Stimulation-dependent Changes Research Articles

E.3 fMRI correlates of symptom-specific improvement in STN deep brain stimulation

Background: Subthalamic nucleus (STN) deep brain stimulation (DBS) improves the cardinal symptoms of Parkinson’s disease (PD). However, the therapeutic mechanisms are incompletely understood. By leveraging patient-specific brain responses to DBS using functional magnetic resonance imaging (fMRI) acquired during stimulation, we identify and validate symptom-specific networks associated with clinical improvement. Methods: Forty PD patients with STN-DBS were enrolled for fMRI using a 30-sec DBS-ON/OFF cycling paradigm. The four cardinal motor outcomes of PD were chosen a priori and measured using the Movement Disorder Society-Sponsored Revision of the Unified Parkinson’s Disease Rating Scale, part III (MDS-UPDRSIII): axial instability, tremor, rigidity, bradykinesia. Stimulation-dependent changes in blood oxygen level-dependent (BOLD) signal were correlated with each symptom. Results: The relationship between BOLD response and outcomes revealed significant networks of clinical response (p<0.001). Using BOLD responses from the network hubs, each symptom-specific model was significantly predictive of actual improvement: axial instability (R2=0.38, p=0.000026), bradykinesia (R2=0.29, p=0.00033), rigidity (R2=0.40, p=0.000013), tremor (R2=0.26, p=0.00073). Conclusions: Using patient-specific imaging, we provide evidence of an association between DBS-evoked fMRI response and individual symptom improvement. Brain networks associated with clinical improvement were different depending on the PD symptom examined, suggesting the presence of symptom-specific networks of efficacy which may allow personalization of DBS therapy.

Assessing stimulation-dependent changes in LRRK2 and GCase expression/activity and convergence at the lysosome in cryopreserved monocytes.

Both leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GCase) are promising targets for the treatment of Parkinson's disease (PD). Evidence suggests that both proteins are involved in similar biological pathways, including lysosomal function. However, studies to date have largely investigated the enzymes in isolation and any relationship between LRRK2 and GCase remains unclear. Recent data suggest that it is the response to immunological challenges that is altered in PD and that circulating baseline cytokine levels are not useful diagnostic or predictive biomarkers in PD. Taken together, this project aims to optimize a blood collection and cryopreservation protocol to facilitate the collection and study of monocytes from PD patients and healthy controls, with a particular focus on measuring stimulation-dependent changes in LRRK2 and GCase protein activity levels and lysosomal function. Human PBMCs were isolated from whole blood and plated fresh or post-cryopreservation. PBMCs were treated with MLi2 or CBE +/- IFN-γ. Treatment effects on cathepsin activity, LRRK2 and GCase enzymatic activity, and antigen presentation were assessed in monocytes via flow cytometry. Media was collected RESULT: We first assessed the effects of cryopreservation on our read-outs. Despite cryopreservation having a small effect at baseline, both fresh and cryopreserved monocytes gated from total PBMCs exhibited comparable changes in response to IFN-γ. We observed that LRRK2 kinase inhibition decreases cathepsin activity in monocytes in a stimulation-dependent manner. This was coupled with increased LRRK2 expression and alterations in immune cell activation with LRRK2 kinase inhibition. Similarly, an increase in GCase activity levels were observed in monocytes upon IFN-γ treatment. These results show that cryopreservation of PBMCs doesn't affect our ability to measure stimulation-dependent changes in both LRRK2 and GCase expression/activity levels. These assays are therefore to be repeated in idiopathic PD PBMCs. Furthermore, these results suggest a fundamental role of LRRK2, specifically LRRK2 kinase activity, in lysosomal function in immune cells. Given the parallel increases in GCase activity, future research aims to investigate how these two enzymes interact and converge on biological pathways regulating inflammation and the lysosome in patient monocytes.

