Maintenance of optogenetic channel rhodopsin (ChR2) function in aging mice: Implications for pharmacological studies of inhibitory synaptic transmission, quantal content, and calcium homeostasis.

Disruption of synaptic function is believed to represent a common pathway contributing to cognitive decline during aging. Optogenetics is a prodigious tool for studying relationships between function and synaptic circuitry but models utilizing viral vectors present limitations. Careful characterization of the functionality of channel rhodopsin in transgenic models is crucial for determining whether they can be used across aging. This includes verifying the light sensitivity of the protein and confirming its ability to generate action potentials in response to light stimulation. We combined in vitro optogenetic methodology and a reduced synaptic preparation of acutely isolated neurons to determine if the ChR2(H134R)-eYFP vGAT mouse model is well-suited for aging studies. We used neurons from young (2-6 mo), middle-aged (10-14 mo) and aged (17-25 mo) bacterial artificial chromosome (BAC) transgenic mouse line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R in GABAergic cell populations. Cellular physiology and calcium dynamics were assessed in basal forebrain (BF) neurons using patch-clamp recording and fura-2 microfluorimetry, alongside 470 nm light stimulation of the transgenic ChR2 channel to characterize a wide array of physiological functions known to decline with age. We found ChR2 expression is functionally maintained across aging, while spontaneous and optically evoked inhibitory postsynaptic currents, and quantal content were decreased. Aged mice also showed an increase in intracellular calcium buffering. These results, which are on par with previous observations, demonstrate that the optogenetic vGAT BAC mouse model is well-suited for investigating age-related changes in calcium signaling and synaptic transmission.

Late-Onset, Short-Term Intermittent Fasting Reverses Age-Related Changes in Calcium Buffering and Inhibitory Synaptic Transmission in Mouse Basal Forebrain Neurons.

Aging is often associated with cognitive decline and recurrent cellular and molecular impairments. While life-long caloric restriction (CR) may delay age-related cognitive deterioration as well as the onset of neurologic disease, recent studies suggest that late-onset, short-term intermittent fasting (IF), may show comparable beneficial effects as those of life-long CR to improve brain health. We used a new optogenetic aging model to study the effects of late-onset (>18 months), short-term (four to six weeks) IF on age-related changes in GABAergic synaptic transmission, intracellular calcium (Ca2+) buffering, and cognitive status. We used male mice from a bacterial artificial chromosome (BAC) transgenic mouse line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R [VGAT-ChR2(H134R)-EYFP] in a reduced synaptic preparation that allows for specific optogenetic light stimulation on GABAergic synaptic terminals across aging. We performed quantal analysis using the method of failures in this model and show that short-term IF reverses the age-related decrease in quantal content of GABAergic synapses. Likewise, short-term IF also reversed age-related changes in Ca2+ buffering and spontaneous GABAergic synaptic transmission in basal forebrain (BF) neurons of aged mice. Our findings suggest that late-onset short-term IF can reverse age-related physiological impairments in mouse BF neurons but that four weeks IF is not sufficient to reverse age-related cognitive decline.SIGNIFICANCE STATEMENT Here, we demonstrate plasticity of the aging brain and reversal of well-defined hallmarks of brain aging using short-term intermittent fasting (IF) initiated later in life. Few therapeutics are currently available to treat age-related neurologic dysfunction although synaptic dysfunction occurs during aging and neurologic disease is a topic of intense research. Using a new reduced synaptic preparation and optogenetic stimulation we are able to study age-related synaptic mechanisms in greater detail. Several neurophysiological parameters including quantal content were altered during aging and were reversed with short-term IF. These methods can be used to identify potential therapies to reverse physiological dysfunction during aging.

Amitriptyline Decreases GABAergic Transmission in Basal Forebrain Neurons Using an Optogenetic Model of Aging.

