Age-related alterations to working memory and to pyramidal neurons in the prefrontal cortex of rhesus monkeys begin in early middle-age and are partially ameliorated by dietary curcumin.

Layer 3 (L3) pyramidal neurons in aged rhesus monkey lateral prefrontal cortex (LPFC) exhibit significantly elevated excitability in vitro and reduced spine density compared to neurons in young subjects. The time-course of these alterations, and whether they can be ameliorated in middle age by the powerful anti-oxidant curcumin is unknown. We compared the properties of L3 pyramidal neurons from the LPFC of behaviorally characterized rhesus monkeys over the adult lifespan using whole-cell patch clamp recordings and neuronal reconstructions. Working memory (WM) impairment, neuronal hyperexcitability, and spine loss began in middle age. There was no significant relationship between neuronal properties and WM performance. Middle-aged subjects given curcumin exhibited better WM performance and less neuronal excitability compared to control subjects. These findings suggest that the appropriate time frame for intervention for age-related cognitive changes is early middle age, and points to the efficacy of curcumin in delaying WM decline. Because there was no relationship between excitability and behavior, the effects of curcumin on these measures appear to be independent.

Dendritic spine changes associated with normal aging.

Given the rapid rate of population aging and the increased incidence of cognitive decline and neurodegenerative diseases with advanced age, it is important to ascertain the determinants that result in cognitive impairment. It is also important to note that much of the aged population exhibit 'successful' cognitive aging, in which cognitive impairment is minimal. One main goal of normal aging studies is to distinguish the neural changes that occur in unsuccessful (functionally impaired) subjects from those of successful (functionally unimpaired) subjects. In this review, we present some of the structural adaptations that neurons and spines undergo throughout normal aging and discuss their likely contributions to electrophysiological properties and cognition. Structural changes of neurons and dendritic spines during aging, and the functional consequences of such changes, remain poorly understood. Elucidating the structural and functional synaptic age-related changes that lead to cognitive impairment may lead to the development of drug treatments that can restore or protect neural circuits and mediate cognition and successful aging.

Age-related increase of sI(AHP) in prefrontal pyramidal cells of monkeys: relationship to cognition.

Reduced excitability, due to an increase in the slow afterhyperpolarization (and its underlying current sI(AHP)), occurs in CA1 pyramidal cells in aged cognitively-impaired, but not cognitively-unimpaired, rodents. We sought to determine whether similar age-related changes in the sI(AHP) occur in pyramidal cells in the rhesus monkey dorsolateral prefrontal cortex (dlPFC). Whole-cell patch-clamp recordings were obtained from layer 3 and layer 5 pyramidal cells in dlPFC slices prepared from young (9.6 ± 0.7 years old) and aged (22.3 ± 0.7 years old) behaviorally characterized subjects. The amplitude of the sI(AHP) was significantly greater in layer 3 (but not layer 5) cells from aged-impaired compared with both aged-unimpaired and young monkeys, which did not differ. Aged layer 3, but not layer 5, cells exhibited significantly increased action potential firing rates, but there was no relationship between sI(AHP) and firing rate. Thus, in monkey dlPFC layer 3 cells, an increase in sI(AHP) is associated with age-related cognitive decline; however, this increase is not associated with a reduction in excitability.

Structural and functional changes in tau mutant mice neurons are not linked to the presence of NFTs.

In the rTg4510 mouse model, expression of the mutant human tau variant P301L leads to development of neurofibrillary tangles (NFTs), neuronal death, and memory impairment, reminiscent of the pathology observed in human tauopathies. In the present study, we examined the effects of mutant tau expression on the electrophysiology and morphology of individual neurons using whole-cell patch-clamp recordings and biocytin filling of pyramidal cells in cortical slices prepared from rTg4510 (TG) and wild-type (WT) littermate mice. Among the TG cells, 42% contained a clear Thioflavin-S positive inclusion in the soma and were categorized as NFT positive (NFT+), while 58% had no discernable inclusion and were categorized as NFT negative (NFT-). The resting membrane potential (V(r)) was significantly depolarized (+8 mV) in TG cells, and as a consequence, evoked repetitive action potential (AP) firing rates were also significantly increased. Further, single APs were significantly shorter in duration in TG cells and the depolarizing voltage deflection or "sag" evoked by hyperpolarization was significantly greater in amplitude. In addition to these functional electrophysiological changes, TG cells exhibited significant morphological alterations, including loss or significant atrophy of the apical tuft, reduced dendritic complexity and length, and reduced spine density. Importantly, NFT- and NFT+ TG cells were indistinguishable with regard to both morphological and electrophysiological properties. Our observations show that expression of mutated tau results in significant structural and functional changes in neurons, but that these changes occur independent of mature NFT formation.