Partial Least Squares Analysis in Electrical Brain Activity

Abstract: Partial least squares (PLS) method has been designed for handling two common problems in the data that are encountered in most of the applied sciences including the neuroimaging data: 1) Collinearity problem among explanatory variables (X) or among dependent variables (Y); 2) Small number of observations with large number of explanatory variables. The idea behind this method is to explain as much as possible covariance between two blocks of X and Y variables by a small number of uncorrelated variables. Apart from the other applied sciences in which PLS are used, in the application of imaging data PLS has been used to identify task dependent changes in activity, changes in the relations between brain and behavior, and to examine functional connectivity of one or more brain regions. The aim of this paper is to give some information about PLS and apply on electroencephalography (EEG) data to identify stimulation dependent changes in EEG activity.

Olfactory Stimulation Regulates the Birth of Neurons That Express Specific Odorant Receptors

SUMMARYIn mammals, olfactory sensory neurons (OSNs) are born throughout life, ostensibly solely to replace damaged OSNs. During differentiation, each OSN precursor “chooses,” out of hundreds of possibilities, a single odorant receptor (OR) gene, which defines the identity of the mature OSN. The relative neurogenesis rates of the hundreds of distinct OSN “subtypes” are thought to be constant, as they are determined by a stochastic process in which each OR is chosen with a fixed probability. Here, using histological, single-cell, and targeted affinity purification approaches, we show that closing one nostril in mice selectively reduces the number of newly generated OSNs of specific subtypes. Moreover, these reductions depend on an animal’s age and/or environment. Stimulation-dependent changes in the number of new OSNs are not attributable to altered rates of cell survival but rather production. Our findings indicate that the relative birth rates of distinct OSN subtypes depend on olfactory experience.

Measurement of mast cell surface molecules by high-throughput immunophenotyping using transcription (HIT).

Here we describe the application of a highly multiplexed proteomic assay, called HIT (high-throughput immunophenotyping using transcription), to analyze human mast cell surface antigens at rest and during stimulation. HIT allows analysis of up to 100 analytes, including surface antigens and intracellular phosphoproteins, transcription factors, and cytokines, in a single experiment. Briefly, anti-mouse monovalent Fab fragments are covalently conjugated with barcoded oligonucleotides to generate a panel of conjugates. The oligonucleotide-Fab fragment conjugates are bound to monoclonal primary antibodies, creating a cocktail of up to 48 unique barcoded primary antibodies. As few as 100,000 mast cells are stained with the cocktail and the barcodes of the bound primary antibodies are amplified by in vitro transcription with fluorescently labeled NTPs. The resulting barcoded transcripts are quantified using a microarray spotted with oligonucleotides that are complementary to the barcoded transcripts. Differences in levels of the barcoded transcripts correlate well with actual protein levels and are capable of detecting stimulation-dependent changes in protein levels. HIT is an invaluable, broad-spectrum approach for characterizing mast cell surface antigens, signaling molecules, transcription factors, and cytokines.

Distinct Stages of Stimulated FcεRI Receptor Clustering and Immobilization Are Identified through Superresolution Imaging

Recent advances in fluorescence localization microscopy have made it possible to image chemically fixed and living cells at 20 nm lateral resolution. We apply this methodology to simultaneously record receptor organization and dynamics on the ventral surface of live RBL-2H3 mast cells undergoing antigen-mediated signaling. Cross-linking of IgE bound to FcεRI by multivalent antigen initiates mast cell activation, which leads to inflammatory responses physiologically. We quantify receptor organization and dynamics as cells are stimulated at room temperature (22°C). Within 2 min of antigen addition, receptor diffusion coefficients decrease by an order of magnitude, and single-particle trajectories are confined. Within 5 min of antigen addition, receptors organize into clusters containing ∼100 receptors with average radii of ∼70 nm. By comparing simultaneous measurements of clustering and mobility, we determine that there are two distinct stages of receptor clustering. In the first stage, which precedes stimulated Ca2+ mobilization, receptors slow dramatically but are not tightly clustered. In the second stage, receptors are tightly packed and confined. We find that stimulation-dependent changes in both receptor clustering and mobility can be reversed by displacing multivalent antigen with monovalent ligands, and that these changes can be modulated through enrichment or reduction in cellular cholesterol levels.

