Long-term sensory deprivation selectively rearranges functional inhibitory circuits in mouse barrel cortex

Long-term whisker removal alters the balance of excitation and inhibition in rodent barrel cortex, yet little is known about the contributions of individual cells and synapses in this process. We studied synaptic inhibition in four major types of neurons in live tangential slices that isolate layer 4 in the posteromedial barrel subfield. Voltage-clamp recordings of layer 4 neurons reveal that fast decay of synaptic inhibition requires α1-containing GABA_A receptors. After 7 weeks of deprivation, we found that GABA_A-receptor-mediated inhibitory postsynaptic currents (IPSCs) in the inhibitory low-threshold-spiking (LTS) cell recorded in deprived barrels exhibited faster decay kinetics and larger amplitudes in whisker-deprived barrels than those in nondeprived barrels in age-matched controls. This was not observed in other cell types. Additionally, IPSCs recorded in LTS cells from deprived barrels show a marked increase in zolpidem sensitivity. To determine if the faster IPSC decay in LTS cells from deprived barrels indicates an increase in α1 subunit functionality, we deprived α1(H101R) mutant mice with zolpidem-insensitive α1-containing GABA_A receptors. In these mice and matched wild-type controls, IPSC decay kinetics in LTS cells were faster after whisker removal; however, the deprivation-induced sensitivity to zolpidem was reduced in α1(H101R) mice. These data illustrate a change of synaptic inhibition in LTS cells via an increase in α1-subunit-mediated function. Because α1 subunits are commonly associated with circuit-specific plasticity in sensory cortex, this switch in LTS cell synaptic inhibition may signal necessary circuit changes required for plastic adjustments in sensory-deprived cortex.     

Spontaneous activity competes with externally evoked responses in sensory cortex

The interaction between spontaneous and externally evoked neuronal activity is fundamental for a functional brain. Increasing evidence suggests that bursts of high-power oscillations in the 15- to 30-Hz beta-band represent activation of internally generated events and mask perception of external cues. Yet demonstration of the effect of beta-power modulation on perception in real time is missing, and little is known about the underlying mechanism. Here, we used a closed-loop stimulus-intensity adjustment system based on online burst-occupancy analyses in rats involved in a forepaw vibrotactile detection task. We found that the masking influence of burst occupancy on perception can be counterbalanced in real time by adjusting the vibration amplitude. Offline analysis of firing rates (FRs) and local field potentials across cortical layers and frequency bands confirmed that beta-power in the somatosensory cortex anticorrelated with sensory evoked responses. Mechanistically, bursts in all bands were accompanied by transient synchronization of cell assemblies, but only beta-bursts were followed by a reduction of FR. Our closed loop approach reveals that spontaneous beta-bursts reflect a dynamic state that competes with external stimuli.

The brain is constantly active, even at resting states in the absence of external stimuli (1). Spontaneously active resting-state networks (RSNs) were found in memory, visual, auditory, tactile, and sensorimotor regions, with activity patterns similar to task-evoked responses (2, 3). Functional connectivity studies in humans suggest that a default network, spontaneously activated at resting states and deactivated upon increased cognitive demands, antagonizes a network involved in active attention to external sensory input (4–8). However, whether the activity in different networks is anticorrelated is under debate, and their antagonizing mechanisms and influence on local circuits remain unknown (9).Here, we utilized the occupancy of high-power bursts in the beta-band (15 to 30 Hz) of local field potentials (LFPs) as an indicator of spontaneous activity to investigate its influence on detection in real time. Several lines of evidence relate the RSN to beta-bursts. First, spontaneous correlated oscillatory activity in beta (termed “beta-connectome”) (10) was reported in anatomical regions corresponding to the RSN (11, 12). A recent study derived this beta-connectome from burst occupancy (10). Second, beta-oscillations are dominant during the resting state (13) and bursts are responsible for virtually all beta-band power modulation (14). Third, task-dependent desynchronization of beta was observed in the somatosensory (15, 16), visual (17), auditory (18), and motor (19) cortices, resembling RSN deactivation (4, 7). The task-dependent averaged power modulation was attributed to changes in burst rates in rodents, nonhuman primates, and humans (14, 20, 21). Fourth, the burst duration (50 to a few hundred milliseconds) (22) is similar to “packets” of neural activity, which are conceived as messages initiated in a particular cortical region and spread as a wave over the cortex. Most of these packets are generated spontaneously, and spontaneous and sensory-evoked packets are remarkably similar (23).We found that bursts in all bands indicate transient synchronization of flexible neuronal networks, but only beta-bursts were followed by a reduction in population firing rate (FR). High occupancies of beta-bursts predicted reduced detection, and this effect can be counterbalanced bidirectionally in real time by adjusting the stimulus intensity according to burst occupancy.     