The antidepressant drug amitriptyline is used in the treatment of clinical depression and a variety of neurological conditions such as anxiety, neuropathic pain disorders and migraine. Antidepressants are associated with both therapeutic and untoward effects, and their use in the elderly has tripled since the mid-1990s. Because of this widespread use, we are interested in testing the acute effects of amitriptyline on synaptic transmission at therapeutic concentrations well below those that block voltage-gated calcium channels. We found that 3 µM amitriptyline reduced the frequency of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) and reduced quantal content in mice at ages of 7-10 mo. and 23-25 mo., suggesting a presynaptic mechanism of action that does not diminish with age. We employed a reduced synaptic preparation of the basal forebrain (BF) and a new optogenetic aging model utilizing a bacterial artificial chromosome (BAC) transgenic mouse line with stable expression of the channelrhodopsin-2 (ChR2) variant H134R specific for GABAergic neurons [VGAT-ChR2(H134R)-EYFP]. This model enables optogenetic light stimulation of specific GABAergic synaptic terminals across aging. Age-related impairment of circadian behavior was used to confirm predictable age-related changes associated with this model. Our results suggest that low concentrations of amitriptyline act presynaptically to reduce neurotransmitter release and that this action is maintained during aging.

Molecular, physiological and behavioral characterization of the heterozygous Df[h15q13]/+ mouse model associated with the human 15q13.3 microdeletion syndrome.

The human 15q13.3 microdeletion syndrome (DS) is caused by a heterozygous microdeletion (MD) affecting six genes: FAN1; MTMR10; TRPM1; KLF13; OTUD7A; and CHRNA7. Carriers are at risk for intellectual disability, epilepsy, autism spectrum disorder, and schizophrenia. Here we used the Df[h15q13]/+ mouse model with an orthologous deletion to further characterize molecular, neurophysiological, and behavioral parameters that are relevant to the 15q13.3 DS. First, we verified the expression and distribution of the α7 nicotinic acetylcholine receptor (nAChR), a gene product of the CHRNA7, in cortical and subcortical areas. Results revealed similar mRNA distribution pattern in wildtype (WT) and heterozygous (Het) mice, with about half the number of α7 nAChR binding sites in mutants. Hippocampal recordings showed similar input/output responses of field excitatory post-synaptic potentials and theta-burst induced long-term potentiation in WT and Het mice. Het males exhibited impaired spatial learning acquisition in the Barnes Maze. Indicative of increased seizure susceptibility, Het mice developed secondary seizures after 6-Hz corneal stimulation, and had significantly increased sensitivity to the chemoconvulsant pentylenetetrazol resulting in increased spiking in hippocampal EEG recordings. Basal mRNA expression of brain derived neurotrophic factor and activity regulated immediate early genes (c-fos, Arc, Erg-1 and Npas4) during adolescence, a critical period of brain maturation, was unaffected by genotype. Thus, the MD did not show gross neuroanatomical, molecular, and neurophysiological abnormalities despite deficits in spatial learning and increased susceptibility to seizures. Altogether, our results verify the phenotypic profile of the heterozygous Df[h15q13]/+ mouse model and underscore its translational relevance for human 15q13.3 DS.

Neonatal nicotine exposure increases excitatory synaptic transmission and attenuates nicotine-stimulated GABA release in the adult rat hippocampus.

