Healthy brain aging: Interplay between reactive species, inflammation and energy supply.

Brains' high energy expenditure with preferable utilization of glucose and ketone bodies, defines the specific features of its energy homeostasis. The extensive oxidative metabolism is accompanied by a concomitant generation of high amounts of reactive oxygen, nitrogen, and carbonyl species, which will be here collectively referred to as RONCS. Such metabolism in combination with high content of polyunsaturated fatty acids creates specific problems in maintaining brains' redox homeostasis. While the levels of products of interaction between RONCS and cellular components increase slowly during the first two trimesters of individuals' life, their increase is substantially accelerated towards the end of life. Here we review the main mechanisms controlling the redox homeostasis of the mammalian brain, their age-dependencies as well as their adaptive potential, which might turn out to be much higher than initially assumed. According to recent data, the organism seems to respond to the enhancement of aging-related toxicity by forming a new homeostatic set point. Therefore, further research will focus on understanding the properties of the new set point(s), the general nature of this phenomenon and will explore the limits of brains' adaptivity.

In vivo functional properties of juxtaglomerular neurons in the mouse olfactory bulb.

Juxtaglomerular neurons represent one of the largest cellular populations in the mammalian olfactory bulb yet their role for signal processing remains unclear. We used two-photon imaging and electrophysiological recordings to clarify the in vivo properties of these cells and their functional organization in the juxtaglomerular space. Juxtaglomerular neurons coded for many perceptual characteristics of the olfactory stimulus such as (1) identity of the odorant, (2) odorant concentration, (3) odorant onset, and (4) offset. The odor-responsive neurons clustered within a narrow area surrounding the glomerulus with the same odorant specificity, with ~80% of responding cells located ≤20 µm from the glomerular border. This stereotypic spatial pattern of activated cells persisted at different odorant concentrations and was found for neurons both activated and inhibited by the odorant. Our data identify a principal glomerulus with a narrow shell of juxtaglomerular neurons as a basic odor coding unit in the glomerular layer and underline the important role of intraglomerular circuitry.

Large-scale oscillatory calcium waves in the immature cortex.

Two-photon imaging of large neuronal networks in cortical slices of newborn rats revealed synchronized oscillations in intracellular Ca2+ concentration. These spontaneous Ca2+ waves usually started in the posterior cortex and propagated slowly (2.1 mm per second) toward its anterior end. Ca2+ waves were associated with field-potential changes and required activation of AMPA and NMDA receptors. Although GABAA receptors were not involved in wave initiation, the developmental transition of GABAergic transmission from depolarizing to hyperpolarizing (around postnatal day 7) stopped the oscillatory activity. Thus we identified a type of large-scale Ca2+ wave that may regulate long-distance wiring in the immature cortex.

Developmental profile and synaptic origin of early network oscillations in the CA1 region of rat neonatal hippocampus.

1. By applying fura-2-based fluorometric calcium imaging to neonatal rat hippocampal slices we identified a developmentally regulated spontaneous neuronal activity in the CA1 region of the hippocampus. The activity consisted of bursts of intracellular Ca2+ transients recurring synchronously at a slow rate of 0.4-2 min-1 in the entire population of pyramidal neurones and interneurones. 2. These early network oscillations (ENOs) were present during a restricted period of postnatal development. Thus, they were not detected at the day of birth (P0), at P1-P4 they consisted of bursts of large (up to 1.5 microM) Ca2+ transients, gradually transforming into regularly occurring, smaller Ca2+ transients during the subsequent week. Beyond P15-P16 no ENOs were detected. 3. The ENOs were blocked by tetrodotoxin (TTX) and by a reduction in temperature from 33-35 degrees C to 20-22 degrees C. By combining fluorometric imaging with whole-cell current-clamp recordings, we found that each ENO-related Ca2+ transient was associated with a high-frequency (up to 100 Hz) train of action potentials riding on a depolarizing wave. 4. Recordings in the voltage-clamp mode revealed barrages of synaptic currents that were strictly correlated with the ENO-associated Ca2+ transients in neighbouring pyramidal neurones. Perfusing the cells with an intracellular solution that allowed for a discrimination between GABAA and glutamate receptor-mediated currents showed that these barrages of synaptic currents were predominantly of GABAergic origin. 5. The ENOs were totally blocked by the GABAA receptor antagonist bicuculline and they were also substantially reduced by the glutamatergic antagonists D,L-2-amino-5-phosphonovaleric acid (D, L-APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). 6. Synaptic stimulation and application of the GABAA receptor agonist muscimol mimicked the spontaneous Ca2+ transients in pyramidal neurones. The efficacy of muscimol in evoking Ca2+ transients decreased during development in parallel with the gradual disappearance of the ENOs. 7. The developmental decrease in the amplitude of ENO-associated Ca2+ transients occurred in parallel with the transformation of the excitatory synaptic transmission in the hippocampus from the immature GABAergic to the mature glutamatergic form. Thus, at the beginning of the first postnatal week single-shock synaptic stimulation produced Ca2+ transients that were completely blocked by bicuculline. At the end of the second postnatal week the same type of evoked synaptic stimulation produced a Ca2+ transient that was little affected by bicuculline but was abolished by the combined application of D,L-APV and CNQX. 8. These results demonstrate the presence of periodic and spontaneous Ca2+ transients in the majority of pyramidal cells and interneurones of the neonatal CA1 hippocampal network. These ENOs exhibit a highly region-specific developmental profile and may control the activity-dependent wiring of the synaptic connectivity during early postnatal development.