Emergence of functional subnetworks in layer 2/3 cortex induced by sequential spikes in vivo

During cortical circuit development in the mammalian brain, groups of excitatory neurons that receive similar sensory information form microcircuits. However, cellular mechanisms underlying cortical microcircuit development remain poorly understood. Here we implemented combined two-photon imaging and photolysis in vivo to monitor and manipulate neuronal activities to study the processes underlying activity-dependent circuit changes. We found that repeated triggering of spike trains in a randomly chosen group of layer 2/3 pyramidal neurons in the somatosensory cortex triggered long-term plasticity of circuits (LTPc), resulting in the increased probability that the selected neurons would fire when action potentials of individual neurons in the group were evoked. Significant firing pattern changes were observed more frequently in the selected group of neurons than in neighboring control neurons, and the induction was dependent on the time interval between spikes, N-methyl-D-aspartate (NMDA) receptor activation, and Calcium/calmodulin-dependent protein kinase II (CaMKII) activation. In addition, LTPc was associated with an increase of activity from a portion of neighboring neurons with different probabilities. Thus, our results demonstrate that the formation of functional microcircuits requires broad network changes and that its directionality is nonrandom, which may be a general feature of cortical circuit assembly in the mammalian cortex.Layer 2/3 neurons in the barrel cortex play a central role in integrative cortical processing (1–4). Neurons in layer 2/3 are interconnected with each other, and their axons and dendrites traverse adjacent barrel areas (5, 6). Recent calcium (Ca²⁺) imaging studies in awake animals showed that two very closely localized layer 2/3 pyramidal neurons are independently activated by different whiskers (7). In addition, adjacent layer 2/3 neurons have different receptive field properties; signals from different whiskers may emerge on different spines in the same neurons (8, 9). These findings suggest that the organization of functional subnetworks in somatosensory layer 2/3 is heterogeneous at the single-cell level and that microcircuits are assembled at a very fine scale (10). In vivo whole-cell recording experiments have also shown that most, but not all, layer 2/3 pyramidal neurons receive subthreshold depolarization by single-whisker stimulation with much broader receptive fields than neurons in layer 4 (11, 12). These anatomical and functional data suggest that electric signals relayed to the cortex by whisker activation are greatly intermingled within layer 2/3 neurons, and that studying the mechanisms by which these layer 2/3 neurons make connections may be critical for understanding the cortical network organizing principles underlying somatosensation.A previous modeling study suggested that spike timing-dependent plasticity (STDP) can lead to the formation of functional cortical columns and activity-dependent reorganization of neural circuits (13–16). However, how spikes arising in multiple neurons in vivo influence their connectivity is poorly understood. In this study using two-photon glutamate photolysis, which allowed us to control neuronal activity in a spatially and temporally precise manner, we examined activity-dependent cellular mechanisms during network rearrangement generated by repetitive spike trains in a group of neurons. We found that repetitive spikes on a group of neurons induced the probability of the neurons firing together. This circuit plasticity required spiking at short intervals among neurons and is expressed by N-methyl-D-aspartate (NMDA) receptor- and Calcium/calmodulin-dependent protein kinase II (CaMKII)-dependent long-lasting connectivity changes. The probability of firing was differentially affected by the order of the spike sequence but was not dependent on the physical distance between neurons. Thus, our data show that neuronal connectivity within a functional subnetwork is established in not only a preferred but also a directional manner.     

Spectral mixing of rhythmic neuronal signals in sensory cortex

The ability to compute the difference between two frequencies depends on a nonlinear operation that mixes two periodic signals. Behavioral and psychophysical evidence suggest that such mixing is likely to occur in the mammalian nervous system as a means to compare two rhythmic sensory signals, such as occurs in human audition, and as a means to lock an intrinsic rhythm to a sensory input. However, a neurological substrate for mixing has not been identified. Here we address the issue of nonlinear mixing of neuronal activity in the vibrissa primary sensory cortex of rat, a region that receives intrinsic as well as sensory-driven rhythmic input during natural whisking. In our preparation, the intrinsic signal originates from cortical oscillations that were induced by anesthetics, and the extrinsic input is introduced by periodic stimulation of vibrissae. We observed that the local extracellular current in vibrissa primary sensory cortex contained oscillatory components at the sum and difference of the intrinsic and extrinsic frequencies. In complementary experiments, we observed that the simultaneous stimulation of contralateral and ipsilateral vibrissae at different frequencies also led to current flow at the sum and difference frequencies. We show theoretically that the relative amplitudes of the observed mixture terms can be accounted for by a threshold nonlinearity in the input-output relation of the underlying neurons. In general, our results provide a neurological substrate for the modulation and demodulation of rhythmic neuronal signals for sensory coding and feedback stabilization of motor output.     

