cage or 24, 48, 72 h pedestal controls (means 8.17, 7.50, 2.67, 4.67 and 3.17 min, respectively). At 15 min post-tetanization there were no group differences, however at 60 min post-tetanization the slopes of the field excitatory postsynaptic potentials were significantly diminished for the 24, 48 and 72 h sleep-deprived groups (means 30.44, -1.89, 1.47, respectively) as compared with home cage and pedestal controls (means 59.54, 58.42, respectively). This delay in maximal induction, and the degradation of the maintenance phase of LTP, may represent sleep deprivation-induced impairment of the underlying neurochemical mechanisms normally responsible for memory acquisition.

Effects of matrix metalloproteinase inhibition on short- and long-term plasticity of schaffer collateral/CA1 synapses

It is increasingly evident that matrix metalloproteinases (MMPs), a family of zinc containing extracellular endopeptidases, participate in processes supporting hippocampal synaptic plasticity. The purpose of this study was to further the understanding of MMPs involvement in hippocampal plasticity. Acute hippocampal slices, generated from 20‐ to 30‐day‐old male Sprague–Dawley rats, were subjected to various electrophysiologic stimulatory paradigms to produce either short‐term or long‐term modifications to synaptic efficacy. Slices exposed to broad‐spectrum MMP inhibitor, FN‐439, exhibited impairments in paired‐pulse facilitation, theta‐burst facilitation, and long‐term depression. Additionally, we observed that MMP inhibition impaired both the induction and stability of long‐term potentiation (LTP). Furthermore, evidence indicated that the effect of MMP inhibition on LTP maintenance is dependent upon integrin‐directed adhesion, whereas the effects of MMP inhibition on LTP induction are independent of integrin‐directed adhesion. Together, these data support a generalized role for MMPs in short‐term and long‐term hippocampal plasticity and indicate that MMPs are a necessary facet of integrin‐mediated cell adhesion supporting LTP stabilization.

Biochemical Regulation of Sleep and Sleep Biomarkers

Symptoms commonly associated with sleep loss and chronic inflammation include sleepiness, fatigue, poor cognition, enhanced sensitivity to pain and kindling stimuli, excess sleep and increases in circulating levels of tumor necrosis factor α (TNF) in humans and brain levels of interleukin-1 β (IL1) and TNF in animals. Cytokines including IL1 and TNF partake in non-rapid eye movement sleep (NREMS) regulation under physiological and inflammatory conditions. Administration of exogenous IL1 or TNF mimics the accumulation of these cytokines occurring during sleep loss to the extent that it induces the aforementioned symptoms. Extracellular ATP associated with neuro- and glio-transmission, acting via purine type 2 receptors, e.g., the P2X7 receptor, has a role in glia release of IL1 and TNF. These substances in turn act on neurons to change their intrinsic membrane properties and sensitivities to neurotransmitters and neuromodulators such as adenosine, glutamate and GABA. These actions change the network input-output properties, i.e., a state shift for the network. State oscillations occur locally within cortical columns and are defined using evoked response potentials. One such state, so defined, shares properties with whole animal sleep in that it is dependent on prior cellular activity--it shows homeostasis. The cortical column sleep-like state is induced by TNF and is associated with experimental performance detriments. ATP released extracellularly as a consequence of cellular activity is posited to initiate a mechanism by which the brain tracks its prior sleep-state history to induce/prohibit sleep. Thus, sleep is an emergent property of populations of local neural networks undergoing state transitions. Specific neuronal groups participating in sleep depend upon prior network use driving local network state changes via the ATP-cytokine-adenosine mechanism. Such considerations add complexity to finding biochemical markers for sleepiness.

