David Murchison

Calcium buffering systems and calcium signaling in aged rat basal forebrain neurons

Disturbances of neuronal Ca2+ homeostasis are considered to be important determinants of age‐related cognitive impairment. Cholinergic neurons of the basal forebrain (BF) are principal targets of decline associated with aging and dementia. During the last several years, we have attempted to link these concepts in a rat model of ‘normal’ aging. In this review, we will describe some changes that we have observed in Ca2+ signaling of aged BF neurons and the reversal of one of these changes by dietary caloric restriction. Our evidence supports a scenario in which subtle changes in the properties of voltage‐gated Ca2+ channels result in increased Ca2+ influx during aging. This increased Ca2+, in turn, triggers an increase in rapid Ca2+ buffering in the somatic compartment of aged BF neurons. However, this nominal ‘compensation’, along with other changes in Ca2+ handling machinery (notably mitochondria) alters the Ca2+ signal with age in a way that is dependent on the magnitude of the Ca2+ load. By combining whole‐cell patch clamp electrophysiology, ratiometric Ca2+‐sensitive microfluorimetry and single‐cell reverse transcription‐polymerase chain reaction, we have determined that age‐related rapid buffering changes are present in identified cholinergic BF neurons and that these changes can be prevented by a caloric restriction dietary regimen. Because caloric restriction extends lifespan and retards the progression of age‐related dysfunction, these findings suggest that increased Ca2+ buffering in cholinergic neurons may be relevant to cognitive decline during normal aging. Importantly, calcium homeostatic mechanisms of BF cholinergic neurons are amenable to dietary interventions that could promote cognitive health during aging.

Mitochondria buffer non-toxic calcium loads and release calcium through the mitochondrial permeability transition pore and sodium/calcium exchanger in rat basal forebrain neurons.

Mitochondria participate in intracellular Ca2+ buffering and signalling. They are also major mediators of cell death. Toxic Ca2+ accumulation in mitochondria is widely believed to initiate cell death in many cell types by opening the permeability transition pore (PTP). In non-neuronal cells, the PTP has been implicated as a Ca2+ release mechanism in physiological Ca2+ signalling. In neurons, Ca2+ release from mitochondria has been attributed primarily to mitochondrial Na+/Ca2+ exchange. Using fura-2 ratiometric microfluorimetry in acutely dissociated rat basal forebrain neurons, we show that mitochondria are able to buffer non-toxic Ca2+ loads arising from caffeine-sensitive internal stores or from extracellular influx through voltage gated channels. We also show that these non-toxic Ca2+ loads are reversibly released from mitochondria through the PTP and the Na+/Ca2+ exchanger. Evoked Ca2+ transients have characteristic peak and shoulder features mediated by mitochondrial buffering and release. Depolarizing mitochondria with carbonyl cyanide m-chlorophenylhydrazone (CCCP, 5 microM) causes release of mitochondrial Ca2+ and prevents Ca2+ uptake. In CCCP, the magnitudes of evoked Ca2+ transients are increased, and the peak and shoulder features are eliminated. The PTP antagonist, cyclosporin A, (CSA, 2 microM) and the Na+/Ca2+ exchange blocker, clonazepam, (CLO, 20 microM) reversibly inhibited both the shoulder features of evoked Ca2+ transients and Ca2+ transients associated with CCCP application. We suggest that central neuronal mitochondria buffer and release Ca2+ through the PTP and Na+/Ca2+ exchanger during physiological Ca2+ signalling. We also suggest that CLO blocks both the PTP and the mitochondrial Na+/Ca2+ exchanger.

Modification of ion channels and calcium homeostasis of basal forebrain neurons during aging

In this paper we review the last several years of work from our lab with attention to changes in the properties of basal forebrain neurons during aging. These neurons play a central role in behavioral functions, such as: attention, arousal, cognition and autonomic activity, and these functions can be adversely affected during aging. Therefore, it is fundamental to define the cellular mechanisms of aging in order to understand the basal forebrain and to correct deficits associated with aging. We have examined changes in the physiological properties of basal forebrain neurons during aging with whole-cell and single-channel patch-clamp, as well as, microfluorimetric measurements of intracellular calcium concentrations. These studies contribute to the understanding of integration within the basal forebrain and to the identification of age-related changes within central mammalian neurons. Although extensive functional/behavioral decline is often assumed to occur during aging, our data support an interpretation of compensatory increases in function for excitatory amino acid receptors, GABA(A) receptors, voltage-gated calcium currents and calcium homeostatic mechanisms. We believe that these changes occur to compensate for decrements accruing with age, such as decreased synaptic contacts, ion imbalances or neuronal loss. The basal forebrain must retain functionality into late aging if senescence is to be productive. Thus, it is critical to recognize the potential cellular and subcellular targets for therapeutic interventions intended to correct age-related behavioral deficits.

