Changes in rat cerebral blood volume due to modulation of the 5-HT(1A) receptor measured with susceptibility enhanced contrast MRI.

Brain blood volume changes in the rat in response to 5-HT(1A) agonist and antagonist administration were measured using susceptibility contrast enhanced magnetic resonance imaging (MRI). Administration of the 5-HT(1A) agonist 8-OH-DPAT resulted in decreases in fractional brain blood volumes. Administration of the 5-HT(1A) antagonist WAY-100635 following a dose of 8-OH-DPAT resulted in increases in fractional blood volumes greatest in hippocampus and cortex and smallest in thalamus and caudate-putamen. The magnitude of the regional increases in blood volumes paralleled the distribution of 5-HT(1A) receptors in the rat brain. Administration of WAY-100635 alone resulted in decreases in cortical blood volume and increases in cerebellar blood volume.

[123I]IPCIT and [123I]beta-CIT as SPECT tracers for the dopamine transporter: a comparative analysis in nonhuman primates.

[123I]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([123I]-CIT) and its isopropylester analog [123I]PCIT, both of which are phenyltropane derivatives of cocaine with high affinity for the dopamine (DA) transporter, were compared using single photon emission computed tomography in nonhuman primates. Although IPCIT is significantly more selective for the DA transporter than beta-CIT, striatal distribution volumes of specifically bound tracer were similar for both tracers. Compartmental modeling results were compared with a simple peak equilibrium method used previously by this group. The peak equilibrium method is shown to overestimate striatal distribution volumes, primarily due to a difference in the calculated time of peak specific uptake.

Physiological basis for BOLD MR signal changes due to neuronal stimulation: separation of blood volume and magnetic susceptibility effects.

An NMR method is applied for separating blood volume and magnetic susceptibility effects in response to neuronal stimulation in a rat model. The method uses high susceptibility contrast agents to enhance blood volume induced signal changes. In the absence of exogenous agent, the dominant source of signal change on neuronal activation is associated with the signal increase from the blood oxygen level dependent (BOLD) effect. The relative negative contribution of blood volume changes to BOLD changes is maximally estimated to be 34%. The blood volume changes associated with median nerve stimulation (7 Hz) in the motor cortex are 26+/-7% and the corresponding blood susceptibility changes are 0.021+/-0.006 ppm. These methods can be applied to enhance the sensitivity of fMRI signal response and provide accurate quantitative measures of blood volume response to stimulation.

Alterations of benzodiazepine receptors in type II alcoholic subjects measured with SPECT and [123I]iomazenil.

OBJECTIVE: Alterations in cortical benzodiazepine receptor density have been described in postmortem and in vivo studies of alcoholic subjects. The authors attempted to replicate these findings using single photon emission computed tomography and the benzodiazepine receptor radiotracer [123I]iomazenil. METHOD: They measured the distribution volume of benzodiazepine receptors in 11 recently detoxified patients with type II alcoholism and 11 healthy comparison subjects. The tracer was given as a bolus followed by a continuous infusion to achieve sustained binding equilibrium at the benzodiazepine receptors. Data were analyzed by using a region of interest method (regions of interest were identified on coregistered magnetic resonance imaging scans) and by a pixel-by-pixel method (distribution volume maps were analyzed with statistical parametric mapping for between-group differences). RESULTS: The region of interest analysis revealed that alcoholic patients had significantly lower benzodiazepine distribution volume than comparison subjects in the frontal, anterior cingulate, and cerebellar cortices. Statistical parametric mapping revealed two large excursions in which the distribution volume in alcoholic patients was significantly lower than in comparison subjects: the anterior cingulate, extending into the right middle frontal gyrus, and the left occipital cortex. CONCLUSIONS: Benzodiazepine receptor distribution volume is significantly lower in several cortical regions and the cerebellum in alcoholic subjects than in healthy comparison subjects. These results are consistent with previous reports and might indicate either a toxic effect of alcoholism on benzodiazepine receptors or a vulnerability factor for developing alcoholism.

Physiologic basis for BOLD MR signal changes due to hypoxia/hyperoxia: separation of blood volume and magnetic susceptibility effects.

