Age-related alterations in caffeine-sensitive calcium stores and mitochondrial buffering in rat basal forebrain.

The properties of caffeine- and thapsigargin-sensitive endoplasmic reticulum calcium stores were compared in acutely dissociated basal forebrain neurons from young and aged F344 rats by ratiometric microfluorimetry. The ability of these stores to sequester and release calcium resembles that observed in other central neurons, with an important role of mitochondrial calcium buffering in regulating the response to caffeine. An age-related reduction in the filling state of the stores in resting cells appears to be mediated by increased rapid calcium buffering, which reduces the availability of calcium for uptake into the stores. An age-related decrease in the amplitude of maximal caffeine-induced calcium release was attributed to increased mitochondrial buffering. There were no age-related differences in the sensitivity to caffeine or in the calcium sequestration/release process at the level of the endoplasmic reticulum per se. These findings demonstrate the importance of interactions between cellular calcium buffering mechanisms and provide details regarding age-related changes in calcium homeostasis which have been thought to occur in these and other neurons associated with age-related neuronal dysfunctions.

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.

Pharmacological characterization of ionotropic excitatory amino acid receptors in young and aged rat basal forebrain.

Ionotropic glutamate receptors were characterized in acutely dissociated medial septum/nucleus of diagonal band neurons from one- to four-month- and 24-26-month-old male Fischer 344 rats. Whole-cell patch-clamp recordings were used to study glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, kainate and N-methyl-D-aspartate-induced currents. Pharmacological properties of these ionotropic receptors were studied across different age groups by comparing concentration response curves and EC50 for agonist-induced currents, as well as dissociation constants (Kb) for competitive receptor antagonists. Our results suggest that non-N-methyl-D-aspartate receptors on medial septum/nucleus of diagonal band neurons were predominantly of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate type and display biophysical and pharmacological properties similar to other central neurons. However, peak alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-induced currents were enhanced in aged (35.0+/-4.4 pA/pF) compared to young cells (16.2+/-1.7 pA/pF, P<0.005), and the EC50 shifted to the right (4.4+/-0.6 in young compared to 8.8+/-1.3 microM in aged, P<0.05). The Kb for 6,7-dinitroquinoxaline-2,3-dione inhibition of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-induced currents likewise shifted to the right (0.16+/-0.02 in young and 0.29+/-0.04 microM in aged, P<0.05) suggesting an age-related decrease in affinity for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors. N-Methyl-D-aspartate-induced currents were generated in standard physiological solutions with the addition of 1 microM glycine and the removal of Mg2+. The N-methyl-D-aspartate responses were predictably modulated by magnesium and glycine, and were antagonized by the competitive antagonist 2-amino-5-phosphonovaleric acid. No age-related change in N-methyl-D-aspartate maximum, EC50, magnesium sensitivity, glycine sensitivity or Kb for 2-amino-5-phosphonovaleric acid was observed. Overall, our results suggest that ionotropic glutamate receptors in the medial septum/nucleus of diagonal band have a similar pharmacological profile compared to glutamate receptors in other brain regions. More importantly, these data suggest that while medial septum/nucleus of diagonal band cells maintain N-methyl-D-aspartate receptors during ageing, a significant increase in current density and decrease in receptor affinity for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors, during this same time period, may provide a mechanism for age-related changes in neuronal plasticity and excitotoxicity in the basal forebrain.

Membrane currents in hippocampal neurons.

This chapter reviews properties and functions of endogenous ionic currents in hippocampal neurones. Currents considered are: Na currents INa(fast) and INa(slow); Ca currents; K currents--delayed rectifier IK(DR), transient IK(A), 'delay' current IK(D) and M current IK(M); inward rectifiers IQ, IK(IR) and ICl(V); Ca-activated currents IK(Ca) (IC and IAHP), ICl(Ca) and Ication(Ca); Na-activated currents; and anoxia-induced currents.

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.

Sodium valproate decreases synaptic potentiation and epileptiform activity in hippocampus.

