Raju Metherate

Triple-Transgenic Model of Alzheimer's Disease with Plaques and Tangles: Intracellular Aβ and Synaptic Dysfunction

The neuropathological correlates of Alzheimers disease (AD) include amyloid-beta (Abeta) plaques and neurofibrillary tangles. To study the interaction between Abeta and tau and their effect on synaptic function, we derived a triple-transgenic model (3xTg-AD) harboring PS1(M146V), APP(Swe), and tau(P301L) transgenes. Rather than crossing independent lines, we microinjected two transgenes into single-cell embryos from homozygous PS1(M146V) knockin mice, generating mice with the same genetic background. 3xTg-AD mice progressively develop plaques and tangles. Synaptic dysfunction, including LTP deficits, manifests in an age-related manner, but before plaque and tangle pathology. Deficits in long-term synaptic plasticity correlate with the accumulation of intraneuronal Abeta. These studies suggest a novel pathogenic role for intraneuronal Abeta with regards to synaptic plasticity. The recapitulation of salient features of AD in these mice clarifies the relationships between Abeta, synaptic dysfunction, and tangles and provides a valuable model for evaluating potential AD therapeutics as the impact on both lesions can be assessed.

A Role for Synaptic Zinc in Activity-Dependent Aβ Oligomer Formation and Accumulation at Excitatory Synapses

Soluble amyloid β oligomers (AβOs) interfere with synaptic function and bind with high affinity to synapses, but the mechanism underlying AβO synaptic targeting is not known. Here, we show that the accumulation of synthetic or native Alzheimers disease (AD)-brain oligomers at synapses is regulated by synaptic activity. Electrical or chemical stimulation increased AβO synaptic localization and enhanced oligomer formation at synaptic terminals, whereas inhibition with TTX blocked AβO synaptic localization and reduced AβO synaptic load. The zinc-binding 8-OH-quinoline clioquinol markedly reduced AβO synaptic targeting, which was also reduced in brain sections of animals deficient in the synaptic vesicle zinc transporter ZnT3, indicating that vesicular zinc released during neurotransmission is critical for AβO synaptic targeting. Oligomers were not internalized in recycled vesicles but remained at the cell surface, where they colocalized with NR2B NMDA receptor subunits. Furthermore, NMDA antagonists blocked AβO synaptic targeting, implicating excitatory receptor activity in oligomer formation and accumulation at synapses. In AD brains, oligomers of different size colocalized with synaptic markers in hippocampus and cortex, where oligomer synaptic accumulation correlated with synaptic loss.

Differential modulation of auditory thalamocortical and intracortical synaptic transmission by cholinergic agonist.

To investigate synaptic mechanisms underlying information processing in auditory cortex, we examined cholinergic modulation of synaptic transmission in a novel slice preparation containing thalamocortical and intracortical inputs to mouse auditory cortex. Extracellular and intracellular recordings were made in cortical layer IV while alternately stimulating thalamocortical afferents (via medial geniculate or downstream subcortical stimulation) and intracortical afferents. Either subcortical or intracortical stimulation elicited a fast, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive, monosynaptic EPSP followed by long-duration, polysynaptic activity. The cholinergic agonist carbachol suppressed each of the synaptic potentials to different degrees. At low concentrations (5 microM) carbachol strongly reduced (>60%) the polysynaptic slow potentials for both pathways but did not affect the monosynaptic fast potentials. At higher doses (10-50 microM), carbachol also reduced the fast potentials, but reduced the intracortically-elicited fast potential significantly more than the thalamocortically-elicited fast potential, which at times was actually enhanced. Atropine (0.5 microM) blocked the effects of carbachol, indicating muscarinic receptor involvement. We conclude that muscarinic modulation can strongly suppress intracortical synaptic activity while exerting less suppression, or actually enhancing, thalamocortical inputs. Such differential actions imply that auditory information processing may favor sensory information relayed through the thalamus over ongoing cortical activity during periods of increased acetylcholine (ACh) release.

Thalamocortical inputs trigger a propagating envelope of gamma-band activity in auditory cortex in vitro.