A rapid, topographical platelet activation assay

Platelets are central in maintaining normal haemostasis and are responsible for the cessation of blood loss following vascular injury. Platelet adhesion, leading to thrombus formation, involves rapid and distinct morphological changes culminating in cell aggregation. Rapid and highly specific platelet diagnostic tests are currently being developed to enable monitoring of drug response in patients with cardiovascular diseases. For a diagnostic device to be effective it needs to be rapid and simple, requiring minimal user interaction and providing results with minimal delay. This study describes the development of a rapid, label free platform for assay platelet function and demonstrates its use to monitor the influence of anti-thrombotic drugs. A rapid, single-step cell purification surface immobilises platelets from whole blood onto a protein array at specific locations used to define the assay site. Morphological changes and cell aggregation properties of activated platelets are exploited and combined within a label free, rapid, dye displacement chamber to determine cell morphology. Stimulation-dependent changes in cell morphology are described using both high resolution AFM imaging and a dye displacement platform.

Amyloid Precursor Protein and Proinflammatory Changes Are Regulated in Brain and Adipose Tissue in a Murine Model of High Fat Diet-Induced Obesity

BackgroundMiddle age obesity is recognized as a risk factor for Alzheimer's disease (AD) although a mechanistic linkage remains unclear. Based upon the fact that obese adipose tissue and AD brains are both areas of proinflammatory change, a possible common event is chronic inflammation. Since an autosomal dominant form of AD is associated with mutations in the gene coding for the ubiquitously expressed transmembrane protein, amyloid precursor protein (APP) and recent evidence demonstrates increased APP levels in adipose tissue during obesity it is feasible that APP serves some function in both disease conditions.Methodology/Principal FindingsTo determine whether diet-induced obesity produced proinflammatory changes and altered APP expression in brain versus adipose tissue, 6 week old C57BL6/J mice were maintained on a control or high fat diet for 22 weeks. Protein levels and cell-specific APP expression along with markers of inflammation and immune cell activation were compared between hippocampus, abdominal subcutaneous fat and visceral pericardial fat. APP stimulation-dependent changes in macrophage and adipocyte culture phenotype were examined for comparison to the in vivo changes.Conclusions/SignificanceAdipose tissue and brain from high fat diet fed animals demonstrated increased TNF-α and microglial and macrophage activation. Both brains and adipose tissue also had elevated APP levels localizing to neurons and macrophage/adipocytes, respectively. APP agonist antibody stimulation of macrophage cultures increased specific cytokine secretion with no obvious effects on adipocyte culture phenotype. These data support the hypothesis that high fat diet-dependent obesity results in concomitant pro-inflammatory changes in brain and adipose tissue that is characterized, in part, by increased levels of APP that may be contributing specifically to inflammatory changes that occur.

Disturbances in Body Ownership in Schizophrenia: Evidence from the Rubber Hand Illusion and Case Study of a Spontaneous Out-of-Body Experience