Synapses are lost during aging in the primate prefrontal cortex.

An electron microscopic analysis has been carried out on the effects of age on the numerical density of both excitatory (asymmetric) and inhibitory (symmetric) synapses in the neuropil of layers 2/3 and of layer 5 in area 46 from the frontal cortex of behaviorally tested rhesus monkeys. There is no change in the lengths of synaptic junctions with age or in the percentage distribution of synapses relative to the postsynaptic spines and dendritic shafts. However, in layers 2/3 there is an overall loss of about 30% of synapses from 5 to 30 years of age, and both asymmetric and symmetric synapses are lost at the same rate. In layer 5 the situation is different; the overall loss of synapses is only 20% and this is almost entirely due to a loss of asymmetric synapses, since there is no significant loss of symmetric synapses from this layer with age. When the synapse data are correlated with the overall cognitive impairment shown by the monkeys, it is found that there is a strong correlation between the numerical density of asymmetric synapses in layers 2/3 and cognitive impairment, with a weaker correlation between symmetric synapse loss and cognitive impairment. In layer 5 on the other hand there is no correlation between synapse loss and cognitive impairment. However synapse loss is not the only factor causing cognitive impairment, since in previous studies of area 46 we have found that age-related alteration in myelin in this frontal area also significantly contributes to cognitive decline. The synapse loss is also considered in light of earlier studies, which show that the frequency of spontaneous excitatory synaptic responses is reduced with age in layers 2/3 neurons.

Effects of aging on the electrophysiological properties of layer 5 pyramidal cells in the monkey prefrontal cortex.

A significant decline in executive system function mediated by the prefrontal cortex (PFC) often occurs with normal aging. In vitro slice studies have shown that layer 2/3 pyramidal cells in the monkey PFC exhibit increased action potential (AP) firing rates which may, in part, contribute to this decline. Given that layer 5 cells also play a role in executive system function, it is important to determine if similar age-related changes occur in these cells. Whole-cell patch-clamp recordings in in vitro slices prepared from the PFC of young and aged behaviorally characterized rhesus monkeys were employed to answer this question. Basic membrane and repetitive AP firing properties were unaltered with age. Aged cells exhibited significantly decreased single AP amplitude, duration and fall time and increased slow afterhyperpolarization (sAHP) amplitude, but these changes were not associated with cognitive performance. This study demonstrates that layer 5 pyramidal cells, unlike layer 2/3 pyramidal cells, undergo only modest electrophysiological changes with aging, and that these changes are unlikely to contribute to age-related cognitive decline.

Calcium influx through presynaptic 5-HT3 receptors facilitates GABA release in the hippocampus: in vitro slice and synaptosome studies.

Serotonin 5-hydroxytryptamine type 3 receptors (5HT3R) are Ca2+-permeant, non-selective cation channels that have been localized to presynaptic terminals and demonstrated to modulate neurotransmitter release. In the present study the effect of 5-HT on GABA release in the hippocampus was characterized using both electrophysiological and biochemical techniques. 5-HT elicited a burst-like, 6- to 10-fold increase in the frequency of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) measured with whole-cell voltage-clamp recordings of CA1 neurons in hippocampal slices. When tetrodotoxin was used to block action potential propagation, the 5-HT-induced burst of IPSCs was still observed. Stimulation of hippocampal synaptosomes with 5-HT resulted in a significant increase in the amount of [3H]GABA released by hyperosmotic saline. In both preparations, the 5-HT effect was shown to be mediated by 5HT3Rs, as it was mimicked by the selective 5HT3R agonist m-chlorophenyl biguanide and blocked by the selective 5HT3R antagonist 3-tropanylindole-3-carboxylate hydrochloride. The 5HT3R-mediated increase in GABA release was blocked by 100 microM cadmium or by omitting Ca2+ in external solutions, indicating the Ca2+-dependence of the effect. The high voltage-activated Ca2+ channel blockers omega-conotoxin GVIA and omega-conotoxin MVIIC and 10 microM cadmium had no significant effect on the 5-HT3R-mediated enhancement of GABA release, indicating that Ca2+ influx through the 5-HT3R facilitates GABA release. Taken together, these data provide direct evidence that Ca2+ entry via presynaptic 5HT3Rs facilitates the release of GABA from hippocampal interneurons.

Normal aging results in decreased synaptic excitation and increased synaptic inhibition of layer 2/3 pyramidal cells in the monkey prefrontal cortex.