Neuronal correlates of sensory discrimination in the somatosensory cortex

Monkeys are able to discriminate the difference in frequency between two periodic mechanical vibrations applied sequentially to the fingertips. It has been proposed that this ability is mediated by the periodicity of the responses in the quickly adapting (QA) neurons of the primary somatosensory cortex (S1), instead of the average firing rates. We recorded from QA neurons of S1 while monkeys performed the vibrotactile discrimination task. We found that the periodic mechanical vibrations can be represented both in the periodicity and in the firing rate responses to varying degrees across the QA neuronal population. We then computed neurometric functions by using both the periodicity and the firing rate and sought to determine which of these two measures is associated with the psychophysical performance. We found that neurometric thresholds based on the firing rate are very similar to the animal's psychometric thresholds whereas neurometric thresholds based on periodicity are far lower than those thresholds. These results indicate that an observer could solve this task with a precision similar to that of the monkey, based only on the firing rate produced during the stimulus periods.     

Effect of ageing on Ca2+ uptake via NMDA receptors into barrel cortex slices of spontaneously hypertensive rats

In the normal ageing cortex of the brain there is a group of dying neurons with shrinking dendritic trees and a group of surviving neurons with expanding dendritic trees. The ageing process affects neurotransmitter systems, including glutamate neurons and NMDA receptors. Calcium is an important signaling molecule. It enters brain cells through NMDA receptors and voltage-gated calcium channels. Since NMDA receptors play an important role in brain plasticity, calcium uptake through NMDA receptors can be used as a measure of brain activity. This study therefore sought to determine the effect of ageing on NMDA-stimulated Ca²⁺ uptake into barrel cortex slices of Spontaneously Hypertensive Rats (SHR) compared to control Wistar-Kyoto rats (WKY). Young rats (prepuberty, 4–6 weeks) and adult rats (14–16 weeks) were used in the study. The results show a significant decrease in NMDA-stimulated Ca²⁺ uptake in adult rats compared to their young litter-mates. It can be concluded that ageing negatively affects NMDA-stimulated Ca²⁺ uptake into barrel cortex slices of SHR and WKY.     

Dissociation of experience-dependent and -independent changes in excitatory synaptic transmission during development of barrel cortex

A fundamental problem in the study of cortical development is the extent to which the formation and refinement of synaptic circuitry depends upon sensory experience. The barrel cortex is a useful model system to study experience-dependent cortical development because there is a simple mapping of individual whiskers to the corresponding barrel columns in the cortex. We investigated experience-dependent and -independent changes in glutamatergic synaptic transmission in the barrel cortex during the second postnatal week by comparing synaptic responses from whisker-intact mice at postnatal day (P) 7 and P14 with those from whisker-deprived mice at P14. alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA)-receptor-mediated excitatory synaptic responses were recorded from layer 2/3 pyramidal cells in vitro during voltage-clamp in response to stimulation in layer 4. We observed that the ratio of synaptic AMPA- to NMDA-receptor-mediated current (A/N ratio) increased with developmental age. The development of the A/N ratio was unchanged by deprivation of the whisker input throughout the second postnatal week. In contrast, the NMDA-receptor current decay and sensitivity to the NMDA receptor 2B subunit-selective antagonist ifenprodil was affected strongly by such deprivation. These results demonstrate a concurrent dissociation between sensory experience-dependent and -independent changes of glutamatergic transmission in the barrel cortex during the second postnatal week. Furthermore, they suggest that the development of subunit composition of synaptic receptors is dependent on sensory experience, whereas maturation of the synaptic A/N ratio is independent of such experience. Thus, different components of synaptic development may be governed by different developmental rules.     