Involvement of cytokines in slow wave sleep

Cytokines such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL1β) play a role in sleep regulation in health and disease. TNFα or IL1β injection enhances non-rapid eye movement sleep. Inhibition of TNFα or IL1β reduces spontaneous sleep. Mice lacking TNFα or IL1β receptors sleep less. In normal humans and in multiple disease states, plasma levels of TNFα covary with EEG slow wave activity (SWA) and sleep propensity. Many of the symptoms induced by sleep loss, for example, sleepiness, fatigue, poor cognition, enhanced sensitivity to pain, are elicited by injection of exogenous TNFα or IL1β. IL1β or TNFα applied unilaterally to the surface of the cortex induces state-dependent enhancement of EEG SWA ipsilaterally, suggesting greater regional sleep intensity. Interventions such as unilateral somatosensory stimulation enhance localized sleep EEG SWA, blood flow, and somatosensory cortical expression of IL1β and TNFα. State oscillations occur within cortical columns. One such state shares properties with whole animal sleep in that it is dependent on prior cellular activity, shows homeostasis, and is induced by TNFα. Extracellular ATP released during neuro- and gliotransmission enhances cytokine release via purine type 2 receptors. An ATP agonist enhances sleep, while ATP antagonists inhibit sleep. Mice lacking the P2X7 receptor have attenuated sleep rebound responses after sleep loss. TNFα and IL1β alter neuron sensitivity by changing neuromodulator/neurotransmitter receptor expression, allowing the neuron to scale its activity to the presynaptic neurons. TNFαs role in synaptic scaling is well characterized. Because the sensitivity of the postsynaptic neuron is changed, the same input will result in a different network output signal and this is a state change. The top-down paradigm of sleep regulation requires intentional action from sleep/wake regulatory brain circuits to initiate whole-organism sleep. This raises unresolved questions as to how such purposeful action might itself be initiated. In the new paradigm, sleep is initiated within networks and local sleep is a direct consequence of prior local cell activity. Whole-organism sleep is a bottom-up, self-organizing, and emergent property of the collective states of networks throughout the brain.

Sleep loss changes microRNA levels in the brain: a possible mechanism for state-dependent translational regulation.

MicroRNAs (miRNAs) are small ( approximately 22 nucleotides) non-coding RNA strands that base pair with mRNA to degrade it or inhibit its translation. Because sleep and sleep loss induce changes in many mRNA species, we hypothesized that sleep loss would also affect miRNA levels in the brain. Rats were sleep-deprived for 8h then decapitated; hippocampus, prefrontal and somatosensory cortices and hypothalamus tissues were harvested and frozen in liquid nitrogen. miRNA was extracted and then characterized using microarrays. Several let-7 miRNA microarray results using hippocampus and prefrontal cortex samples were verified by PCR. From the array data it was determined that about 50 miRNA species were affected by sleep loss. For example, in the hippocampus of sleep-deprived rats, miRNA expression increased compared to cage control samples. In contrast, the majority of miRNA species in the somatosensory and prefrontal cortices decreased, while in the hypothalamus miRNA species were both up- and down-regulated after sleep deprivation. The number of miRNA species affected by sleep loss, their differential expression in separate brain structures and their predicted targets suggest that they have a role in site-specific sleep mechanisms. Current results are, to our knowledge, the first demonstration of the homeostatic process, sleep, altering brain miRNA levels.

Influence of hippocampectomy on habituation, exploratory behavior, and spatial memory in rats.

Two frequently cited functions of the hippocampus are mediation of spatial memories and habituation. The present investigation employed head-shake response (HSR) as the habituated behavior in intact and bilaterally hippocampectomized rats. This HSR appears to be minimally influenced by spatial cues. These rats were further tested on two behavioral paradigms that make use of spatial cues, namely open field object exploration, and the Morris water maze. The results indicate that hippocampectomized rats revealed habituation of the HSR, but not to objects within the open field. In agreement with previous reports, hippocampectomized rats were severely impaired both in acquiring and recalling the location of the submerged platform in the Morris water maze task. In a separate experiment independent groups of rats were trained on one of these three paradigms, and tissues were collected from hippocampal, prefrontal, and piriform cortices for the measurement of matrix metalloproteinases (MMPs) as markers of neural plasticity. There were significant MMP-9 elevations in the prefrontal and piriform cortices of rats tested using the object exploration task, in the prefrontal and hippocampal cortices of rats that solved the Morris water maze task, but minimal MMP changes in any tissues taken from HSR habituated rats. These results question the hypothesis that habituation is solely mediated by the hippocampus in favor of a process that utilizes different brain structures and degrees of neural plasticity dependent upon task requirements.

Delta wave power: an independent sleep phenotype or epiphenomenon?