Developmental exposure to nicotine has been linked to long-lasting changes in synaptic transmission which may contribute to behavioral abnormalities seen in offspring of women who smoke during pregnancy. Here, we examined the long-lasting effects of developmental nicotine exposure on glutamatergic and GABAergic neurotransmission, and on acute nicotine-induced glutamate and GABA release in the adult hippocampus, a structure important in cognitive and emotional behaviors. We utilized a chronic neonatal nicotine treatment model to administer nicotine (6 mg/kg/day) to rat pups from postnatal day (P) 1-7, a period that falls developmentally into the third human trimester. Using whole-cell voltage clamp recordings from CA1 pyramidal neurons in hippocampal slices, we measured excitatory and inhibitory postsynaptic currents in neonatally control- and nicotine-treated young adult males. Neonatal nicotine exposure significantly increased AMPA receptor-mediated spontaneous and evoked excitatory signaling, with no change in glutamate release probability in adults. Conversely, there was no increase in spontaneous GABAergic neurotransmission in nicotine-males. Chronic neonatal nicotine treatment had no effect on acute nicotine-stimulated glutamate release in adults, but acute nicotine-stimulated GABA release was significantly attenuated. Thus, neonatal nicotine exposure results in a persistent net increase in excitation and a concurrent loss of nicotinic acetylcholine receptor (nAChR)-mediated regulation of presynaptic GABA but not glutamate release, which would exacerbate excitation following endogenous or exogenous nAChR activation. Our data underscore an important role for nAChRs in hippocampal excitatory synapse development, and suggest selective long-term changes at specific presynaptic nAChRs which together could explain some of the behavioral abnormalities associated with maternal smoking.

Characterization of age-related changes in synaptic transmission onto F344 rat basal forebrain cholinergic neurons using a reduced synaptic preparation.

Basal forebrain (BF) cholinergic neurons participate in a number of cognitive processes that become impaired during aging. We previously found that age-related enhancement of Ca(2+) buffering in rat cholinergic BF neurons was associated with impaired performance in the water maze spatial learning task (Murchison D, McDermott AN, Lasarge CL, Peebles KA, Bizon JL, and Griffith WH. J Neurophysiol 102: 2194-2207, 2009). One way that altered Ca(2+) buffering could contribute to cognitive impairment involves synaptic function. In this report we show that synaptic transmission in the BF is altered with age and cognitive status. We have examined the properties of spontaneous postsynaptic currents (sPSCs) in cholinergic BF neurons that have been mechanically dissociated without enzymes from behaviorally characterized F344 rats. These isolated neurons retain functional presynaptic terminals on their somata and proximal dendrites. Using whole cell patch-clamp recording, we show that sPSCs and miniature PSCs are predominately GABAergic (bicuculline sensitive) and in all ways closely resemble PSCs recorded in a BF in vitro slice preparation. Adult (4-7 mo) and aged (22-24 mo) male rats were cognitively assessed using the water maze. Neuronal phenotype was identified post hoc using single-cell RT-PCR. The frequency of sPSCs was reduced during aging, and this was most pronounced in cognitively impaired subjects. This is the same population that demonstrated increased intracellular Ca(2+) buffering. We also show that increasing Ca(2+) buffering in the synaptic terminals of young BF neurons can mimic the reduced frequency of sPSCs observed in aged BF neurons.

Long-lasting distortion of GABA signaling in MS/DB neurons after binge-like ethanol exposure during initial synaptogenesis.

Using a well-established model of binge-like ethanol treatment of rat pups on postnatal days (PD) 4-9, we found that maturation of GABAA receptor (GABAAR) miniature postsynaptic currents (mPSCs) was substantially blunted for medial septum/diagonal band (MS/DB) neurons in brain slices on PD 11-16. Ethanol reduced mPSC amplitude, frequency, and decay kinetics, while attenuating or exaggerating allosteric actions of zolpidem and allopregnanolone, respectively. The impact of ethanol in vivo was long lasting as most changes in MS/DB GABAAR mPSCs were still observed as late as PD 60-85. Maturing MS/DB neurons in naïve brain slices PD 4-16 showed increasing mPSC frequency, decay kinetics, and zolpidem sensitivity that were nearly identical to our earlier findings in cultured septal neurons (DuBois et al., 2004, 2006). These rapidly developing mPSC parameters continued to mature through the first month of life then stabilized throughout the remainder of the lifespan. Finally, equivalent ethanol-induced alterations in GABAAR mPSC signaling were present in MS/DB neurons from both male and female animals. Previously, we showed ethanol treatment of cultured embryonic day 20 septal neurons distorts the maturation of GABAAR mPSCs predicting that early stages of GABAergic transmission in MS/DB neurons are vulnerable to intoxication injury (DuBois et al., 2004, 2006). Since the overall character, timing, and magnitude of GABAergic mPSC developmental- and ethanol-induced changes in the in vivo model so closely mirror chronologically equivalent adaptations in cultured septal neurons, this suggests that such parallel models of ethanol impairment of GABAergic synaptic development in vivo and in vitro should be useful for translational studies exploring the efficacy and mechanism of action of potential therapeutic interventions from the cellular to whole animal level.