Interaction of the AMPA receptor subunit GluR2/3 with PDZ domains regulates hippocampal long-term depression

The interaction of PDZ domain-containing proteins with the C termini of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors has been suggested to be important in the regulation of receptor targeting to excitatory synapses. Recent studies have shown that the rapid internalization of AMPA receptors at synapses may mediate, at least in part, the expression of long-term depression (LTD). We have previously shown that phosphorylation of Ser-880 on the AMPA receptor GluR2 subunit differentially regulated the interaction of GluR2 with the PDZ domain-containing proteins GRIP1 and PICK1. Here, we show that induction of LTD in hippocampal slices increases phosphorylation of Ser-880 within the GluR2 C-terminal PDZ ligand, suggesting that the modulation of GluR2 interaction with GRIP1 and PICK1 may regulate AMPA receptor internalization during LTD. Moreover, postsynaptic intracellular perfusion of GluR2 C-terminal peptides that disrupt GluR2 interaction with PICK1 inhibit the expression of hippocampal LTD. These results suggest that the interaction of GluR2 with PICK1 may play a regulatory role in the expression of LTD in the hippocampus.     

Brain-derived neurotrophic factor-dependent unmasking of "silent" synapses in the developing mouse barrel cortex

Brain-derived neurotrophic factor (BDNF) is a critical modulator of central synaptic functions such as long-term potentiation in the hippocampal and visual cortex. Little is known, however, about its role in the development of excitatory glutamatergic synapses in vivo. We investigated the development of N-methyl-D-aspartate (NMDA) receptor (NMDAR)-only synapses (silent synapses) and found that silent synapses were prominent in acute thalamocortical brain slices from BDNF knockout mice even after the critical period. These synapses could be partially converted to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-containing ones by adding back BDNF alone to the slice or fully converted to together with electric stimulation without affecting NMDAR transmission. Electric stimulation alone was ineffective under the BDNF knockout background. Postsynaptically applied TrkB kinase inhibitor or calcium-chelating reagent blocked this conversion. Furthermore, the AMPAR C-terminal peptides essential for interaction with PDZ proteins postsynaptically prevented the unmasking of silent synapses. These results suggest that endogenous BDNF and neuronal activity synergistically activate AMPAR trafficking into synaptic sites.     

Dynamic synaptic modification threshold: computational model of experience-dependent plasticity in adult rat barrel cortex.

Previous electrophysiological experiments have documented the response of neurons in the adult rat somatic sensory ("barrel") cortex to whisker movement after normal experience and after periods of experience with all but two whiskers trimmed close to the face (whisker "pairing"). To better understand how the barrel cortex adapts to changes in the flow of sensory activity, we have developed a computational model of a single representative barrel cell based on the Bienenstock, Cooper, and Munro (BCM) theory of synaptic plasticity. The hallmark of the BCM theory is the dynamic synaptic modification threshold, theta M, which dictates whether a neuron's activity at any given instant will lead to strengthening or weakening of the synapses impinging on it. The threshold theta M is proportional to the neuron's activity averaged over some recent past. Whisker pairing was simulated by setting input activities of the cell to the noise level, except for two inputs that represented untrimmed whiskers. Initially low levels of cell activity, resulting from whisker trimming, led to low values for theta M. As certain synaptic weights potentiated, due to the activity of the paired inputs, the values of theta M increased and after some time their mean reached an asymptotic value. This saturation of theta M led to the depression of some inputs that were originally potentiated. The changes in cell response generated by the model replicated those observed in in vivo experiments. Previously, the BCM theory has explained salient features of developmental experience-dependent plasticity in kitten visual cortex. Our results suggest that the idea of a dynamic synaptic modification threshold, theta M, is general enough to explain plasticity in different species, in different sensory systems, and at different stages of brain maturity.     

Effects of potassium conductance inhibitors on spontaneous diastolic depolarization and abnormal automaticity in human atrial fibers

Summary The capability of generating spontaneous diastolic depolarization and automaticity was investigated in vitro by means of standard microelectrode techniques in 50 human atrial preparations. Samples were classified within two groups: (i) group 1 was composed of 12 well-polarized preparations exhibiting action potentials that were fast responses (mean maximum diastolic potential:–75.5 mV and Vmax greater than 100V);(ii) group 2 was composed of 38 partially-depolarized samples (mean maximum distolic potential: –50.3 mV and Vmax less than 10 V/s) and was further divided into two subgroups. Subgroup 2A consisted of 20 spontaneously beating preparations and subgroup 2B consisted of 18 non-automatic partially-depolarized specimens. Highly-polarized fibers from group 1, although exhibiting a slight diastolic depolarization which was almost entirely suppressed by 2 mM cascium, never presented spontaneous activity under our experimental conditions. 90% of automatic fibers from subgroup 2A were sampled from dilated atria. In automatic preparations, diastolic depolarization was usually separated into two phases: an initial phase, also present in non-automatic fibers, and a late phase. Changes in the initial phase were not accompanied by concomitant changes in the spontaneous rate. Abnormal automaticity was clearly related to the late diastolic phase (absent in non-automatic fibers), the generation of which appeared to be specific property of automatic fibers. The use of K conductance inhibitors (caesium, 4-aminopyridine, barium, low K solutions) provided indirect evidence that neither delayed outward i_x current nor i_t type inward current are principally responsible for abnormal automaticity.     