Executive system function, mediated largely by the prefrontal cortex (PFC), often declines significantly with normal aging in humans and non-human primates. The neural substrates of this decline are unknown, but age-related changes in the structural properties of PFC neurons could lead to altered synaptic signaling and ultimately to PFC dysfunction. The present study addressed this issue using whole-cell patch clamp assessment of excitatory and inhibitory postsynaptic currents (PSCs) in layer 2/3 pyramidal cells in in vitro slices of the PFC from behaviorally characterized young (< or =12 years old) and aged (> or =19 years old) rhesus monkeys. Behaviorally, aged monkeys were significantly impaired in performance on memory and executive system function tasks. Physiologically, the frequency of spontaneous glutamate receptor-mediated excitatory PSCs was significantly reduced in cells from aged monkeys, while the frequency of spontaneous GABAA receptor-mediated inhibitory PSCs was significantly increased. In contrast, there was no effect of age on the frequency, amplitude, rise time or decay time of action potential-independent miniature excitatory and inhibitory PSCs. The observed change in excitatory-inhibitory synaptic balance likely leads to significantly altered signaling properties of layer 2/3 pyramidal cells in the PFC with age.

Development and modulation of GABA(A) receptor-mediated neurotransmission in the CA1 region of prenatally protein malnourished rats.

Prenatal protein malnutrition has been demonstrated to result in alterations in the serotonergic and GABAergic neurotransmitter systems in the rat hippocampus. In the present study, whole-cell patch clamp recordings of CA1 pyramidal cells were employed in an effort to gain insight into the specific cellular locus and functional consequences of the previously reported changes. Hippocampal slices were prepared from Sprague-Dawley rats whose dams were fed either a normal (25% casein) or low (6% casein) protein diet during pregnancy. The development of GABA(A) receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) and their modulation by the benzodiazipine agonist zolpidem were compared in cells from the two nutritional groups at postnatal days 7, 14, 21 and >90. The modulation of mIPSCs by serotonin was also examined in cells from 21 day old rats. No significant differences were observed in the characteristics of mIPSCs in cells from control vs. prenatally protein malnourished rats at any of the ages studied, although there was a trend for a higher frequency of mIPSCs in adult (>p90) prenatally protein malnourished rats. At all ages, zolpidem produced a significant increase in the mean decay time of mIPSCs that was not significantly different in cells from the two nutritional groups. Serotonin application resulted in a significant increase in the frequency of mIPSCs in CA1 pyramidal cells but there was no significant difference between cells from the two nutritional groups in the characteristics of this effect. These data demonstrate that the previously observed alterations in the serotonergic and GABAergic systems that result from prenatal protein malnutrition do not have significant functional consequences at a single cell level in the CA1 region of the rat hippocampus as measured in vitro.

Morphology and electrophysiology of dentate granule cells in the rhesus monkey: comparison with the rat.

The morphologic and electrophysiologic properties of dentate granule cells in the young adult rhesus monkey (Macaca mulatta) were examined for the first time with whole-cell patch clamp recordings and intracellular biocytin filling in in vitro hippocampal slice preparations. Data from monkeys were compared with data generated in an identical manner from adult Sprague-Dawley rats. Intracellularly filled monkey and rat granule cells were identical in numerous morphologic parameters, including area of somata, total dendritic length, dendritic spread, segment number and length, and branching pattern. The single statistically significant difference in morphology was the vertical extent of the dendritic tree (distance from soma to fissure), which was 20% greater in the monkey. The passive membrane properties (resting membrane potential, input resistance, and membrane time constant) measured under current clamp conditions were virtually identical. The thresholds and amplitudes of action potentials were the same, but significant differences were seen in the kinetics of single action potentials. Monkey granule cell action potentials were significantly longer in duration (with slower rise and fall times) than action potentials in rat cells. These differences were likely due to a much smaller fast after hyperpolarization in the monkey as compared with the rat cells. Thus, with the exception of action potential properties, the principal finding of this study is that there is significant conservation of both form and function in dentate granule cells in these two species, despite the enormous phylogenetic separation. This suggests that granule cell properties may be extremely stable across diverse mammalian species.

Exocytotic Ca2+ channels in mammalian central neurons.

Intracellular Ca2+ initiates physiological events as diverse as gene transcription, muscle contraction, cell division and exocytosis. Predictably, the metabolic machinery that elicits and responds to changes in intracellular Ca2+ is correspondingly heterogeneous. This review focuses on one element of this complex web that is of particular importance to neurobiologists: identifying which members of the voltage-dependent Ca(2+)-channel superfamily are responsible for the Ca2+ that enters nerve terminals and elicits vesicular release of chemical transmitters.