Chronic neonatal nicotine exposure increases excitation in the young adult rat hippocampus in a sex-dependent manner.

Smoking during pregnancy exposes the fetus to nicotine, resulting in nicotine-stimulated neurotransmitter release. Recent evidence suggests that the hippocampus develops differently in males and females with delayed maturation in males. We show that chronic nicotine exposure during the first postnatal week has sex-specific long-term effects. Neonatal rat pups were chronically treated with nicotine (6mg/kg/day) (CNN) from postnatal day 1 to 7 or milk only (Controls), and hippocampal slices were prepared from Control- and CNN-treated young adults. Field excitatory postsynaptic potentials (fEPSPs) or population spikes (PSs) were recorded from the CA1 hippocampus following CA1 s. radiatum stimulation. Input/Output curves constructed from fEPSP data indicated that CNN-males, but not females, had significantly increased excitatory responses compared to Controls (p<0.05, n=10 Con, n=11 CNN). Long-term potentiation (LTP) was not significantly changed by CNN. In the presence of bicuculline, which blocks inhibitory GABA(A) receptors, an epileptiform burst consisting of a series of PSs was evoked. The amplitude of the first PS was significantly larger in CNN-males and females compared to Controls (males: p<0.01, n=8 Con, n=8 CNN; females: p<0.05, n=9 Con, n=7 CNN). Only CNN-males also had significantly larger second PSs (p<0.05, n=8 con, n=8 CNN). Epileptiform activity evoked by zero Mg(2+) incubation did not differ in amplitude or duration of bursts in CNN-males or females compared to Controls. These data indicate that neonatal nicotine exposure has long lasting effects and results in increased excitation within the CA1 hippocampus in adulthood, with males showing increased sensitivity to nicotine's effects.

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.

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.

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.

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.

Functional compensation by other voltage-gated Ca2+ channels in mouse basal forebrain neurons with Ca(V)2.1 mutations.

Tottering (tg/tg) and leaner (tg(la)/tg(la)) mutant mice exhibit distinct mutations in the gene encoding the voltage-activated Ca(2+) channel alpha(1A) subunit (CACNA1A), the pore-forming subunit of the Ca(V)2.1 (P/Q type) Ca(2+) channels. These mice exhibit absence seizures and deficiencies in motor control and other functions. Previous work in cerebellar Purkinje neurons has shown that these mutations cause dramatic reductions in calcium channel function. Because Purkinje cell somata primarily express the Ca(V)2.1 channels, the general decrease in Ca(V)2.1 channel function is observed as a profound decrease in whole-cell current. In contrast to Purkinje cells, basal forebrain (BF) neurons express all of the Ca(2+) channel alpha(1) subunits, with Ca(V)2.1 contributing approximately 30% to the whole-cell current in wild-type (+/+) mice. Here, we show that whole-cell Ba(2+) current densities in BF neurons are not reduced in the mutant genotypes despite a reduction in the Ca(V)2.1 contribution. By blocking the different Ca(2+) channel subtypes with specific pharmacological agents, we found a significant increase in the proportion of Ca(V)1 Ca(2+) current in mutant phenotypes. There was no change in tissue mRNA expression of calcium channel subtypes Ca(V)2.1, Ca(V)2.2, Ca(V)1.2, Ca(V)1.3, and Ca(V)2.3 in the tottering and leaner mutant mice. These results suggest that Ca(V)1 channels may functionally upregulate to compensate for reduced Ca(V)2.1 function in the mutants without an increase in Ca(v)1 message. Single-cell reverse transcription polymerase chain reaction (RT-PCR) experiments in a subset of sampled neurons revealed that approximately 90% of the cells could be considered cholinergic based on choline acetyltransferase (ChAT) mRNA expression.