BackgroundA weakened sense of self may contribute to psychotic experiences. Body ownership, one component of self-awareness, can be studied with the rubber hand illusion (RHI). Watching a rubber hand being stroked while one's unseen hand is stroked synchronously can lead to a sense of ownership over the rubber hand, a shift in perceived position of the real hand, and a limb-specific drop in stimulated hand temperature. We aimed to assess the RHI in schizophrenia using quantifiable measures: proprioceptive drift and stimulation-dependent changes in hand temperature.MethodsThe RHI was elicited in 24 schizophrenia patients and 21 matched controls by placing their unseen hand adjacent to a visible rubber hand and brushing real and rubber hands synchronously or asynchronously. Perceived finger location was measured before and after stimulation. Hand temperature was taken before and during stimulation. Subjective strength of the illusion was assessed by a questionnaire.ResultsAcross groups, the RHI was stronger during synchronous stimulation, indicated by self-report and proprioceptive drift. Patients reported a stronger RHI than controls. Self-reported strength of RHI was associated with schizotypy in controls Proprioceptive drift was larger in patients, but only following synchronous stimulation. Further, we observed stimulation-dependent changes in skin temperature. During right hand stimulation, temperature dropped in the stimulated hand and rose in the unstimulated hand. Interestingly, induction of RHI led to an out-of-body experience in one patient, linking body disownership and psychotic experiences.ConclusionsThe RHI is quantitatively and qualitatively stronger in schizophrenia. These findings suggest that patients have a more flexible body representation and weakened sense of self, and potentially indicate abnormalities in temporo-parietal networks implicated in body ownership. Further, results suggest that these body ownership disturbances might be at the heart of a subset of the pathognomonic delusions of passivity.

Area-specific modulation of neural activation comparing escitalopram and citalopram revealed by pharmaco-fMRI: A randomized cross-over study

Area-specific and stimulation-dependent changes of human brain activation by selective serotonin reuptake inhibitors (SSRI) are an important issue for improved understanding of treatment mechanisms, given the frequent prescription of these drugs in depression and anxiety disorders. The aim of this neuroimaging study was to investigate differences in BOLD-signal caused by administration of the SSRIs escitalopram and citalopram using pharmacological functional magnetic resonance imaging (pharmaco-fMRI). Eighteen healthy subjects participated in a placebo-controlled, randomized, double-blind study in cross-over repeated measures design. Each volunteer performed facial emotional discrimination and a sensorimotor control paradigm during three scanning sessions. Citalopram (20 mg/d), escitalopram (10 mg/d) and placebo were administered for 10 days each with a drug-free period of at least 21 days. Significant pharmacological effects on BOLD-signal were found in the amygdala, medial frontal gyrus, parahippocampal, fusiform and middle temporal gyri. Post-hoc t-tests revealed decreased BOLD-signal in the right amygdala and left parahippocampal gyrus in both pharmacological conditions, compared to placebo. Escitalopram, compared to citalopram, induced a decrease of BOLD-signal in the medial frontal gyrus and an increase in the right fusiform and left parahippocampal gyri. Drug effects were concentrated in brain regions with dense serotonergic projections. Both escitalopram and citalopram attenuated BOLD-signal in the amygdala and parahippocampal cortex to emotionally significant stimuli compared to control stimuli. We believe that reduced reactivity in the medial frontal gyrus found for escitalopram compared to citalopram administration might explain the response differences between study drugs as demonstrated in previous clinical trials.

Sensory Deafferentation Transsynaptically Alters Neuronal GluR1 Expression in the External Plexiform Layer of the Adult Mouse Main Olfactory Bulb

Altered distribution of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluR1 has been linked to stimulation-dependent changes in synaptic efficacy, including long-term potentiation and depression. The main olfactory bulb (OB) remains plastic throughout life; how GluR1 may be involved in this plasticity is unknown. We have previously shown that neonatal naris occlusion reduces numbers of interneuron cell bodies that are immunoreactive for GluR1 in the external plexiform layer (EPL) of the adult mouse OB. Here, we show that immunoreactivity of mouse EPL interneurons for GluR1 is also dramatically reduced following olfactory deafferentation in adulthood. We further show that expression of glutamic acid decarboxylase (GAD) 65, 1 of 2 GAD isoforms expressed by adult gamma-aminobutyric acidergic interneurons, is reduced, but to a much smaller extent, and that in double-labeled cells, immunoreactivity for the Ca(2+)-binding protein parvalbumin (PV) is also reduced. In addition, GluR1 expression is reduced in presumptive tufted cells and interneurons that are negative for GAD65 and PV. Consistent with previous reports, sensory deafferentation resulted in little neuronal degeneration in the adult EPL, indicating that these differences were not likely due to death of EPL neurons. Together, these results suggest that olfactory input regulates expression of the GluR1 AMPA receptor subunit by tufted cells that may in turn regulate GluR1 expression by interneurons within the OB EPL.