An NMR method is presented for separating blood volume and magnetic susceptibility effects in response to respiratory challenges such as hypoxia and hyperoxia. The technique employs high susceptibility contrast agents to enhance blood volume induced signal changes. The results show that for a rat model the dominant source of signal variation upon changing breathing gas from 100% oxygen to 10% oxygen/90% nitrogen is the change in blood magnetic susceptibility associated with the BOLD effect. The results imply that signal changes associated with respiratory challenges can be regarded as indicators of local blood oxygenation in vivo.

Functional magnetic resonance imaging of median nerve stimulation in rats at 2.0 T.

An animal model of sensory activation using fMRI at 2.0 T has been developed, demonstrating that fMRI studies on animals need not be limited to high field magnets. These methods produced reliable image intensity changes of 2% using median nerve stimulation in rats at 3 Hz and propofol as the anesthetic agent. At 6 Hz the activation was slightly but not statistically significantly greater. The feasibility of fMRI studies in animals using propofol suggests that it may be a useful anesthetic for fMRI studies in agitated adult patients or in children.

Characterization of the dopamine transporter in nonhuman primate brain: homogenate binding, whole body imaging, and ex vivo autoradiography using [125I] and [123I]IPCIT.

IPCIT [2 beta-carboisopropoxy-3 beta-(4-iodophenyl)tropane; also designated RTI-121] is the isopropyl ester of beta-CIT [2 beta-carbomethoxy-3 beta-(4-iodophenyl) tropane]. Although beta-CIT binds to dopamine (DA), serotonin (5-HT) and norepinephrine (NE) transporters, IPCIT has been reported to be selective for the DA transporter. IPCIT was labeled with 125I and its receptor binding to membranes prepared from baboon striatum was compared with that of [125I] beta-CIT. These studies confirmed the relative selectivity of IPCIT for the DA transporter in comparison to 5-HT and NE transporters. The nonspecific binding of [125I]IPCIT was almost four times greater than that of [125I] beta-CIT. The biodistribution of IPCIT was examined in two baboons with whole body imaging for 24-30 h after administration of 3 mCi of 123I-labeled tracer. The brain uptake peaked within the first hour at 9.2% of the injected dose and the majority of activity in the body cleared through the hepatobiliary system. The distribution of activity within the brain was examined with ex vivo autoradiography in one monkey injected with [123I]IPCIT. Activity was concentrated in the caudate and putamen and had values of 5 and 7 microCi/cm3 per microCi/g, respectively. The distribution in brain regions receiving moderately dense serotonergic innervation (e.g. superior colliculus and thalamus) had levels of activity equivalent to that in cerebellum. This study confirmed the in vitro and in vivo selectivity of IPCIT for the DA transporter but also showed that [125I]IPCIT had higher in vitro nonspecific binding than [125I] beta-CIT.

Serotonergic mechanisms of cocaine effects in humans.

The objective of this study was to investigate the role of serotonin (5-HT) in mediating the effects of cocaine in humans. To accomplish this, 12 subjects each participated in two randomized, double-blind test sessions separated by 1 week. In one session, subjects underwent acute depletion of the 5-HT amino acid precursor tryptophan (TRP), followed by a test dose of intranasal cocaine. In the other session, the cocaine test dose was preceded by sham depletion. Subject ratings of cocaine "high" were significantly lower following active TRP depletion than after the sham procedure. Subjects also showed an earlier but less sustained rise in self-rated nervousness during active TRP depletion. These findings are consistent with the hypothesis that 5-HT may be involved in mediating the euphorigenic and modulating the anxiogenic effects of cocaine in humans, either directly or through actions on other (e.g., dopaminergic) systems.

Comparison of [123I]beta-CIT and [123I]IPCIT as single-photon emission tomography radiotracers for the dopamine transporter in nonhuman primates.