Low voltage-activated calcium and fast tetrodotoxin-resistant sodium currents define subtypes of cholinergic and noncholinergic neurons in rat basal forebrain.

Neurons of the basal forebrain (BF) possess unique combinations of voltage-gated membrane currents. Here, we describe subtypes of rat basal forebrain neurons based on patch-clamp analysis of low-voltage activated (LVA) calcium and tetrodotoxin-resistant (TTX-R) sodium currents combined with single-cell RT-PCR analysis. Neurons were identified by mRNA expression of choline acetyltransferase (ChAT+, cholinergic) and glutamate decarboxylase (GAD67, GABAergic). Four cell types were encountered: ChAT+, GAD+, ChAT+/GAD+ and ChAT-/GAD- cells. Both ChAT+ and ChAT+/GAD+ cells (71/75) displayed LVA currents and most (34/39) expressed mRNA for LVA Ca(2+) channel subunits. Ca(v)3.2 was detected in 31/34 cholinergic neurons and Ca(v)3.1 was expressed in 6/34 cells. Three cells expressed both subunits. No single neurons showed Ca(v)3.3 mRNA expression, although BF tissue expression was observed. In young rats (2-4 mo), ChAT+/GAD+ cells displayed larger LVA current densities compared to ChAT+ neurons, while these latter neurons displayed an age-related increase in current densities. Most (29/38) noncholinergic neurons (GAD+ and ChAT-/GAD-) possessed fast TTX-R sodium currents resembling those mediated by Na(+) channel subunit Na(v)1.5. This subunit was expressed predominately in noncholinergic neurons. No cholinergic cells (0/75) displayed fast TTX-R currents. The TTX-R currents were faster and larger in GAD+ neurons compared to ChAT-/GAD- neurons. The properties of ChAT+/GAD+ neurons resemble those of ChAT+ neurons, rather than of GAD+ neurons. These results suggest novel features of subtypes of cholinergic and noncholinergic neurons within the BF that may provide new insights for understanding normal BF function.

Differential expression of T-type calcium channels in P/Q-type calcium channel mutant mice with ataxia and absence epilepsy.

Mutations in P/Q-type calcium channels generate common phenotypes in mice and humans, which are characterized by ataxia, paroxysmal dyskinesia, and absence seizures. Subsequent functional changes of T-type calcium channels in thalamus are observed in P/Q-type calcium channel mutant mice and these changes play important roles in generation of absence seizures. However, the changes in T-type calcium channel function and/or expression in the cerebellum, which may be related to movement disorders, are still unknown. The leaner mouse exhibits severe ataxia, paroxysmal dyskinesia, and absence epilepsy due to a P/Q-type calcium channel mutation. We investigated changes in T-type calcium channel expression in the leaner mouse thalamus and cerebellum using quantitative real-time polymerase chain reaction (qRT-PCR) and quantitative in situ hybridization histochemistry (ISHH). qRT-PCR analysis showed no change in T-type calcium channel alpha 1G subunit (Cav3.1) expression in the leaner thalamus, but a significant decrease in alpha 1G expression in the whole leaner mouse cerebellum. Interestingly, quantitative ISHH revealed differential changes in alpha 1G expression in the leaner cerebellum, where the granule cell layer showed decreased alpha 1G expression while Purkinje cells showed increased alpha 1G expression. To confirm these observations, the granule cell layer and the Purkinje cell layer were laser capture microdissected separately, then analyzed with qRT-PCR. Similar to the observation obtained by ISHH, the leaner granule cell layer showed decreased alpha 1G expression and the leaner Purkinje cell layer showed increased alpha 1G expression. These results suggest that differential expression of T-type calcium channels in the leaner cerebellum may be involved in the observed movement disorders.