Release and sequestration of calcium by ryanodine-sensitive stores in rat hippocampal neurones.

1. The properties of ryanodine-sensitive Ca2+ stores in CA1 pyramidal cells were investigated in rat hippocampal slices by using whole-cell patch-clamp recordings combined with fura-2-based fluorometric digital imaging of cytoplasmic Ca2+ concentration ([Ca2+]i). 2. Brief pressure applications of caffeine onto the somata of pyramidal cells caused large transient increases in [Ca2+]i (Ca2+ transients) of 50-600 nM above baseline. 3. The Ca2+ transients evoked by caffeine at -60 mV were not associated with an inward current, persisted after blocking voltage-activated Ca2+ currents and were completely blocked by bath-applied ryanodine. Similar transients were also evoked at +60 mV. Thus, these transients reflect Ca2+ release from intracellular ryanodine-sensitive Ca2+ stores. 4. The Ca2+ transients evoked by closely spaced caffeine pulses rapidly decreased in amplitude, indicating progressive depletion of the Ca2+ stores. The amplitude of the Ca2+ transients recovered spontaneously with an exponential time constant of 59 s. Recovery was accelerated by depolarization-induced elevations in [Ca2+]i and blocked by cyclopiazonic acid (CPA) and thapsigargin, indicating that store refilling is mediated by endoplasmic reticulum Ca(2+)-ATPases. 5. Even without prior store depletion the caffeine-induced Ca2+ transients disappeared after 6 min exposure to CPA, suggesting that ryanodine-sensitive Ca2+ stores are maintained at rest by continuous Ca2+ sequestration. 6. Caffeine-depleted Ca2+ stores did not refill in Ca(2+)-free saline, suggesting that the refilling of the stores depends upon Ca2+ influx through a 'capacitative-like' transmembrane influx pathway operating at resting membrane potential. The refilling of the stores was also blocked by Ni2+ and gallopamil (D600). 7. Elevations of basal [Ca2+]i produced by bath-applied KCl markedly potentiated (up to 6-fold) the caffeine-induced Ca2+ transients. The degree of potentiation was positively related to the increase in basal [Ca2+]i. The Ca2+ transients remained potentiated up to 9 min after reversing the KCl-induced [Ca2+]i increase. Thus, the ryanodine-sensitive Ca2+ stores can 'overcharge' when challenged with an increase in [Ca2+]i and slowly discharge excess Ca2+ after basal [Ca2+]i returns to its resting level. 8. Pressure applications of caffeine onto pyramidal cell dendrites evoked local Ca2+ transients similar to those separately evoked in the respective somata. Thus, dendritic ryanodine-sensitive Ca2+ stores are also loaded at rest and can function as independent compartments. 9. In conclusion, the ryanodine-sensitive Ca2+ stores in hippocampal pyramidal neurones contain a releasable pool of Ca2+ that is maintained by a Ca2+ entry pathway active at subthreshold membrane potentials. Ca2+ entry through voltage-gated Ca2+ channels transiently overcharges the stores. Thus, by acting as powerful buffers at rest and as regulated sources during activity, Ca2+ stores may control the waveform of physiological Ca2+ signals in CA1 hippocampal pyramidal neurones.

Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene.