Single-cell RT-PCR detects shifts in mRNA expression profiles of basal forebrain neurons during aging

The medial septum and nucleus of the diagonal band (MS/nDB) contain cholinergic and GABAergic neuronal populations that have been identified based on immunohistochemical staining and/or electrophysiological properties. We explored the molecular diversity of MS/nDB neurons using single-cell reverse transcription-polymerase chain reaction (scRT-PCR) to assess gene expression profiles during aging in individual neurons acutely isolated from young (2-4 months) and aged (26-27 months) F344 rats. Neuronal gene expression profiles were characterized by detection of mRNAs for choline acetyltransferase (ChAT, cholinergic) and glutamate decarboxylase (GAD67, GABAergic), as well as mRNAs for calcium binding proteins (CaBPs) calbindin-D28k, calretinin and parvalbumin. Four major neuronal populations were identified: ChAT-positive (ChAT+) cells, GAD-positive (GAD+) cells, ChAT+/GAD+ cells and ChAT negative/GAD negative (ChAT-/GAD-) cells. With age, the percentage of cells expressing ChAT mRNA decreased from 53% in young to 40%, and the expression of GAD67 mRNA was reduced from 56 to 35% of the cells tested. The percentage of cells with detectable levels of both ChAT and GAD67 mRNA was reduced from 24% in young to 9% in aged. Concomitantly, the percentage of ChAT-/GAD- cells increased from 15 to 34% with age. Of the CaBPs, calretinin expression was observed most frequently in this study, and its detection decreased from 33 to 22% of the cells with age. Observations concerning the CaBPs were confirmed using in situ hybridization. These results suggest a shift in the mRNA expression profiles of MS/nDB neuronal populations during aging and exemplify the molecular diversity of cholinergic and GABAergic cells.

Homeostatic compensation maintains Ca2+ signaling functions in Purkinje neurons in the leaner mutant mouse

Several human neurological disorders have been associated with mutations in the gene coding for the 1 subunit of the P/Q type voltage-gated calcium channel (α1A/CaV2.1). Mutations in this gene also occur in a number of neurolog-ically afflected mouse strains, including leaner (tgla/tgla). Because the P-type calcium current is very prominent in cerebellar Purkinje neurons, these cells from mice with α1 subunit mutations make excellent models for the investigation of the functional consequences of native mutations in a voltage-gated calcium channel of mammalian central nervous system. In this review, we describe the impact of altered channel function on cellular calcium homeostasis and signaling. Remarkably, calcium buffering functions of the endoplasmic reticulum and calcium-binding proteins appear to be regulated in order to compensate for altered calcium influx through the mutant channels. Although this compensation may serve to maintain calcium signaling functions, such as calcium-induced calcium release, it remains uncertain whether such compensation alleviates or contributes to the behavioral phenotype.

Enhanced Calcium Buffering in F344 Rat Cholinergic Basal Forebrain Neurons Is Associated With Age-Related Cognitive Impairment

Alterations in neuronal Ca(2+) homeostasis are important determinants of age-related cognitive impairment. We examined the Ca(2+) influx, buffering, and electrophysiology of basal forebrain neurons in adult, middle-aged, and aged male F344 behaviorally assessed rats. Middle-aged and aged rats were characterized as cognitively impaired or unimpaired by water maze performance relative to young cohorts. Patch-clamp experiments were conducted on neurons acutely dissociated from medial septum/nucleus of the diagonal band with post hoc identification of phenotypic marker mRNA using single-cell RT-PCR. We measured whole cell calcium and barium currents and dissected these currents using pharmacological agents. We combined Ca(2+) current recording with Ca(2+)-sensitive ratiometric microfluorimetry to measure Ca(2+) buffering. Additionally, we sought changes in neuronal firing properties using current-clamp recording. There were no age- or cognition-related changes in the amplitudes or fractional compositions of the whole cell Ca(2+) channel currents. However, Ca(2+) buffering was significantly enhanced in cholinergic neurons from aged cognitively impaired rats. Moreover, increased Ca(2+) buffering was present in middle-aged rats that were not cognitively impaired. Firing properties were largely unchanged with age or cognitive status, except for an increase in the slow afterhyperpolarization in aged cholinergic neurons, independent of cognitive status. Furthermore, acutely dissociated basal forebrain neurons in which choline acetyltransferase mRNA was detected had the electrophysiological profiles of identified cholinergic neurons. We conclude that enhanced Ca(2+) buffering by cholinergic basal forebrain neurons may be important during aging.