Loss of N-cadherin and alpha-catenin in the proximal tubules of aging male Fischer 344 rats.

Aging is associated with a loss of renal reserve, and increased sensitivity to either xenobiotic or physiologic insult. Given the critical role of the cadherin/catenin complex in establishing and maintaining the integrity and polarity of tubular epithelial cells, it was hypothesized that aging was associated with alterations in renal cadherin/catenin complexes. Histological assessment of aged (24 months) kidneys harvested from male Fischer 344 rats demonstrates mild degeneration of proximal tubules, multifocal chronic lymphocytic infiltration, moderate development of protein casts inside tubules, and tubular dilatation or degeneration. Western blot analysis revealed that N-cadherin protein expression is not constant over 24 months. N-cadherin expression increased from 4 to 9 months, with peak levels at 9 and 13 months. A decrease in expression was seen at 19 months and an almost complete loss of expression was seen at 24 months. In contrast, the expression of E- and Ksp-cadherin was constant over 24 months. A loss of alpha-catenin at was seen at 19 and 24 months in the absence of changes in beta-, gamma-, and p120-catenin. This pattern of N-cadherin expression (increase followed by decrease) was confirmed by real-time PCR analysis, which demonstrated a similar pattern as the Western blot, suggesting that the loss of N-cadherin protein was due to decreased gene expression. The loss of N-cadherin was specific for the kidney, as no changes in N-cadherin expression in the liver, brain, or testes were seen during aging. The conclusion that loss of N-cadherin expression is a critical component of the renal dysfunction associated with aging is supported by the finding that caloric restriction attenuates the loss of N-cadherin, as well as the finding that a significant loss of N-cadherin is seen in the kidneys of ZDF x SHHF rats, a genetic model of end-stage renal disease. Cadherin and catenin expression was further analyzed by immunofluorescence. A significant loss of staining of both N-cadherin and alpha-catenin was seen in the proximal tubules of rats at 24 months. Interestingly, this corresponded with delocalization of the alpha-1 subunit of the Na+K+-ATPase, i.e. aberrant staining on cell-cell borders and some indication of apical staining in proximal tubules. Taken together, these data suggest that aging is associated with decreased expression of N-cadherin and alpha-catenin and is associated with a loss of cell polarity.

Reduced mitochondrial buffering of voltage-gated calcium influx in aged rat basal forebrain neurons.

Alterations of neuronal Ca(2+) homeostatic mechanisms could be responsible for many of the cognitive deficits associated with aging in mammals. Mitochondrial participation in Ca(2+) signaling is now recognized as a prominent feature in neuronal physiology. We combined voltage-clamp electrophysiology with Ca(2+)-sensitive ratiometric microfluorimetry and laser scanning confocal microscopy to investigate the participation in Ca(2+) buffering of in situ mitochondria in acutely dissociated basal forebrain neurons from young and aged F344 rats. By pharmacologically blocking mitochondrial Ca(2+) uptake, we determined that mitochondria were not involved in rapid buffering of small Ca(2+) influx through voltage-gated Ca(2+) channels (VGCCs) in the somatic compartment. For larger Ca(2+) influx, aged mitochondria showed a significant buffering deficit. Evidence obtained with the potentiometric indicator, JC-1, suggests a significantly reduced mitochondrial membrane potential in aged neurons. These results support the interpretation that there is a fundamental difference in the way young and aged neurons buffer Ca(2+), and a corresponding difference in the quality of the Ca(2+) signal experienced by young and aged neurons for different intensities of cytoplasmic Ca(2+) influx.

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 alpha1 subunit of the P/Q type voltage-gated calcium channel (alpha1A/Ca(v)2.1). Mutations in this gene also occur in a number of neurologically affected mouse strains, including leaner (tg(la)/tg(la)). Because the P-type calcium current is very prominent in cerebellar Purkinje neurons, these cells from mice with alpha1 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.