Intracellular calcium-binding proteins are abundantly expressed in many neuronal populations. Previous evidence suggests that calcium-binding proteins can modulate various neuronal properties, presumably by their action as calcium buffers. The importance of calcium-binding proteins for nervous system function in an intact integrated system is, however, less clear. To investigate the physiological role of a major endogenous calcium-binding protein, calbindin D28k (calbindin) in vivo, we have generated calbindin null mutant mice by gene targeting. Surprisingly, calbindin deficiency does not affect general parameters of development and behavior or the structure of the nervous system at the light microscopic level. Null mutants are, however, severely impaired in tests of motor coordination, suggesting functional deficits in cerebellar pathways. Purkinje neurons, the only efferent of the cerebellar cortex, and inferior olive neurons, the source of the climbing fiber afferent, have previously been shown to express calbindin. Correlated with this unusual type of ataxia, confocal calcium imaging of Purkinje cells in cerebellar slices revealed marked changes of synaptically evoked postsynaptic calcium transients. Their fast, but not their slow, decay component had larger amplitudes in null mutant than in wild-type mice. We conclude that endogenous calbindin is of crucial importance for integrated nervous system function.

Molecular determinants of NMDA receptor function in GABAergic neurones of rat forebrain.

1. The functional and molecular properties of NMDA receptors (NMDA-Rs) were studied in single, visually identified GABAergic medial septal neurones of the rat forebrain using patch clamp, fluorometric Ca2+ measurements and the single-cell reverse transcription-polymerase chain reaction (RT-PCR) technique. 2. Large neurones close to the mid-line of the medial septal region were shown by the expression of mRNA for a form of glutamate decarboxylase (GAD65) to be almost exclusively GABAergic. A variety of NR2 subunit combinations were detected in the same population of neurones. When tested for NR2A-C, all but one neurone were shown to express mRNA for NR2B. The NR2B subunit mRNA was usually detected together with NR2A or NR2C. mRNA for NR2D was detected in most neurones from a separate batch of cells tested only for this subunit. 3. Single channel measurements in outside-out patches combined with RT-PCR on the same cell showed that NMDA-R channels from these neurones had main single channel conductance levels of 42 pS in 2 mM Ca2+ and 49 pS in 1 mM Ca2+. In addition, a number of other conductance levels were observed, with values in 2 mM Ca2+ of 51, 31, 19 and 13 pS. No clear difference was observed in the pattern of conductance levels displayed by neurones in which different subunit combinations were detected. 4. Whole-cell agonist-induced currents were strongly reduced by the NMDA-R antagonist ifenprodil, at a concentration that mainly affects receptors containing NR2B in recombinant systems. Currents activated by NMDA had a high sensitivity to extracellular Mg2+. 5. The fraction of the total cation current through NMDA-R that was carried by Ca2+, measured using a combination of patch clamp and fluorometry in neurones loaded with a high concentration of the Ca2+ indicator fura-2, was found to be approximately 12%. 6. NMDA-R-mediated excitatory synaptic currents (EPSCs) had similar time courses to those in neurones in other brain regions. The decay kinetics were biexponential, with respective mean values for the fast (tau f) and slow (tau 8) time constants of 79 and 300 ms at -60 mV, and 66 and 284 ms at +40 mV. EPSCs were greatly reduced by ifenprodil (3 microM). 7. In conclusion, NMDA receptors in GABAergic medial septal neurones display a characteristic functional profile. The NR2 subunit mRNA detected and the single channel conductance levels observed suggest that, in addition to NR2B, which is present in nearly all cells, NR2A, NR2C and NR2D are also expressed. However, most of the functional properties of NMDA-Rs in these neurones, including the strong inhibition by ifenprodil and Mg2+, the high fractional Ca2+ current, and the time course of the synaptic currents, are more consistent with those known for NR2B than for the other NR2 subunits. These results suggest that the NR2B subunit dominates over other NR2 subunits in determining the functional properties of NMDA-Rs in these neurones.

Activity-dependent wiring of the developing hippocampal neuronal circuit.

In the developing hippocampus, functional excitatory synaptic connections seem to be recruited from a preformed, initially silent synaptic network. This functional synapse induction requires presynaptic action potentials paired with postsynaptic depolarization, thus obeying Hebb's rule of association. During early postnatal development the hippocampus exhibits an endogenous form of patterned neuronal activity that is driven by GABAergic depolarization. We propose that this recurrent activity promotes the input-specific induction of functional synapses in the CA1 region. Thus, activity-dependent synaptic reorganization in the developing hippocampus appears to be dominated by an active recruitment of new synapses rather than an active elimination of redundant connections.

Fractional Ca2+ currents through somatic and dendritic glutamate receptor channels of rat hippocampal CA1 pyramidal neurones.