Tracking chromaffin granules on their way through the actin cortex.

Quantitative time-lapse evanescent-wave imaging of individual fluorescently labelled chromaffin granules was used for kinetic analysis of granule trafficking through a approximately 300-nm(1/e2) optical section beneath the plasma membrane. The mean squared displacement (MSD) was used to estimate the three-dimensional diffusion coefficient (D(3)). We calculated the granules' speed, frame-to-frame displacement and direction and their autocorrelation to identify different stages of approach to the membrane. D(3) was about 10,000 times lower than expected for free diffusion. Granules located approximately 60 nm beneath the plasma membrane moved on random tracks (D(3) approximately 10(-10) cm(2)s(-1)) with several reversals in direction before they approached their docking site at angles larger than 45 degrees. Docking was observed as a loss of vesicle mobility by two orders of magnitude within <100 ms. For longer observation times the MSD saturated, as if the granules' movement was confined to a volume only slightly larger than the granule. Rarely, the local random motion was superimposed with a directed movement in a plane beneath the membrane. Stimulation of exocytosis selectively depleted the immobile, near-membrane granule population and caused a recruitment of distant granules to sites at the plasma membrane. Their absolute mobility levels were not significantly altered. Application of latrunculin or jasplakinolide to change F-actin polymerisation caused a change in D(3) of the mobile granule population as well as a reduction of the rate of release, suggesting that granule mobility is constrained by the filamentous actin meshwork and that stimulation-dependent changes in actin viscosity propel granules through the actin cortex.

Activation of metabotropic glutamate receptors is necessary for transneuronal regulation of ribosomes in chick auditory neurons

Elimination of auditory nerve activity results in atrophy and death of nucleus magnocellularis (NM) neurons in the chick. One early event in the degeneration of NM neurons is a disruption of their ribosomes. This experiment examines the role of metabotropic glutamate receptors in afferent regulation of ribosomes. The auditory nerve on one side of a chick brainstem slice was stimulated in vitro. Rapid stimulation-dependent changes in ribosomes were visualized by immunolabeling using an antibody, called Y10B, that recognizes ribosomal RNA. In normal media, NM neurons on the stimulated side of the slice show greater Y10B labeling than the unstimulated NM neurons on the opposite side of the same slice. The role of metabotropic glutamate receptors was evaluated by unilaterally stimulating the auditory nerve in media containing the metabotropic glutamate receptor antagonist (RS)-α-methyl-4-carboxyphenyl-glycine (MCPG). Addition of MCPG to the bath did not block EPSPs produced by stimulating the auditory nerve. However, MCPG did prevent the stimulation-dependent regulation of ribosomes in NM neurons (as indexed by Y10B labeling). These data suggest that glutamate may play a trophic role in the young auditory system through activation of metabotropic glutamate receptors.

Effects of GABA(A) inhibition on the expression of long-term potentiation in CA1 pyramidal cells are dependent on tetanization parameters.