The actions of sodium valproate (NaVP) were studied in the in vitro hippocampus using extracellular, intracellular and voltage-clamp recording techniques. In the CA1 region, concentrations of 30-200 microM NaVP reduced the amplitude but not the time course of post-tetanic potentiation (PTP) of dendritic field excitatory postsynaptic potentials (EPSPs). Epileptiform discharges were studied intracellularly in CA3 cells after pharmacological blockade of synaptic inhibition and repeated tetanic stimulation. NaVP (100 microM) blocked evoked paroxysmal depolarizing shift (PDS) discharges through a mechanism of increasing the threshold for burst-firing. When the PDS current was studied under voltage-clamp, application of NaVP (100 microM) resulted in a graded reduction of the PDS waveform. All of the actions of NaVP may result from inhibition of excitatory synaptic transmission following repetitive cell firing. A hypothesis is proposed that NaVP may act to decrease excitatory synaptic potentiation necessary for network synchronization.

Calcium channel types mediating synaptic transmission during aging.

The effects of omega-conotoxin (CTX) GVIA, omega-agatoxin (Aga) IVA, and the dihydropyridine nicardipine were studied on synaptic transmission in the hippocampus during aging. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the CA1 region in slices from young and aged Fischer 344 rats. Peptide toxins reduced synaptic transmissions similarly in both age groups while nicardipine showed no effect. These results suggests that the well documented age-related changes in synaptic transmissions in the hippocampus cannot be explained by changes in the types of Ca2+ channel mediating synaptic transmission.

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.

Sucrose-gap recordings of nerve-evoked potentials in mammalian parasympathetic ganglia.

The sucrose-gap recording technique was used to study mammalian parasympathetic ganglionic transmission. Both a fast nicotinic depolarization potential and a slow muscarinic hyperpolarizing potential were recorded in vesical pelvic ganglia (VPG). A long afterhyperpolarization caused by an electrical response of through-fibers was also recorded. However, a slow excitatory postsynaptic potential (S-EPSP) was not readily observed and may be masked by the long afterhyperpolarization. In addition, the slow inhibitory postsynaptic potential (S-IPSP) of the VPG was due to a direct effect of acetylcholine (ACh). Thus, sucrose-gap recordings of VPG potentials are similar to those obtained in sympathetic ganglia, but the mechanism for transmission of the S-IPSP may be different in the respective ganglia.

Effects of acute and chronic ethanol treatment on pre- and postsynaptic responses to baclofen in rat hippocampus.

Interactions between the GABAB receptor and acute or chronic ethanol treatment were studied using extracellular and intracellular electrophysiological recording techniques. Bath application of the GABAB receptor agonist, (-)-baclofen (0.1-100 microM) induced concentration-dependent inhibition of extracellularly recorded dendritic excitatory postsynaptic potentials (EPSPs) in the CA1 region of hippocampal slices. Responses to baclofen were unchanged relative to control either by simultaneous application of ethanol (10-60 mM) or by previous chronic ethanol exposure. The membrane potential of CA1 pyramidal neurons was reversibly hyperpolarized an average of 5 mV by pressure ejection of baclofen (1 mM). Bath application of ethanol (30 mM) alone occasionally caused a small depolarization of resting membrane potentials in CA1 neurons but failed to increase hyperpolarizing responses to pressure-ejected baclofen. However, in slices from chronic ethanol-treated animals hyperpolarizing responses to bath-applied baclofen (10 microM) were reduced by approximately 30% relative to controls. These results suggest that GABAB-mediated responses in CA1 hippocampal pyramidal neurons are relatively resistant to the acute effects of ethanol, but that continuous exposure to ethanol sufficient to induce physical dependence may evoke an adaptive reduction in some GABAB receptor mediated responses.

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.

Interaction of ethanol and allosteric modulators with GABAA-activated currents in adult medial septum/diagonal band neurons.