1. The Ca2+ permeability of non-NMDA and NMDA receptor channels was studied using a fluorometric flux measurement approach in somata and dendrites of CA1 pyramidal neurones in rat hippocampal slices. For this purpose, the Ca2+ fraction of the total cation current (named 'fractional Ca2+ current') was measured directly from the change in the Ca(2+)-sensitive fura-2 fluorescence at 380 nm excitation wavelength. 2. The fractional Ca2+ current through the somatic NMDA receptor channels was 10.69 +/- 2.13% (mean +/- S.D.) and that through dendritic receptor channels was 10.70 +/- 1.96%. The fractional Ca2+ current was not dependent on the extracellular Mg2+ concentration and its voltage dependence was in agreement with the Goldman-Hodgkin-Katz current equation. 3. AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) or kainate applications produced small but significant Ca2+ entry. Fractional Ca2+ currents of 0.58 +/- 0.34% were measured for somatic AMPA applications, 0.68 +/- 0.20% for somatic kainate applications, 0.66 +/- 0.25% for dendritic AMPA applications and 0.61 +/- 0.16% for dendritic kainate applications. 4. The expression pattern of glutamate receptor subunits encoding messenger ribonucleic acids (mRNAs) was analysed with the single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) approach applied to CA1 pyramidal neurones. The AMPA receptor subunits GluR-A, GluR-B and GluR-C, and the NMDA receptor subunits NR2A and NR2B were found to be abundantly expressed in all CA1 pyramidal neurones tested. 5. This study establishes the fractional Ca2+ current through somatic and dendritic NMDA and non-NMDA receptor channels in CA1 pyramidal neurones. The dendritic, presumably synaptic, NMDA receptor channels are highly Ca2+ permeable and have a fractional Ca2+ current closely resembling that of somatic extrasynaptic NMDA receptor channels. Both somatic and dendritic non-NMDA receptor channels are of the 'low Ca2+ permeable' type and have a fractional Ca2+ current that is about twenty times smaller than that of NMDA receptor channels.

Fractional calcium current through neuronal AMPA-receptor channels with a low calcium permeability.

The Ca(2+)-permeation properties of AMPA-receptor (AMPA-R) channels in Purkinje neurons in rat cerebellar slices were studied using a combination of whole-cell patch-clamp recordings, Fura-2 fluorometry, and single-cell reverse-transcription (RT)-PCR. Several lines of evidence indicate that Purkinje neurons, at both early and late stages of postnatal development, express exclusively AMPA-R channels with a low Ca2+ permeability. First, no Ca2+ signal was detected during application of either AMPA or kainate to Purkinje neurons loaded with the Ca2+ indicator Fura-2 AM. In contrast, kainate application induced large Ca2+ transients in Bergmann glia cells. Second, in ion substitution experiments, when Ca2+ is the only extracellular permeant cation, the reversal potential corresponds to that expected for AMPA-R channels with a low permeability for Ca2+. Third, using a fluorometric flux-measurement approach (Schneggenburger et al., 1993a), we found that the Ca2+ fraction of the total cation current through AMPA-R channels is approximately 0.6%. This value is approximately sixfold lower than that found for recombinant AMPA-R lacking the AMPA-R subunit GluR2. Furthermore, single-cell RT-PCR experiments revealed the presence of the AMPA-R subunits GluR1, GluR2, and GluR3 in Purkinje neurons in cerebellar slices at developmental stages corresponding to those studied electrophysiologically. The expression of GluR2 in all cells tested (n = 14) is consistent with the subunit composition predicted from studies of recombinant AMPA-R channels with a low permeability for Ca2+ (Burnashev et al., 1992b). In conclusion, this study establishes that cerebellar Purkinje neurons at all postnatal developmental stages possess AMPA-R channels with a low permeability for Ca2+.

Ryanodine receptor-mediated intracellular calcium release in rat cerebellar Purkinje neurones.