Abstract To investigate how auditory cortex responds to thalamic inputs, we have used electrophysiological and anatomical techniques to characterize a brain slice containing functionally linked thalamocortical and intracortical pathways. In extracellular recordings, stimulation of thalamic afferents elicited a short-latency field potential and current sink in layer IV of the cortex, followed by 100–500 ms of polysynaptic activity containing rapid (gamma-band, 20–80 Hz) fluctuations. Paired intracellular and extracellular recordings showed that a short-latency excitatory postsynaptic potential (EPSP) corresponded to the fast extracellular potential, and that a slow intracellular depolarization with superimposed rapid fluctuations corresponded to the polysynaptic extracellular activity. Pharmacological manipulations demonstrated that glutamate receptors contributed to mono- and polysynaptic activity, and that the gamma-band fluctuations contained intermixed rapid depolarizations and Cl–-mediated inhibition. The spread of evoked activity through auditory cortex was determined by extracellular mapping away from the excitatory focus (the site of the largest amplitude fast response). The short-latency potential traversed auditory cortex at 1.25 m/s and decreased over 1–2 mm, likely reflecting sequential activation of cells contacted by thalamocortical arbors. In contrast, polysynaptic activity did not decrease but propagated as a spatially restricted wave at a 57-fold slower velocity (0.022 m/s). Thus, stimulation of the auditory thalamocortical pathway in vitro elicited a fast glutamatergic potential in layer IV, followed by polysynaptic activity, including gamma-band fluctuations, that propagated through the cortex. Propagating activity may form transient neural assemblies that contribute to auditory information processing.

Nicotinic control of axon excitability regulates thalamocortical transmission

The thalamocortical pathway, a bundle of myelinated axons that arises from thalamic relay neurons, carries sensory information to the neocortex. Because axon excitation is an obligatory step in the relay of information from the thalamus to the cortex, it represents a potential point of control. We now show that, in adult mice, the activation of nicotinic acetylcholine receptors (nAChRs) in the initial portion of the auditory thalamocortical pathway modulates thalamocortical transmission of information by regulating axon excitability. Exogenous nicotine enhanced the probability and synchrony of evoked action potential discharges along thalamocortical axons in vitro, but had little effect on synaptic release mechanisms. In vivo, the blockade of nAChRs in the thalamocortical pathway reduced sound-evoked cortical responses, especially those evoked by sounds near the acoustic threshold. These data indicate that endogenous acetylcholine activates nAChRs in the thalamocortical pathway to lower the threshold for thalamocortical transmission and to increase the magnitude of sensory-evoked cortical responses. Our results show that a neurotransmitter can modulate sensory processing by regulating conduction along myelinated thalamocortical axons.

Neonatal nicotine exposure impairs nicotinic enhancement of central auditory processing and auditory learning in adult rats

Children of women who smoke cigarettes during pregnancy display cognitive deficits in the auditory–verbal domain. Clinical studies have implicated developmental exposure to nicotine, the main psychoactive ingredient of tobacco, as a probable cause of subsequent auditory deficits. To test for a causal link, we have developed an animal model to determine how neonatal nicotine exposure affects adult auditory function. In adult control rats, nicotine administered systemically (0.7 mg/kg, s.c.) enhanced the sensitivity to sound of neural responses recorded in primary auditory cortex. The effect was strongest in cortical layers 3 and 4, where there is a dense concentration of nicotinic acetylcholine receptors (nAChRs) that has been hypothesized to regulate thalamocortical inputs. In support of the hypothesis, microinjection into layer 4 of the nonspecific nAChR antagonist mecamylamine (10 µm) strongly reduced sound‐evoked responses. In contrast to the effects of acute nicotine and mecamylamine in adult control animals, neither drug was as effective in adult animals that had been treated with 5 days of chronic nicotine exposure (CNE) shortly after birth. Neonatal CNE also impaired performance on an auditory‐cued active avoidance task, while having little effect on basic auditory or motor functions. Thus, neonatal CNE impairs nicotinic regulation of cortical function, and auditory learning, in the adult. Our results provide evidence that developmental nicotine exposure is responsible for auditory–cognitive deficits in the offspring of women who smoke during pregnancy, and suggest a potential underlying mechanism, namely diminished function of cortical nAChRs.