Long-term potentiation (LTP) of excitatory synaptic responses of principal neurons in the hippocampus is accompanied by changes in GABAergic inhibition mediated by interneurons. The impact of inhibition on LTP of excitatory postsynaptic responses in CA1 pyramidal cells was assessed by monitoring changes in field potentials evoked by Schaffer collateral stimulation in hippocampal slices in vitro. First, to determine the effect of inhibition on population EPSPs, slices were exposed to the GABA(A) receptor antagonist bicuculline (10 microM). Both the slope and amplitude of field EPSPs (fEPSPs) were significantly enhanced by bicuculline indicating that inhibition modulates excitatory postsynaptic responses of pyramidal cells. To assess if stimulation-dependent changes in inhibition influence LTP of excitatory responses of pyramidal cells, LTP was examined in the presence and absence of bicuculline (20 microM) following either 100 Hz tetanization, or theta-patterned stimulation (short bursts delivered at 5 Hz). In normal medium, 100 Hz stimulation produced marked short-term potentiation that decayed 5-10 min post-tetanus and both stimulation paradigms produced similar LTP at 30 min post-tetanus. In comparison, LTP of the fEPSP slope and amplitude was significantly enhanced after theta-patterned stimulation, but not after 100 Hz stimulation, in bicuculline. The greater potentiation of field responses following theta-patterned stimulation in the presence of bicuculline indicates that a larger potentiation of excitatory responses was unmasked during suppression of inhibitory inputs. These results suggest that a long-lasting enhancement of inhibition in pyramidal cells was also induced following theta-patterned stimulation in normal ACSF. Since suppression of inhibition did not uncover a significantly larger potentiation following 100 Hz tetanization, the influence of inhibition on LTP of excitatory responses appears to be stimulation-dependent. In conclusion, theta-patterned stimulation appears to be more effective at inducing plasticity within inhibitory circuits, and this plasticity may partially offset concurrent increases in the excitability of the CA1 network.

Repetitive stimulation induced potentiation of excitatory transmission in the rat dorsal horn: an in vitro study.

1. The effects of repetitive stimulation of primary afferents in lumbar dorsal roots on synaptic transmission in the dorsal horn (DH) were studied in a rat spinal cord slice-dorsal root ganglion (DRG)-peripheral nerve trunk preparation by the use of intracellular recording from neurons (n = 115) of the spinal dorsal horn (depth 147 +/- 139, mean +/- SD). All DH neurons were excited synaptically by electrical stimulation of the dorsal root or the peripheral nerve trunk. The electrical shocks were calibrated to produce activation either of large fibers (10-20 V, 0.02 ms) or the whole fiber population including unmyelinated afferents (supramaximal stimulus: > 35 V, 0.5 ms). Postsynaptic potentials induced by low intensity repetitive stimulation of primary afferents at frequencies below 5 Hz failed to produce a prolonged change in the resting membrane potential. In 97/115 DH neurons, slow excitatory postsynaptic potentials (EPSP)--evoked by high intensity low-frequency repetitive stimulation (0.1-2 Hz) of primary afferents--summated, producing a prolonged cumulative depolarization. In the remaining 18/115 DH neurons, high intensity low-frequency stimulation produced a cumulative hyperpolarizing response. 2. In 22 of 97 neurons that responded to high intensity repetitive stimulation with a cumulative depolarization, wind-up in the firing of action potentials was recorded. In all but two experiments, neurons that responded with wind-up to stimulation of one root responded with wind-up to stimulation of the adjacent dorsal root. In 14/22 wind-up neurons, the synaptic response to high intensity stimulation of primary afferents was composed of a short latency EPSP, followed by an inhibitory postsynaptic potential (IPSP), followed by a slow EPSP. The decrease of the amplitude and duration of the IPSP obtained during train stimulation did not seem to contribute to facilitation of transmission induced by repetitive stimulation. 3. The wind-up in firing of action potentials was followed by a prolonged potentiation of synaptic transmission in tetanized synapses. A test of other, adjacent primary afferents revealed that these synapses in the neurons in the superficial laminae had not undergone potentiation. This "synaptic specificity" of post-wind-up potentiation suggested that the mechanism for the induction of stimulation-dependent changes in the excitability of the DH neuron is presynaptic to the recorded-from neuron. 4. In a concentration of 0.5 microM and higher, tetrodotoxin (TTX) applied to sensory neurons selectively blocked action potentials in large myelinated primary afferents.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition by noradrenaline and adrenaline of the increase in glucose and lactate output and decrease in flow after sympathetic nerve stimulation in perfused rat liver: possible involvement of protein kinase C.