Neuronal avalanches organize as nested theta- and beta/gamma-oscillations during development of cortical layer 2/3

Maturation of the cerebral cortex involves the spontaneous emergence of distinct patterns of neuronal synchronization, which regulate neuronal differentiation, synapse formation, and serve as a substrate for information processing. The intrinsic activity patterns that characterize the maturation of cortical layer 2/3 are poorly understood. By using microelectrode array recordings in vivo and in vitro, we show that this development is marked by the emergence of nested - and beta/gamma-oscillations that require NMDA- and GABA(A)-mediated synaptic transmission. The oscillations organized as neuronal avalanches, i.e., they were synchronized across cortical sites forming diverse and millisecond-precise spatiotemporal patterns that distributed in sizes according to a power law with a slope of -1.5. The correspondence between nested oscillations and neuronal avalanches required activation of the dopamine D(1) receptor. We suggest that the repetitive formation of neuronal avalanches provides an intrinsic template for the selective linking of external inputs to developing superficial layers.     

Lymphocytes in β-thalassemia/HbE: subpopulations and mitogen responses

Abstract: Lymphocyte subpopulations and proliferative responses to mitogens of 24 β-thalassemia/HbE patients were studied and compared with those of 23 healthy controls. Results of the study were analyzed in correlation with clinical aspects i.e. severity of disease (anemia), frequency of infections and iron status. T(CD3⁺) lymphocytes were found to increase in thalassemic patients compared to normal controls. The CD4- or CD8-positive lymphocytes and CD4/CD8 ratio were not statistically different from normals. Without mitogen, lymphocytes from thalassemic patients incorporated more [³H]Tdr than those from normal controls. Stimulation index (SI) of these cells after various mitogens were lower than in normal subjects. The observations were more obvious in patients with severe disease (severe anemia) and those who had frequent infections. These findings suggest that lymphocytes from thalassemic patients are activated in vivo. Whether these cells are less efficient in response to new or previously unexposed antigens remains to be proven.     

Transformation of acoustic information to sensory decision variables in the parietal cortex

The process by which sensory evidence contributes to perceptual choices requires an understanding of its transformation into decision variables. Here, we address this issue by evaluating the neural representation of acoustic information in the auditory cortex-recipient parietal cortex, while gerbils either performed a two-alternative forced-choice auditory discrimination task or while they passively listened to identical acoustic stimuli. During task engagement, stimulus identity decoding performance from simultaneously recorded parietal neurons significantly correlated with psychometric sensitivity. In contrast, decoding performance during passive listening was significantly reduced. Principal component and geometric analyses revealed the emergence of low-dimensional encoding of linearly separable manifolds with respect to stimulus identity and decision, but only during task engagement. These findings confirm that the parietal cortex mediates a transition of acoustic representations into decision-related variables. Finally, using a clustering analysis, we identified three functionally distinct subpopulations of neurons that each encoded task-relevant information during separate temporal segments of a trial. Taken together, our findings demonstrate how parietal cortex neurons integrate and transform encoded auditory information to guide sound-driven perceptual decisions.

