William H. Griffith

Electrophysiology of AChE-positive neurons in basal forebrain slices

We have utilized a guinea pig in vitro brain slice preparation of the medial septum (MS) and the vertical and horizontal limbs of the nucleus of the diagonal band of Broca (nDBB) to identify and classify different cell types within cholinergic nuclei. Utilizing a double-labeling technique which pairs intracellular injection of the fluorescent dye Lucifer yellow with acetylcholinesterase (AChE) histochemistry, we were able to correlate electrophysiological characteristics with a specific cholinergic cell marker. We report that at least two cell groups can be identified electrophysiologically within the MS/nDBB complex, and one population of neurons demonstrates distinct electrophysiological characteristics that are highly correlated with positive AChE-staining.

Spatial reference and working memory across the lifespan of male Fischer 344 rats.

Loss of mnemonic function is among the earliest and most disconcerting consequences of the aging process. This study was designed to provide a comprehensive profile of spatial mnemonic abilities in male Fischer 344 (F344) rats across the lifespan. Young, middle-aged, and aged F344 rats were trained in spatial reference and working memory versions of the water maze task. There was a progressive age-related decline in spatial reference memory across the lifespan. Reliable individual differences were observed among aged rats, with some aged rats performing as well as young cohorts and others performing outside this range. An age-related delay-dependent decline was observed on a working memory version of the water maze task although no relationship between performance on reference and working memory tasks was present. Notably, middle-aged rats were impaired relative to young on both tasks. Together these data demonstrate that individual differences in spatial reference memory exist among aged F344 rats and provide novel data demonstrating an unrelated decline in working memory across the lifespan, suggesting that age-related mnemonic dysfunction may occur across multiple brain systems.

Circadian Regulation and Function of Voltage-Dependent Calcium Channels in the Suprachiasmatic Nucleus

Individual neurons within the suprachiasmatic nuclei (SCNs) are capable of functioning as autonomous clocks and generating circadian rhythms in the expression of genes that form the molecular clockworks. Limited information is available on how these molecular oscillations in individual clock cells are coordinated to provide for the ensemble rhythmicity that is normally observed from the entire SCN. Because calcium influx via voltage-dependent calcium channels (VDCCs) has been implicated in the regulation of gene expression and synchronization of rhythmicity across the population of SCN clock cells, we first examined the rat SCN and an immortalized line of SCN cells (SCN2.2) for expression and circadian regulation of different VDCC α1 subunits. The rat SCN and SCN2.2 cells exhibited mRNA expression for all major types of VDCC α1 subunits. Relative levels of VDCC expression in the rat SCN and SCN2.2 cells were greatest for L-type channels, moderate for P/Q- and T-type channels, and minimal for R- and N-type channels. Interestingly, both rat SCN and SCN2.2 cells showed rhythmic expression of P/Q- and T-type channels. VDCC involvement in the regulation of molecular rhythmicity in SCN2.2 cells was then examined using the nonselective antagonist, cadmium. The oscillatory patterns of rPer2 and rBmal1 expression were abolished in cadmium-treated SCN2.2 cells without affecting cellular morphology and viability. These findings raise the possibility that the circadian regulation of VDCC activity may play an important role in maintaining rhythmic clock gene expression across an ensemble of SCN oscillators.

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.

Age-related decline in cholinergic synaptic transmission in hippocampus

Age-dependent changes in central nervous system (CNS) cholinergic synaptic transmission were studied in three age groups of Sprague-Dawley and Fischer 344 rats: 1- to 2-month-old, 8- to 10-month-old, and 18- to 23-month-old. Utilizing intracellular recording techniques and the in vitro hippocampal slice preparation, we report an age-related decline in central cholinergic transmission as a function of age. Slow excitatory postsynaptic potentials (slow EPSPs) were reduced approximately 60% in aged (18- to 23-month-old) compared to younger (1- to 2-month-old) animals. The response of the postsynaptic membrane to the muscarinic agonist, carbachol (0.3 microM), was also reduced with age. These changes were not accompanied by a global decline in muscarinic receptor function since two additional measures of cholinergic function were not changed with age. Both presynaptic inhibition of fast excitatory synaptic transmission and postsynaptic inhibition of the afterhyperpolarization (AHP) following a train of spikes were not changed during aging. Our results suggest that a primary functional decline in central cholinergic mechanisms during aging may be a specific reduction in central cholinergic synaptic transmission.

Acute tolerance to ethanol inhibition of NMDA-mediated EPSPs in the CA1 region of the rat hippocampus

The time course of ethanol-induced inhibition of NMDA-mediated synaptic activity was studied in brain slices using extracellular electrophysiological techniques in the CA1 region of the hippocampus. Bath application of 60 mM ethanol inhibited NMDA-mediated field excitatory postsynaptic potentials (EPSPs) by at least 45% in 7/11 of the slices tested, with the remaining 4 slices inhibited by 8.7-35%. Most slices inhibited by at least 45% showed a significant reduction in ethanol inhibition over a 15 min ethanol exposure period, suggesting the development of acute tolerance. In a second set of experiments, tolerance to ethanol-induced inhibition of NMDA-mediated EPSPs that developed over time during the first ethanol exposure persisted during a second ethanol exposure. In contrast to ethanol, inhibition of EPSPs by the NMDA antagonist DL-2-amino-5-phosphonopentanoic acid (APV) remained stable during a comparable application of the drug. These results suggest that acute tolerance can develop to ethanol inhibition of NMDA mediated synaptic activity in the hippocampus.

Deficits across multiple cognitive domains in a subset of aged Fischer 344 rats.

Rodent models of cognitive aging routinely use spatial performance on the water maze to characterize medial temporal lobe integrity. Water maze performance is dependent upon this system and, as in the aged human population, individual differences in learning abilities are reliably observed among spatially characterized aged rats. However, unlike human aging in which cognitive deficits rarely occur in isolation, few non-spatial learning deficits have been identified in association with spatial impairment among aged rats. In this study, a subset of male aged Fischer 344 rats was impaired both in water maze and odor discrimination tasks, whereas other aged cohorts performed on par with young adult rats in both settings. The odor discrimination learning deficits were reliable across multiple problems. Moreover, these deficits were not a consequence of anosmia and were specific to olfactory learning, as cognitively impaired aged rats performed normally on an analogous non-olfactory discrimination task. These are among the first data to describe an aging model in which individual variability among aged rat cognition occurs across two independent behavioral domains.

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.

Chronic neonatal nicotine increases anxiety but does not impair cognition in adult rats.

Developmental nicotine exposure has been implicated in the association between maternal smoking and adverse outcomes in offspring. The 3rd trimester of human pregnancy is equivalent to the 1st postnatal week in rodents; both are periods of active brain growth during which nicotinic acetylcholine receptors are transiently upregulated. Chronic neonatal nicotine (CNN; 6 mg/kg/day) from postnatal Days 1 to 7 was given orally to rat pups to evaluate long-term behavioral effects. Males and females were tested as adolescents or as young adults. CNN significantly decreased center time, ambulatory behavior, and rearing in the open-field test and decreased the number of entrances and time spent in the open arm of the elevated plus-maze in both sexes and ages. CNN did not change performance in the T maze or in the water maze in adult males or females. Motor coordination was not altered. In summary, CNN had long-term effects on anxiety-like behavior but did not affect spatial learning and memory functions. This finding is particularly important when evaluating the benefits of nicotine-replacement therapies during human pregnancies, which may improve smoking cessation rates but could result in long-term behavioral consequences.

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.