In perfused rat liver stimulation of the hepatic nerve plexuses increased via alpha 1-receptors glucose and lactate output decreased flow and caused an overflow of noradrenaline into the hepatic vein. Infusion of noradrenaline and adrenaline also elicited similar metabolic and hemodynamic alterations via alpha 1-receptors, whereas infusion of isoproterenol via beta 2-receptors enhanced glucose output and slightly reduced lactate release without affecting flow. The influence of circulating catecholamines on the nerve stimulation-dependent changes was investigated. Noradrenaline (100 nmol/L) or adrenaline (40 nmol/L) but not isoproterenol (1 mumol/L), which themselves caused about half-maximal alterations, strongly inhibited the nerve stimulation-induced increase in glucose and lactate output and decrease in flow but had no effect on noradrenaline overflow. The protein kinase C activator (4 beta)phorbol 12-myristate, 13-acetate (100 nmol/L) but not its analog (4 alpha)phorbol 12,13-didecanoate (100 nmol/L) strongly inhibited the metabolic and hemodynamic changes caused by nerve stimulation or noradrenaline infusion. The protein kinase C inhibitor H7 (20 mumol/L) partially prevented the inhibition of the nerve actions by noradrenaline. The results lead us to conclude that noradrenaline and adrenaline inhibited the metabolic and hemodynamic nerve actions by means of a mechanism involving protein kinase C rather than presynaptic alpha-receptors or beta-receptors. The catecholamines apparently increased via alpha 1-receptors inositol 1,4,5-trisphosphate, which in turn enhanced cytosolic Ca2+ and thus altered metabolism and in part hemodynamics, and diacylglycerol, which in turn activated protein kinase C and thus feedback inhibited the signal chain from alpha 1-receptors via G proteins to phospholipase C.

The cytosolic calcium transient modulates the action potential of rat ventricular myocytes.

1. The modulation of the action potential by the cytosolic Ca2+ (Cai2+) transient was studied in single isolated rat ventricular myocytes loaded with the acetoxymethyl ester form of the Ca(2+)-sensitive fluorescent dye Indo-1. Stimulation following rest and exposure to ryanodine were used to change the amount of Ca2+ released from the sarcoplasmic reticulum and thus the size of the Cai2+ transient. The Cai2+ transient was measured as the change, upon stimulation, in the ratio of Indo-1 fluorescence at 410 nm to that at 490 nm (410/490) and action potentials or membrane currents were recorded using patch-type microelectrodes. 2. When stimulation was initiated following rest, the magnitude of the Cai2+ transient decreased in a beat-dependent manner until a steady state was reached. The negative staircase in the Cai2+ transient was accompanied by a similar beat-dependent decrease in the duration of the action potential, manifested primarily as a gradual loss of the action potential plateau (approximately -45 mV). A slow terminal phase of repolarization of a few millivolts in amplitude was found to parallel the terminal decay of the Cai2+ transient. 3. The terminal portion of phase-plane loops of membrane potential (Vm) vs. Indo-1 ratio from all of the beats of a stimulus train followed a common linear trajectory even though the individual beats differed markedly in the duration and amplitude of the action potential and Cai2+ transient. 4. When the stimulation dependence of the Cai2+ transient was titrated away with submaximal exposure to ryanodine, the stimulation-dependent changes in the action potential plateau and terminal phase of repolarization were also eliminated. The same effect was noted in cells which, fortuitously, did not show a staircase in the Cai2+ transient following a period of rest. 5. When action potentials were triggered immediately following spontaneous release of Ca2+ from the sarcoplasmic reticulum, which results in a small depolarization at the resting potential, phase-plane loops (Vm vs. Indo-1 ratio) of the spontaneous events followed the same linear trajectory as the terminal phase of repolarization in the loops of the stimulated beats. 6. Following repolarization from brief voltage clamp pulses (to minimize time and voltage-dependent currents associated with depolarization), an inward current was observed that rose and fell in phase with the Cai2+ transient. This current was present at -70 mV, near the resting potential, and at -40 mV, a potential relevant to the plateau of the action potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Rest- and stimulation-dependent changes in exchangeable calcium content in rabbit ventricular myocardium.