Intrinsic electrophysiology of neurons in thalamorecipient layers of developing rat auditory cortex

During early postnatal life, several critical events contribute to the functional development of rat sensory neocortex. Thalamocortical innervation of sensory cortex is completed during the first postnatal week and extrathalamic innervation develops over the first several weeks. In auditory cortex, acoustic-evoked potentials first occur in week 2 and develop most rapidly over weeks 2-3. Thus, rapid functional maturation of cortical circuits in sensory cortex occurs during the second and third postnatal weeks. The electrophysiological properties of cortical neurons that receive afferent inputs during this time may play an important role in development and function. In this study we examined the intrinsic electrophysiology, including spiking patterns, of neurons in layers II/III and IV of auditory cortex during postnatal weeks 2 and 3. Many neurons displayed characteristics consistent with previous descriptions of response classes (regular spiking, fast spiking, intrinsic bursting). In addition, we identified two groups, Rectifying and On-spiking neurons, that were characterized by (i) brief spike trains in response to maintained intracellular depolarizations, and (ii) striking outward rectification upon depolarization. Unusually brief spike trains (1-2 spikes) and short spike latencies (<10 ms) further distinguished On-spiking from Rectifying cells. Biocytin labeling demonstrated that On-spiking and Rectifying cells could be either pyramidal or nonpyramidal neurons. The intrinsic physiology of these cell groups may play an important role in auditory cortex function.

Synaptic Potentials and Effects of Amino Acid Antagonists in the Auditory Cortex

Neurons of in vitro guinea pig and rat auditory cortex receive a complex synaptic pattern of afferent information. As many as four synaptic responses to a single-stimulus pulse to the gray or white matter can occur; an early-EPSP followed, sequentially, by an early-IPSP, late-EPSP, and late-IPSP. Paired pulse stimulation and pharmacological studies show that the early-IPSP can modify information transmission that occurs by way of the early-EPSP. Each of these four synaptic responses differed in estimated reversal potential, and each was differentially sensitive to antagonism by pharmacological agents. DNQX (6,7-dinitroquinoxaline-2,3-dione), a quisqualate/kainate receptor antagonist, blocked the early-EPSP, and the late-EPSP was blocked by the NMDA receptor antagonist APV (D-2-amino-5-phosphonovalerate). The early-IPSP was blocked by the GABA-a receptor antagonist bicuculline, and the late-IPSP by the GABA-b receptor antagonists 2-OH saclofen or phaclofen. Presentation of stimulus trains, even at relatively low intensities, could produce a long-lasting APV-sensitive membrane depolarization. Also discussed is the possible role of these synaptic potentials in auditory cortical function and plasticity.

Nicotine exposure during a postnatal critical period alters NR2A and NR2B mRNA expression in rat auditory forebrain

Chronic nicotine exposure (CNE) can alter brain development and is thought to produce deficits in auditory function. Previously, we found that CNE during the second postnatal week, but not before or after, increases the duration of excitatory postsynaptic potentials (EPSPs) mediated by N-methyl-D-aspartate receptors (NMDARs) in rat auditory cortex. It was proposed that a potential mechanism underlying increased EPSP duration could be over-stimulation of presynaptic nicotinic acetylcholine receptors, leading to prolonged glutamate release. Since glutamatergic activity regulates levels of postsynaptic NMDAR subunits, here we examine the effects of CNE on mRNA expression for the NR2A and NR2B subunits in auditory cortex and thalamus. Two days of CNE (postnatal days 8-9), produced no effects, but 5 days (postnatal days 8-12) enhanced cortical NR2A mRNA levels and reduced thalamic NR2B mRNA levels for up to 2 weeks. These effects are consistent with the hypothesis that CNE during a postnatal critical period disrupts auditory cortex development by over-stimulating glutamatergic synapses.

Regulation of glutamate synapses by nicotinic acetylcholine receptors in auditory cortex

Acetylcholine plays an important role in regulating the processing of sensory stimuli, and understanding its specific cellular actions is critical to understanding how sensory cortex develops and functions in different behavioral states. Here we review recent work on the cellular effects of nicotinic receptor activation in auditory cortex and describe how these actions could affect systems-level auditory function. In particular, we describe a novel function of nicotinic acetylcholine receptors to regulate glutamate synapses containing N-methyl-D-aspartate receptors during early postnatal development. The transient regulation of developing glutamate synapses also defines a window of vulnerability during which exposure to exogenous nicotine disrupts synapse development. Thus, it appears that nicotinic regulation of glutamate synapses is a critical feature of auditory cortex development.