1. Ryanodine receptor-mediated Ca2+ release was investigated in Purkinje neurones of rat cerebellar slices by using whole-cell patch-clamp recordings combined with fluorometric digital imaging of cytoplasmic Ca2+ concentration ([Ca2+]i). 2. Caffeine caused a transient increase in [Ca2+]i in the somata and dendrites of Purkinje neurones. Caffeine-induced Ca2+ transients were not associated with a membrane inward current and persisted in Ca(2+)-free external solutions, indicating that they are caused by Ca2+ released from intracellular stores. The amplitudes of the caffeine-mediated elevations in [Ca2+]i were strongly dependent on the baseline level of [Ca2+]i. 3. Intracellular application of Ruthenium Red through the patch pipette blocked caffeine-induced Ca2+ transients in Purkinje neurones. Ryanodine when applied either intra- or extracellularly caused a use-dependent block of caffeine-induced Ca2+ release. 4. Depolarization-induced Ca2+ transients were strongly prolonged by caffeine. Several lines of evidence suggest that these prolongations reflect Ca(2+)-induced Ca2+ release. 5. Despite the presence of skeletal muscle type ryanodine receptors in Purkinje neurones, depolarizing pulses failed to induce any changes in [Ca2+]i when the influx of Ca2+ through voltage-gated channels was prevented by using Ca(2+)-free solution, or when applying blockers of voltage-gated Ca2+ channels. 6. Dendritic Ca2+ transients produced by stimulation of the excitatory climbing fibre synaptic input were also prolonged by caffeine, indicating that ryanodine receptor-mediated release of Ca2+ may be involved in synaptic signalling in cerebellar Purkinje neurones. 7. Ryanodine receptor-mediated release of Ca2+ in cerebellar Purkinje neurones can be explained by a model in which release of Ca2+ is strongly facilitated by the co-operative action of Ca2+, caffeine and/or ryanodine. Our results suggest that Ca2+ release in these central neurones becomes prominent only during episodes of intensive electrical activity associated with increased Ca2+ entry.

Glutamate induces long-term increase in the frequency of single N-methyl-D-aspartate channel openings in hippocampal CA1 neurons examined in situ.

Long-term potentiation is currently a leading candidate for a physiological memory mechanism in CNS. The interpretation of this phenomenon is contradictory in many respects. However, there is clear evidence that long-term potentiation is critically dependent on activation of N-methyl-D-aspartate receptors. Recently it has been shown that extracellularly applied glutamate also induces long-lasting changes in the properties of synaptic transmission in the hippocampus that can be attributed to long-term potentiation. The involvement of presynaptic mechanisms has been reported. Here we demonstrate a definite increase in both the open time and open-state probability of N-methyl-D-aspartate-operated channels, induced by prolonged application of glutamate to hippocampal slices.

Glutamate and theta-rhythm stimulation selectively enhance NMDA component of EPSC in CA1 neurons of young rats.

Using in situ whole-cell patch clamp of hippocampal CA1 pyramidal neurons we demonstrate that glutamate initiates processes resulting in an increase in the amplitude of the excitatory post-synaptic current (EPSC). In adult animals both, NMDA and non-NMDA components of the EPSC increase in parallel. In young animals only the NMDA component is increased. A similar enhancement of the EPSC can be achieved by the stimulation of excitatory synaptic inputs to CA1 neurons with the frequency of the theta-rhythm. EPSCs remain enhanced for more than 60 min. The selective enhancement of the NMDA component in young animals is inhibited by preincubation of slices with the NO-synthase blocker, N omega-nitro-L-arginine (NA) or by the NO-scavenger, hemoglobin.

Trans-ACPD selectively inhibits excitability of hippocampal CA1 neurones.

The selective agonist of metabotropic glutamate receptors, t-ACPD (trans-1-aminocyclopentyl-1,3-dicarboxylic acid) (100-250 microM), reversibly inhibited extracellularly recorded EPSP (excitatory postsynaptic potentials) in the CA1 layer of rat hippocampus. This effect was accompanied by depression of electrical excitability of CA1 neurons as revealed by their antidromic stimulation. The excitability of CA3 neurons remained uneffected. Peculiarly, excitatory postsynaptic currents recorded in voltage clamped, internally perfused CA1 cells remained unaltered. Selective depolarization of CA1 neurons may account for the phenomena described.

Adenosine-dependent enhancement by methylxanthines of excitatory synaptic transmission in hippocampus of rats.

Whole-cell patch-clamp was used to investigate synaptic transmission in hippocampal slices. Excitatory post-synaptic currents (EPSCs) were facilitated by low (less than or equal to 1 microM) adenosine (Ado) concentrations, while high concentrations had well-known inhibitory effects on the EPSC. When added on the background of preapplied Ado, methylxanthines caused a large potentiation of EPSCs. At saturation, the enhanced EPSC could exceed the control almost by an order of magnitude. Pertussis toxin strongly impaired the ability of Ado to block EPSCs but did not augment the facilitatory effect. The two components of the EPSC mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors were facilitated simultaneously and in equal proportions.