Ca2+ shifts in the isolated, perfused ventricular muscle of rabbit hearts were investigated with the aid of 45Ca under the conditions of complete equilibration of preparations with 45Ca containing solution. The "cellular" 45Ca content was calculated by subtraction of 45Ca2+ dissolved in the free water of extracellular space from the total tissue 45Ca2+ content. The "cellular" content of 45Ca in stimulated (60 per min) preparation was 0.887 +/- 0.067 mmol/kg wet weight (w.w.). Six minutes of rest resulted in the drop of this content to 0.503 +/- 0.054 mmol/kg w.w. despite continued perfusion with 45Ca containing solution. Contractile force (CF) decreased at that time to 23% of control. The first post-rest contraction (RSC) resulted in a gain of 0.073 mmol45Ca/kg w.w. Both the content of 45Ca and CF returned to the pre-rest values when stimulation was resumed. The difference between the 45Ca content of post-rest stimulated and rested preparations (0.384 mmol/kg w.w.) is equivalent to Ca2 fraction (Ca2), previously described in guinea pig ventricular myocardium (17, 12). However, the volume of rabbit Ca2 is only about 42% of that in guinea pig. Caffeine in concentration 12.5 mM, which did not displace Ca2 from guinea pig ventricular muscle, decreased Ca2 in the rabbit ventricle by 44%. CCCP, a protonofore destroying the mitochondrial protone gradient essential for Ca2+ uptake and maintainability, displaced Ca2 completely from rabbit ventricles. These results, although far from conclusive, do suggest that both the mitochondria and sarcoplasmic reticulum might be the site of the rate-dependent Ca2 fraction. The physiological meaning of differences in Ca2 content between rabbit, guinea pig, and rat ventricular myocardium is discussed.

Stimulation-dependent changes in synaptic effects on unit activity of medial preoptic nucleus neurones in rats.

Stimulation-dependent changes in synaptic effects were observed in medial preoptic nucleus neurones during stimulation of the amygdala or pyriform cortex in anaesthetized female rats. The changes occurred after 35--240 triple pulse stimuli repeated at 0.89 Hz. Median eminence stimulation did not produce any synaptic change. These data show the existence of synaptic plasticity in the neural pathway from the amygdala and pyriform cortex to the medial preoptic nucleus.

Phosphorylase kinase phosphorylates a brain protein which is influenced by repetitive synaptic activation

SYNAPTIC systems from mammalian brain can now be studied in situ using a brain slice preparation which displays most of the electrophysiological characteristics observed in intact preparations1,2. We have been particularly interested in analysis of long-term potentiation (LTP), a marked and quasi-permanent increase in synaptic efficacy which is found in the hippocampal formation following a brief (1 s) train of repetitive stimulation3–6. Previous work from this laboratory demonstrated a strong correlation between induction of such LTP and the apparent phosphorylation of a 40,000 molecular weight protein in synaptic plasma membranes (SPM)7,8. Both LTP and the stimulation-dependent changes in the 40,000-MW protein were dependent on calcium and this cation has also recently been shown (ref. 9 and unpublished observations) to enhance the endogenous phosphorylation of a protein with a molecular weight of ∼40,000. If potentiation and the phosphorylation of the 40,000-MW protein are related, with calcium being required for both, then it is possible that a calcium-dependent protein kinase might be an intermediary enzyme in the two processes. We have therefore investigated the effect of exogeneous phosphorylase kinase (PBK; EC 2.7.1.38), a calcium-sensitive enzyme found in brain10,11, on the phosphorylation of the 40,000-MW protein. Since several laboratories have demonstrated cyclic AMP-dependent phosphorylation of several SPM proteins12–15, it was also of interest to test for the possibility that this nucleotide might be involved in the phosphorylation of the 40,000-MW protein. We report here that PBK produces a marked and highly specific phosphorylation of the 40,000-MW protein that we have previously shown to be affected by high frequency potentiating stimulation.