Molecules and Membrane Activity: Single-Cell RT-PCR and Patch-Clamp Recording from Central Neurons

This chapter summarizes methods for characterizing mRNA expression and electrophysiological properties of central neurons using patch-clamp recording and single-cell reverse-transcription/polymerase chain reaction (scRT-PCR). A simple scRT-PCR protocol can be used to identify neurons by the expression of phenotypic marker mRNAs. The combination of these methods allows for the correlation of functional properties with molecular expression. Somewhat more complex methods are available for quantitation of mRNA expression. Both traditional gel-based PCR identification and real-time fluorescent PCR identification methods can be employed. Advantages and requirements of various methods are discussed. Different types of tissue preparations are presented with emphasis on methods used in our laboratories for acutely dissociated or cultured basal forebrain and amygdala neurons. The basal forebrain contains a heterogeneous population of cholinergic and GABAergic neurons, while the amygdala displays neurons with a complex receptor subunit composition. Investigation of neurons with this type of molecular diversity benefits from techniques such as scRT-PCR for cell identification. We also illustrate how these PCR methods can be combined with more complex experimental protocols, such as calcium buffering measurements using fluorescent dyes in dissociated neurons from aged animals. The capacity to combine scRT-PCR with a variety of experimental protocols allows the identification of unique cell types and relationships between physiology and gene expression.

OPTOGENETIC QUANTAL ANALYSIS OF BASAL FOREBRAIN SYNAPTIC TRANSMISSION WITH PHARMACOLOGICAL TESTING OF SYNAPTIC ACTIVITY WITH GLUTAMATE AND CALCIUM MODULATORS

mechanisms downstream of seven transmembrane receptors. Abnormally elevated levels of PLD activity are well-established in Alzheimer’s Disease (AD), implicating the two isoforms of mammalian phosphatidyl choline cleaving PLD (PC-PLD1 and PC-PLD2). Therefore, we took a systematic approach of investigating isoform-specific expression in human synaptosomes and further investigated the possibility of therapeutic intervention using preclinical studies. Methods: Synaptosomal Western blot analysis on post-mortem human hippocampus, temporal cortex and frontal cortex of AD patient brains/age-matched controls and 3XTg-AD mice hippocampus [mouse model with overexpression of human amyloid precursor protein (APP), presenilin1 gene (PSEN1) and microtubule-associated protein tau (MAPT) causing neuropathology progressing comparable to that in human AD patients] were used to detect the levels of neuronal PLD1 expression. Mouse hippocampal long-term potentiation (LTP) of PLD1-dependent changes were studied using pharmacological approaches in ex vivo slice preparations from wildtype and transgenic mouse models. Lastly, PLD1-dependent changes in novel object recognition (NOR) memory were assessed following PLD1 inhibition. Results:We observed elevated synaptosomal PLD1 in hippocampus/temporal cortex from post-mortem tissues of AD patients compared to age-matched controls and age-dependent hippocampal PLD1 increase in 3XTg-AD mice. PLD1 inhibition blocked effects of oligomeric Ab (oAb) or toxic oligomeric tau species (otau) on high-frequency stimulation (HFS) LTP and NOR deficits in wildtype mice. Lastly, PLD1 inhibition blocked LTP deficits normally observed in aging 3XTg-ADmice.Conclusions:Using human studies, we propose a novel role for PLD1-dependent signaling as a critical mechanism underlying oligomer-driven synaptic dysfunction and consequent memory disruption in AD. We, further, provide the first set of preclinical studies towards future therapeutics targeting PLD1 in slowing down/stopping the progression of AD-related memory deficits as a complementary approach to immunoscavenging clinical trials that are currently in progress.