Behavioral and electrophysiological studies suggest that neurons in the medial septum may express ethanol sensitive GABAA receptors. In the present study, patch-clamp recordings of whole-cell currents were used to directly characterize the ethanol sensitivity of GABAA receptors on acutely dissociated neurons, isolated from the medial septum/nucleus of the diagonal band (MS/nDB) of the adult rat brains. MS/nDB neurons displayed inward currents in response to GABA applied rapidly with a large-bore dual pipette system. The currents were mediated by the activation of GABAA receptors, since they reversed near the calculated reversal potential for chloride and were completely blocked by bicuculline. GABA responses were concentration dependent with an EC50 of 8.7 microM GABA and a slope of 1.35 suggesting cooperativity. Pharmacologically relevant concentrations of ethanol (3-300 mM) neither significantly increased nor decreased mean responses to GABA in neurons from Sprague Dawley or High Alcohol Sensitivity (HAS) rats. Mean GABA currents were significantly increased by 300 mM ethanol in neurons from 'ethanol sensitive' Fischer 344, ACI and Wistar Kyoto inbred rats. In subsets of neurons, 12.5 to 57.1% of those tested from these 5 rats strains, ethanol (30-300 mM) significantly increased GABA currents by > or = 20%. An additional, 10 percent of cells from Sprague Dawley rats showed ethanol-induced inhibition of GABA-activated current by < or = 20%. Allosteric modulators pentobarbital (10 microM), midazolam (1 microM) and lanthanum (300 microM), enhanced, while zinc (30 microM) decreased GABA-activated currents in all neurons, consistent with the well-known actions of these agents. These results suggest that GABAA receptors on MS/dDB neurons are pharmacologically similar to those on other neurons with respect to regulation by allosteric modulators. On the other hand, ethanol sensitivity of GABAA receptors varies considerably from cell to cell ranging from significant enhancement to inhibition of GABA-activated current.

The protective effect of phentolamine against cardiac arrhythmias in the rat.

Norepinephrine was given by continuous infusion at a rate of 80 microgram/kg/min for 2 min. Norepinephrine caused the appearance of cardiac arrhythmias, primarily ventricular extrasystoles. The prior administration of phentolamine, even in a dose which did not antagonize the pressor effect of norephinephrine, prevented extrasystole production by norepinephrine. Tolazoline and yohimbine, in doses which did not affect the vasopressor action of norepinephrine, also suppressed extrasystole production. It is concluded that the antiarrhythmic effect of phentolamine, does not require antagonism of alpha-adrenoceptors of the type found in the vasculature, but may involve effects on a different type of alpha-adrenoceptors in the heart.

Muscarinic agonists block a late-afterhyperpolarization in medial septum/diagonal band neurons in vitro.

Intracellular recordings were made from neurons located in the medial septum (MS), and nucleus of the diagonal band (nDB) from slices of guinea pig brain. These forebrain nuclei contain both cholinergic and noncholinergic neurons that project to the cortex and hippocampus and are involved in many cortical functions. Muscarinic agonists (bethanechol, 2-30 microM) had the specific action to reduce a long-duration afterhyperpolarization (long-AHP) while leaving other shorter duration AHPs intact. Since the long-AHP was observed in both cholinergic and non-cholinergic neurons, muscarinic agonists were not selective for any one cell type. Block of a long-AHP was not associated with a consistent increase in cell excitability and therefore can not fully explain the excitatory actions of acetylcholine (ACh) observed in vivo within the MS/nDB.

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.

Ethanol inhibition of reduced frequency-dependent rundown of calcium currents in acutely dissociated MS/nDB neurons from chronic in vivo lead-exposed adult rats.

Although it is well known that lead (Pb2+0 acutely blocks voltage-gated calcium currents (VGCCs) in mammalian neurons, little is known about the long-term effects of continuous exposure to this metal on VGCCs. In the present study, the effects of chronic lead exposure on VGCCs (with barium ions as the charge carrier) were studied using whole-cell patch-clamp electrophysiological techniques in acutely dissociated medial septum (MS)/nucleus diagonal band (nDB) neurons. Neither peak, end current amplitudes, nor the current-voltage relationship were affected by chronic lead exposure. However, VGCCs repetitively evoked at frequent 6 s intervals displayed diminished whole-cell current rundown after 2 min of stimulation in cells from chronic Pb-exposed rats compared to cells from control Na-exposed rats. Because rundown after repetitive stimulation at a slower rate (20 s intervals) was not different between Pb-exposed and Na-exposed, reduced rundown at 6 s intervals was probably due to decreased slow inactivation of voltage-gated calcium channels. Interestingly, acute application of 60 mM ethanol reversed the reduced rundown in cells from Pb-exposed rats while having no effect on cells from Na-exposed rats. Clearly, acute ethanol treatment antagonized the effect of chronic lead exposure, unlike the additive interaction we observed previously with synaptic plasticity (Grover and Frye, 1996). Acute application of 1 microM Pb2+ completely blocked VGCCs similarly in neurons from Na-exposed and Pb-exposed rats. These findings do not suggest that major adaptive changes in VGCCs have occurred during chronic in vivo exposure to lead. But, subtle changes in channel efficiency only revealed under conditions of repetitive stimulation may exist, and are reversed by ethanol. These subtle changes may be sufficient to influence neuroplasticity such as LTP.