Single-photon emission tomographic (SPET) imaging with the radiotracer [123I]2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane ([123I]beta-CIT) has been reported to be a useful in vivo measure of dopamine (DA) transporters. However, in addition to its high DA transporter affinity, beta-CIT also binds with high affinity to serotonin (5-HT) transporters. 2 beta-Carboisopropoxy-3 beta-(4-iodophenyl)tropane (IPCIT) has been demonstrated by in vitro studies to have higher selectivity for the DA transporter. We compared [123I]beta-CIT and [123I]IPCIT SPET imaging and plasma metabolite analyses in baboons to evaluate the potential advantages of [123I]IPCIT for quantitative in vivo measurements of DA transporter densities. Both tracers had low levels (2% of total plasma 123I activity) of lipophilic radiolabeled metabolites at 420 min. [123I]IPCIT had significantly higher binding to plasma proteins. The average percent free (nonprotein bound) [123I]beta-CIT and [123I]IPCIT were 52% +/- 7% and 14% +/- 6%, respectively. Region of interest uptake data were normalized by injected dose and body weight. Consistent with the high density of 5-HT transporters in the midbrain and the lower 5-HT transporter affinity of IPCIT, the normalized peak specific midbrain uptake of [123I]beta-CIT (1.7 +/- 0.5) was higher than that of [123I]IPCIT (0.4 +/- 0.2). Consistent with its greater lipophilicity, [123I]IPCIT had higher nonspecific uptake, such that normalized cerebellar uptake of [123I]IPCIT was about twice that of [123I]beta-CIT. The ratio of specific to nonspecific uptake in striatum was greater for [123I]beta-CIT compared to [123I]IPCIT; however, striatal binding potentials and distribution volumes were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Active and inactive enantiomers of 2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane: comparison using homogenate binding and single photon emission computed tomographic imaging.

2 beta-Carbomethoxy-3 beta-(4-iodophenyl)tropane (beta-CIT; also designated RTI-55) is an analog of cocaine that has been developed as a single photon emission computed tomography radiotracer that labels dopamine and serotonin transporters. We have prepared the 125I- and 123I-labeled ([1R] "active" and [1S] "inactive") enantiomers of beta-CIT. Total homogenate binding of the 125I-labeled inactive isomer to baboon caudate and cortex was approximately equal to nonspecific binding of the active isomer in cortex and much lower than total binding of the active isomer in caudate. However, inactive isomer homogenate binding in caudate was somewhat higher than in cortex, and during single photon emission computed tomography scanning in vivo striatal (1S)-[123I]beta-CIT uptake was also slightly greater than in cortex. Following intravenous administration of the 123I-labeled enantiomers, the plasma clearances of the active and inactive enantiomers were not significantly different. Single photon emission computed tomography imaging demonstrated that a bolus dose of nonradioactive (1R)-beta-CIT rapidly displaced the uptake of (1R)-[123I]beta-CIT. In contrast, the brain uptake of (1S)-[123I]beta-CIT was not displaced by nonradioactive (1R)-beta-CIT using either a bolus ("kinetic") or bolus plus constant infusion ("equilibrium") paradigm for administration of the radiotracer. In scans with bolus administration of radiotracer, peak striatal uptake of the active isomer was approximately twice that of the inactive isomer. In comparison to the 123I-labeled active tracer, the inactive tracer showed earlier times to peak activity and faster washouts of activity in all brain regions. These studies demonstrate beta-CIT stereoselectivity using both homogenate binding and in vivo imaging and suggest that the inactive enantiomer may be a useful measure of the kinetics of both blood-brain barrier transport and nonspecific binding.

Single photon emission computed tomographic imaging demonstrates loss of striatal dopamine transporters in Parkinson disease.

[123I][(1R)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane] ([123I]beta-CIT) labels dopamine transporters and is, therefore, a marker of neurons that degenerate in Parkinson disease. Single photon emission computed tomography imaging with [123I]beta-CIT showed that radioactivity in striatal regions in healthy subjects increased during a 2-day imaging study, whereas that in Parkinsonian patients peaked earlier at reduced levels relative to healthy subjects. Kinetic analyses of radioactivity in plasma and brain suggest that this decrease was due to an approximately 65% loss of target sites in patients compared with healthy subjects; greater losses occurred in putamen than in caudate. All patients showed lateralized differences in striatal uptake, with greater losses in the striatum contralateral to the side of the body with initial symptoms. These preliminary results suggest that [123I]beta-CIT is a marker for the loss of striatal dopamine terminals in patients with Parkinson disease. Single photon emission computed tomographic imaging with [123I]beta-CIT may be useful for early diagnosis of the disorder, for monitoring the progression of the disease, and for distinguishing the idiopathic disorder from other Parkinsonian syndromes with more widespread pathology.

Kinetic analysis of single sodium channels from canine cardiac Purkinje cells.