Integrating sensory information over time is one of the fundamental attributes that is required for accurate perceptual decisions (1, 2). This process is supported by the transformation of stimulus representations into decision variables. In the case of auditory stimuli, prior to the formation of decision variables, the central representations of acoustic cues are gradually reconfigured along the auditory neuraxis. Thus, auditory neurons become more selective to contextually relevant acoustic features as one ascends the central pathway into the auditory cortex (3). Ultimately, individual acoustic components merge into auditory objects to guide perception (4). Similarly, primary visual cortex neurons are selective to the stimulus orientation (5, 6), whereas higher cortices are selective for more complex characteristics (7–9). A hierarchical progression of sensory information processing is also seen across the somatosensory ascending pathway where receptive fields grow more complex (10). These hierarchically transformed neural signals are ultimately decoded downstream of sensory cortices for stimulus-dependent decisions (4, 11–14).Studies in both nonhuman primates and rodents suggest that the parietal cortex integrates sensory inputs and transforms them into decision signals (15–19). The parietal cortex receives direct projections from primary or secondary sensory cortices (20, 21), has been causally implicated in the performance of perceptual decision-making tasks (22–25), and its activity typically reflects action selection (26, 27). Furthermore, parietal neurons gradually increase their spiking activity over time epochs that scale with the accumulation of sensory evidence (11, 28–31). Thus, while parietal cortex activity reflects decision variables, the manner in which relevant sensory stimuli are represented prior to this transformation remains uncertain.To dissociate encoding of stimuli from encoding of decision, we recorded neural activity from the parietal cortex while gerbils performed an auditory discrimination task (25), and again during passive listening sessions, using the same acoustic stimuli in the absence of behavioral decision. While some visual studies have explored visual selectivity of parietal cortex neurons under passive fixation conditions (32, 33), a direct comparison between the decoding of visual stimuli versus decision would require that eye fixation be controlled during stimulus presentation. In contrast, auditory tasks can be performed without the need to maintain head position during a trial, permitting us to directly compare the sound-driven responses of parietal cortex neurons during task engagement versus their responses to identical stimuli during passive listening. Thus, we predicted that if parietal cortex activity during task performance did not reflect the transition into decision-related variables, then all analyses of neural processing would be similar to those displayed during the passive listening condition. We found that during task performance, decoded parietal cortex population activity based on stimulus identity correlated with behavioral discrimination across similar timescales. Furthermore, principal component analysis (PCA) performed on parietal cortex responses revealed neural trajectories (i.e., the change in parietal cortex population activity over time) that demonstrated the temporal progression of low-dimensional encoding of acoustic information that transitioned to encoding of behavioral choices. During passive listening sessions, decoding performance from parietal cortex population activity based on stimulus identity was poorer than decoding during task performance, but scaled with stimulus duration. In addition, the PCA revealed neural trajectories that differentiated between each stimulus condition, but did not reflect a decision variable. Thus, the parietal cortex could accumulate auditory evidence for the purpose of forming a decision variable during task performance. Finally, our clustering analysis based on neuronal response properties suggest subpopulations of parietal neurons that may reflect separate temporal segments of individual trials during decision-making. We propose that the parietal cortex integrates and transforms bottom-up sensory information into decision variables during task performance.     

Mitochondrial membrane sensitivity to depolarization in acute myeloblastic leukemia is associated with spontaneous in vitro apoptosis, wild-type TP53, and vicinal thiol/disulfide status.

Nonresponse to remission-induction chemotherapy, which remains a major problem in acute myeloblastic leukemia (AML), has been linked to cellular resistance to apoptosis. Because the apoptosis induced by chemotherapeutic drugs is mediated by loss of mitochondrial transmembrane potential (MTP), it was postulated that sensitivity to mitochondrial membrane depolarization might be heterogeneous in AML. Using the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (mClCCP), the mitochondrial membrane sensitivity to depolarization (mClCCP concentrations that inhibit 50% of the transmembrane potential [IC(50)]) in AML blasts was measured and demonstrated marked interclonal heterogeneity, with the existence of comparatively sensitive (median mClCCP IC(50), 4 microM) and resistant (median mClCCP IC(50), 10 microM) clones. Furthermore, the mClCCP IC(50) was inversely associated with spontaneous in vitro apoptosis (P =.001). It was high in cases with mutant TP53 and correlated with the total cellular level of the multidrug resistance-associated protein (P =.019) but not of bcl-2, bax, or bcl-x. It was also found that the dithiol oxidant diamide, in contrast to the monovalent thiol oxidant diethyl maleate, increased the sensitivity of mitochondrial membranes to mClCCP. To confirm that TP53 directly affects MTP in leukemic cells and to establish the role of vicinal thiol oxidation in the TP53-dependent pathway, CEM 4G5 leukemia cells with forced, temperature-dependent expression of TP53 were studied. Monobromobimane, which inhibits mitochondrial membrane depolarization by preventing dithiol cross-linking, inhibited depolarization and apoptosis in 4G5 cells. It was concluded that in leukemia, TP53 and vicinal thiol/disulfide status are determinants of mitochondrial membrane sensitivity to depolarization, which is in turn associated with spontaneous apoptosis.