Electroencephalographic (EEG) δ waves during non-rapid eye movement sleep (NREMS) after sleep deprivation are enhanced. That observation eventually led to the use of EEG δ power as a parameter to model process S in the two-process model of sleep. It works remarkably well as a model parameter because it often co-varies with sleep duration and intensity. Nevertheless there is a large volume of literature indicating that EEG δ power is regulated independently of sleep duration. For example, high amplitude EEG δ waves occur in wakefulness after systemic atropine administration or after hyperventilation in children. Human neonates have periods of sleep with an almost flat EEG. Similarly, elderly people have reduced EEG δ power, yet retain substantial NREMS. Rats provided with a cafeteria diet have excess duration of NREMS but simultaneously decreased EEG δ power for days. Mice challenged with influenza virus have excessive EEG δ power and NREMS. In contrast, if mice lacking TNF receptors are infected, they still sleep more but have reduced EEG δ power. Sleep regulatory substances, e.g., IL1, TNF, and GHRH, directly injected unilaterally onto the cortex induce state-dependent ipsilateral enhancement of EEG δ power without changing duration of organism sleep. IL1 given systemically enhances duration of NREMS but reduces EEG δ power in mice. Benzodiazepines enhance NREMS but inhibit EEG δ power. If duration of NREMS is an indicator of prior sleepiness then simultaneous EEG δ power may or may not be a useful index of sleepiness. Finally, most sleep regulatory substances are cerebral vasodilators and blood flow affects EEG δ power. In conclusion, it seems unlikely that a single EEG measure will be reliable as a marker of sleepiness for all conditions.

AT4 receptor activation increases intracellular calcium influx and induces a non-N-methyl-d-aspartate dependent form of long-term potentiation

The angiotensin 4 receptor (AT4) subtype is heavily distributed in the dentate gyrus and CA1-CA3 subfields of the hippocampus. Neuronal pathways connecting these subfields are believed to be activated during learning and memory processing. ur laboratory previously demonstrated that application of the AT4 agonist, Norleucine1-angiotensin IV, enhanced baseline synaptic transmission and long-term potentiation, whereas perfusion with the AT4 antagonist, Norleucine1-Leu3-psi(CH2-NH2)3-4-angiotensin IV disrupted long-term potentiation stabilization in area CA1. The objective of the present study was to identify the mechanism(s) responsible for Norleucine1-angiotensin IV-induced increase in hippocampal long-term potentiation. Hippocampal slices perfused with Norleucine1-angiotensin IV for 20 min revealed a notable increase in baseline responses in a non-reversible manner and were blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt. Infusions of Norleucine1-angiotensin IV prior to, but not after theta burst stimulation, significantly enhanced long-term potentiation compared with control slices. Further, N-methyl-D-aspartate receptor-independent long-term potentiation could be induced by tetanization during the perfusion of Norleucine1-angiotensin IV in the presence of the N-methyl-D-aspartate antagonist, D,L-2-amino-5-phosphonovaleric acid. Blockade of select voltage dependent calcium channels significantly reduced Norleucine1-angiotensin IV-induced increase in baseline responses and subsequent long-term potentiation suggesting that AT4 receptor activation increases intracellular calcium levels via altering voltage dependent calcium channels and triggers an N-methyl-D-aspartate-independent form of long-term potentiation. In support of this notion the application of Nle1-angiotensin IV to cultured rat hippocampal neurons resulted in increased intracellular calcium derived exclusively from extracellular sources. Consistent with these observations Nle1-angiotensin IV was capable of augmenting the uptake of 45Ca2+ into rat hippocampal slices. Taken together, these data indicate that increased calcium influx through postsynaptic calcium channels contribute to Norleucine1-angiotensin IV-induced enhancement of long-term potentiation.

TNFα siRNA reduces brain TNF and EEG delta wave activity in rats

Abstract Tumor necrosis factor alpha (TNFα) is a pleiotropic cytokine with several CNS physiological and pathophysiological actions including sleep, memory, thermal and appetite regulation. Short interfering RNAs (siRNA) targeting TNFα were incubated with cortical cell cultures and microinjected into the primary somatosensory cortex (SSctx) of rats. The TNFα siRNA treatment specifically reduced TNFα mRNA by 45% in vitro without affecting interleukin-6 or gluR1–4 mRNA levels. In vivo the TNFα siRNAα reduced TNFα mRNA, interleukin-6 mRNA and gluR1 mRNA levels compared to treatment with a scrambled control siRNA. After in vivo microinjection, the density of TNFα-immunoreactive cells in layer V of the SSctx was also reduced. Electroencephalogram (EEG) delta wave power was decreased on days 2 and 3 on the side of the brain that received the TNFα siRNA microinjection relative to the side receiving the control siRNA. These findings support the hypothesis that TNFα siRNA attenuates TNFα mRNA and TNFα protein in the rat cortex and that those reductions reduce cortical EEG delta power. Results also are consistent with the notion that TNFα is involved in CNS physiology including sleep regulation.