Single sodium channel events were recorded from cell-attached patches on single canine cardiac Purkinje cells at 10-13 degrees C. Data from four patches containing two to four channels and one patch with one channel were selected for quantitative analysis. The channels showed prominent reopening behavior at voltages near threshold, and the number of reopenings declined steeply with depolarization. Mean channel open time was a biphasic function of voltage with the maximum value (1-1.5 ms) occurring between -50 and -40 mV and lower values at more and at less hyperpolarized levels. Inactivation without opening was also prominent near threshold, and this occurrence also declined with depolarization. The waiting time distributions and the probability of being open showed voltage and time dependence as expected from whole-cell current studies. The results were analyzed in terms of a five-state Markovian kinetic model using both histogram analysis and a maximum likelihood method to estimate kinetic parameters. The kinetic parameters of the model fits were similar to those of GH3 pituitary cells (Horn, R., and C. A. Vandenberg. 1984. Journal of General Physiology. 84:505-534) and N1E115 neuroblastoma cells (Aldrich, R. W., and C. F. Stevens. Journal of Neuroscience. 7:418-431). Both histogram and maximum likelihood analysis implied that much of the voltage dependence of cardiac Na current is in its activation behavior, with inactivation showing modest voltage dependence.

Sodium channels in cardiac Purkinje cells.

Sodium (Na+) currents are responsible for excitation and conduction in most cardiac cells, but their study has been hampered by the lack of a satisfactory method for voltage clamp. We report a new method for low resistance access to single freshly isolated canine cardiac Purkinje cells that permits good control of voltage and intracellular ionic solutions. The series resistance was usually less than 3 omega cm2, similar to that of the squid giant axon. Cardiac Na+ currents resemble those of nerve. However, Na+ current decay is multiexponential. The basis for this was further studied with cell-attached patch clamp recording of single Na+ channel properties. A prominent characteristic of the single channels was their ability to reopen after closure. There was also a long opening state that may be the basis for a small very slowly decaying Na+ current. This rare long opening state may contribute to the Na+ current during the action potential plateau.

Low conductance sodium channels in canine cardiac Purkinje cells.

Low conductance sodium (Na) channels have been observed in nerve, skeletal muscle, and cardiac cells. In cardiac tissues the higher amplitude, more commonly observed Na channel was first investigated in detail by Cachelin et al. (Cachelin, A.B., J.E. de Peyer, S. Kokubun, and H. Reuter, 1983, J. Physiol. (Lond.), 340:389-402). They also reported low amplitude Na channel events. We have studied this low conductance Na channel in single canine cardiac Purkinje cells using cell-attached patches. Patch pipette solutions contained either 140 or 280 mM NaCl, and cells were bathed in a solution of 150 mM KCl to bring their resting potential close to zero. In 140 mM Na+, during steps to -50 mV, the lower and higher openings had amplitudes of 0.57 +/- 0.2 and 1.2 +/- 0.2 pA (means +/- SD of Gaussian fits). In 280 mM Na+ at -50 mV, amplitudes were 0.72 +/- 0.2 and 1.55 +/- 0.2 pA. Over a substantial voltage range, the lower events had amplitudes of about one-third that of the higher events. The frequency of the low conductance openings varied in different patches from zero to 22% of total openings. Histograms of open durations and latencies at several voltages suggested no difference in kinetics between the two channel events. The behavior of the low conductance channels was more consistent with a second population of channels rather than a second open state.

Open sodium channel properties of single canine cardiac Purkinje cells.

Open channel properties of canine cardiac Purkinje cell Na+ channels were studied with single channel cell-attached recording and with whole cell macroscopic current recording in internally perfused cells. Single channel currents and membrane currents increased with an increase in Na+ concentration, but showed evidence of saturation. Assuming first-order binding, the Km for Na+ was 370 mM. PCs/PNa was 0.020 and PK/PNa was 0.094. The current-voltage relationship for single channels showed prominent flattening in the hyperpolarizing direction. This flattening was accentuated by 10 mM Ca2+ and was greatly reduced in O mM Ca2+, indicating that the rectification was a consequence of Ca2+ block of the Na+ channels. A similar instantaneous current-voltage relationship was seen for the whole cell membrane currents. These results demonstrate that the cardiac channel shows substantial Ca2+ block, although it is relatively insensitive to tetrodotoxin. The Na+ and Ca2+ binding properties could be modeled by the four-barrier Eyring rate theory model, with similar values to those reported for the neuroblastoma Na+ channel (Yamamoto, D.,J.Z. Yeh, and T. Narahashi, 1984, Biophys J., 45:337-344).