Sensory neuron N-type calcium currents are inhibited by both voltage-dependent and -independent mechanisms.

The voltage dependence of gamma-aminobutyric-acid- and norepinephrine-induced inhibition of N-type calcium current in cultured embryonic chick dorsal-root ganglion neurons was studied with whole-cell voltage-clamp recording. The inhibitory action of the neurotransmitters was comprised of at least two distinct modulatory components, which were separable on the basis of their differential voltage dependence. The first component, which we term "kinetic slowing", is associated with a slowing of the activation kinetics--an effect that subsides during a test pulse. The kinetic-slowing component is largely reversed at depolarized voltages (i.e., it is voltage-dependent). The second component, which we term "steady-state inhibition", is by definition not associated with a change in activation kinetics and is present throughout the duration of a test pulse. The steady-state inhibition is not reversed at depolarized voltages (i.e., it is voltage-independent). Although the two components can be separated on the basis of their voltage dependence, they appear to be indistinguishable in their time courses for onset and recovery as well as their rates of desensitization following multiple applications of transmitter. Furthermore, neither component requires cell dialysis, as both are observed using perforated-patch as well as whole-cell recording configurations. The co-existence in nerve terminals of both voltage-dependent and -independent mechanisms to modulate calcium channel function could offer a means of differentially controlling synaptic transmission under conditions of low- and high-frequency presynaptic discharge.

Multiple calcium channel types control glutamatergic synaptic transmission in the hippocampus.

N-type calcium channels play a dominant role in controlling synaptic transmission in many peripheral neurons. Transmitter release from mammalian central nerve terminals, however, is relatively resistant to the N channel antagonist omega-conotoxin GVIA. We studied the sensitivity of glutamatergic synaptic transmission in rat hippocampal slices to omega-conotoxin and to omega-Aga-IVA, a P channel antagonist. Both toxins reduced the amplitude of excitatory postsynaptic potentials in CA1 pyramidal neurons, but omega-Aga-IVA was the more rapid and efficacious. These results were corroborated by biochemical studies measuring subsecond, calcium-dependent [3H]glutamate release from hippocampal synaptosomes. Thus, at least two calcium channel types trigger glutamate release from hippocampal neurons, but P-type plays a more prominent role. Eliminating synaptic transmission in the CNS, therefore, may require inhibiting more than a single calcium channel type.

Inhibitory action of muscarinic agonists on neurons in the rat laterodorsal tegmental nucleus in vitro.

1. The effects of the mixed cholinergic agonist carbachol and the muscarinic agonist methacholine (MCh) on neurons of the laterodorsal tegmental nucleus (LDT) were studied with the use of intracellular and whole-cell patch-clamp recordings in a rat brain stem slice preparation. 2. Neurons were classified into one of two categories on the basis of their intrinsic membrane properties: those that displayed a prominent low-threshold calcium burst (LTB, 60%) and those that did not exhibit such a burst (non-LTB, 40%). 3. Neurons from which recordings were obtained were filled with biocytin, visualized with Texas-red avidin, and identified as cholinergic or noncholinergic with NADPH-diaphorase histochemistry. Eighty percent of the LTB neurons that were processed in this manner were cholinergic, and 60% of the non-LTB neurons were cholinergic. 4. Carbachol elicited a membrane hyperpolarization associated with a decrease in input resistance in 95% of the cells tested. Under voltage clamp this response was shown to be due to an outward current that reversed near the equilibrium potential for potassium and displayed marked inward rectification. The conductance/voltage relationship was fit to the Boltzmann equation with a mean V1/2 = -73 +/- 4 (SD) mV and a mean k value of 10 +/- 4. The carbachol-evoked current was fully blocked by extracellular barium. 5. There was no significant effect of carbachol on the transient currents IA or IT. 6. The carbachol-evoked current was mimicked by the specific muscarinic agonist methacholine and blocked by high concentrations of the muscarinic receptor antagonist pirenzepine (IC50 = 580 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of NADPH-diaphorase positive somata in the brainstem of the monitor lizard Varanus exanthematicus.

The distribution and size of presumptive cholinergic somata in the brainstem of the Savanna monitor lizard Varanus exanthematicus were determined using the enzyme histochemical marker NADPH-diaphorase. Numerous neurons were labelled in the lizard brainstem with this technique. A three dimensional computer reconstruction of this population revealed that it shows marked similarity to the laterodorsal tegmental/pedunculopontine tegmental cholinergic cell column, an NADPH-diaphorase positive population in the mesopontine tegmentum of the mammalian brainstem.

Characterization of superior cervical ganglion neurons that project to the submandibular glands, the eyes, and the pineal gland in rats.