Acute ethanol dependence or long-term ethanol treatment and abstinence do not reduce hippocampal responses to carbachol.

In the hippocampus of human alcoholics, prolonged ethanol treatment reduces the number of muscarinic ligand binding sites present at autopsy suggesting a decrease in functional muscarinic receptors. Whether these changes are due to alcohol-induced brain damage or ethanol dependence and represent a reduced level of cholinergic function is unknown. The present studies tested the impact of ethanol dependence or long-term ethanol treatment and subsequent withdrawal on the function of pre- and postsynaptic muscarinic receptors in the CA1 region of the rat hippocampus. Field excitatory postsynaptic potentials (EPSPs) were inhibited in a concentration-dependent manner by 0.1-100 microM carbachol. This presynaptic inhibitory action of carbachol involving muscarinic receptors was not significantly reduced either by ethanol treatment (12 days), causing physical dependence, or by long-term ethanol treatment (97-120 days) and abstinence (3-6 months). Postspike after hyperpolarizations (AHPs) were inhibited in a concentration-dependent manner by carbachol (6-2000 nM). This postsynaptic excitatory action of muscarinic receptors also was not significantly reduced either by 12-day ethanol treatment or by long-term ethanol treatment. Taken together, these results suggest that neither pre- nor postsynaptic muscarinic receptor function measured electrophysiologically is reduced by either ethanol dependence or long-term ethanol consumption and abstinence in the rat as suggested by reduced muscarinic ligand binding in the hippocampus of human alcoholics.

Electrophysiologic characteristics of basal forebrain neurons in vitro.

Our data show that different cell types recorded in vitro can be identified by their intrinsic membrane properties. One type of neuron, namely S-AHP cells, have the ability to fire single action potentials in a rhythmic fashion following sufficient membrane depolarization. The rate is apparently controlled by several voltage-dependent conductances. S-AHP cells are normally quiescent at their resting potentials but will discharge once threshold is reached (-55 to -60 mV). Importantly, S-AHP (or F-AHP) cells will not convert into burst-firing neurons merely with changes in membrane potential. On the other hand, burst-firing cells have the ability to switch to a repetitive-firing pattern following membrane depolarization. All of these data provide a first step in an understanding of the firing rates of basal forebrain neurons, however, our results must be consolidated with existing in vivo studies for a more general understanding of basal forebrain function. Comparing our data to an in vivo preparation of the MS/nDB with synaptic afferents surgically removed may be one approach to correlating in vitro and in vivo studies. Vinogradova et al. (1980) used single unit recording techniques in unanesthetized chronic rabbits and compared the firing rates of cells before and after deafferentation. These authors reported a preservation of burst-firing neurons (25% of the cells) after deafferentation but with a significant reduction in the mean frequency of bursts. In addition a higher percentage of regularly firing cells also occurred following deafferentation (Vinogradova et al., 1980). It is interesting to speculate that these regularly firing cells may correspond to S-AHP cells in our in vitro studies, and some of the burst-firing units may correspond to the burst-firing cells we record in slices. Nevertheless, the in vivo data strongly suggests that endogenous regular spiking as well as rhythmic burst capabilities are present in some MS/nDB cells, however, the firing rates of most MS/nDB neurons are strongly influenced by synaptic afferents (see also Vinogradova et al., 1980; 1987). The endogenous activity in vivo can be explained, in part, by the intrinsic properties elucidated in our in vitro studies. How the synaptic afferents control MS/nDB circuitry and integrative output is premature to speculate without a more thorough understanding of the synaptic mechanisms involved. It is possible that future in vitro studies will help define these mechanisms and again contribute to an understanding of basal forebrain function.

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.