These studies sought to determine whether the cell bodies of rat superior cervical ganglion neurons projecting to three very different target organs differ in terms of their size, number, location within the ganglion and/or neuropeptide content, and whether these features are altered in response to neonatal deafferentiation of the ganglion. A series of retrograde tracer, immunocytochemical, and double-labeling studies revealed differences in the size, number, location and neuropeptide content of superior cervical ganglion neurons that project to the submandibular salivary glands, eyes, or pineal gland. The mean areas of the cell bodies of neurons projecting to the submandibular gland are largest, those projecting to the eye are smallest, and those projecting to the pineal are intermediate in size. Submandibular gland projecting neurons are found throughout the ganglion, while the eye and pineal projecting populations are localized to the rostral quadrants. The different subpopulations of target organ specific superior cervical ganglion neurons are heterogeneous in their content of vasoactive intestinal peptide-, neuropeptide Y- and somatostatin-like immunoreactivity. A greater percentage of submandibular gland than of pineal projecting neurons display vasoactive intestinal peptide-like immunoreactivity, but there are no differences in the percentage of neurons displaying neuropeptide Y- or somatostatin-like immunoreactivity between the target organ specific groups. Neonatal deafferentiation does not result in changes in the size, number or distribution of target organ specific neurons, or in the percentage of immunoreactive neurons in these populations. In conclusion, these studies provide evidence that the size and distribution of neurons and percentage of peptide-containing neurons in the superior cervical ganglion is related to the target organ innervated, but provides no evidence of exclusive target organ-peptide relationships.

Labelling and recording from dissociated target-specific rat superior cervical ganglion neurons.

A population of neurons was retrogradely labelled in the superior cervical ganglia (SCG) of the adult rat following the injection of the fluorescent dye Fast blue into the submandibular salivary glands (SMG). The neurons retained the fluorescent label following dissociation and culture. Electrical and chemosensitive properties of the labelled neurons were studied with the whole-cell patch-clamp technique.

Serotonin hyperpolarizes cholinergic low-threshold burst neurons in the rat laterodorsal tegmental nucleus in vitro.

Serotonergic suppression of cholinergic neuronal activity implicated in the regulation of rapid eye movement sleep and its associated phenomenon, pontogeniculooccipital waves, has long been postulated, but no direct proof has been available. In this study, intracellular and whole-cell patch-clamp recording techniques were combined with enzyme histochemistry to examine the intrinsic electrophysiological properties and response to serotonin (5-HT) of identified cholinergic rat laterodorsal tegmental nucleus neurons in vitro. Sixty-five percent of the recorded neurons demonstrated a prominent low-threshold burst, and of these, 83% were cholinergic. In current-clamp recordings 64% of the bursting cholinergic neurons tested responded to the application of 5-HT with a membrane hyperpolarization and decrease in input resistance. This effect was mimicked by application of the selective 5-HT type 1 receptor agonist carboxamidotryptamine maleate. Whole-cell patch-clamp recordings revealed that the hyperpolarizing response was mediated by an inwardly rectifying K+ current. Application of 5-HT decreased excitability and markedly modulated the discharge pattern of cholinergic bursting neurons: during a 5-HT-induced hyperpolarization these neurons exhibited no rebound burst after hyperpolarizing current input and a burst in response to depolarizing current input. In the absence of 5-HT, the relatively depolarized cholinergic bursting neurons responded to an identical hyperpolarizing current input with a burst and did not produce a burst after depolarizing current input. These data provide a cellular and molecular basis for the hypothesis that 5-HT modulates rapid eye movement sleep phenomenology by altering the firing pattern of bursting cholinergic neurons.

Somatostatin-, vasoactive intestinal polypeptide- and neuropeptide Y-like immunoreactivity in eye- and submandibular gland-projecting sympathetic neurons.

Studies combine the use of the retrograde tracer, fluorogold, and immunocytochemical staining to determine whether superior cervical ganglion (SCG) neurons projecting to the iris or submandibular gland (SMG) in adult male and female rats show distinctive immunoreactivity to somatostatin (SS), vasoactive intestinal polypeptide (VIP), or neuropeptide Y. Overall, more SMG-projecting neurons than eye-projecting neurons contain VIP-like immunoreactivity (VIP-LI), and more eye-projecting neurons than SMG-projecting neurons contain SS-LI and VIP-LI. Thus, postganglionic neurons of the SCG that project to specific target tissues are heterogeneous in their peptide content, and there are differences in the pattern of peptide-immunoreactivity between neurons projecting to these two target tissues. In addition, the results indicate that there may be gender differences in the expression of these neuropeptides.