Zhongchi Luo

Acute Cocaine Induces Fast Activation of D1 Receptor and Progressive Deactivation of D2 Receptor Striatal Neurons: In Vivo Optical Microprobe [Ca2+]i Imaging

Cocaine induces fast dopamine increases in brain striatal regions, which are recognized to underlie its rewarding effects. Both dopamine D1 and D2 receptors are involved in cocaines reward but the dynamic downstream consequences of cocaine effects in striatum are not fully understood. Here we used transgenic mice expressing EGFP under the control of either the D1 receptor (D1R) or the D2 receptor (D2R) gene and microprobe optical imaging to assess the dynamic changes in intracellular calcium ([Ca2+]i) responses (used as marker of neuronal activation) to acute cocaine in vivo separately for D1R- versus D2R-expressing neurons in striatum. Acute cocaine (8 mg/kg, i.p.) rapidly increased [Ca2+]i in D1R-expressing neurons (10.6 ± 3.2%) in striatum within 8.3 ± 2.3 min after cocaine administration after which the increases plateaued; these fast [Ca2+]i increases were blocked by pretreatment with a D1R antagonist (SCH23390). In contrast, cocaine induced progressive decreases in [Ca2+]i in D2R-expressing neurons (10.4 ± 5.8%) continuously throughout the 30 min that followed cocaine administration; these slower [Ca2+]i decreases were blocked by pretreatment with a D2R antagonist (raclopride). Since activation of striatal D1R-expressing neurons (direct-pathway) enhances cocaine reward, whereas activation of D2R-expressing neurons suppresses it (indirect-pathway) (Lobo et al., 2010), this suggests that cocaines rewarding effects entail both its fast stimulation of D1R (resulting in abrupt activation of direct-pathway neurons) and a slower stimulation of D2R (resulting in longer-lasting deactivation of indirect-pathway neurons). We also provide direct in vivo evidence of D2R and D1R interactions in the striatal responses to acute cocaine administration.

Imaging separation of neuronal from vascular effects of cocaine on rat cortical brain in vivo

MRI techniques to study brain function assume coupling between neuronal activity, metabolism and flow. However, recent evidence of physiological uncoupling between neuronal and cerebrovascular events highlights the need for methods to simultaneously measure these three properties. We report a multimodality optical approach that integrates dual-wavelength laser speckle imaging (measures changes in blood flow, blood volume and hemoglobin oxygenation), digital-frequency-ramping optical coherence tomography (images quantitative 3D vascular network) and Rhod(2) fluorescence (images intracellular calcium for measure of neuronal activity) at high spatiotemporal resolutions (30 μm, 10 Hz) and over a large field of view (3×5 mm(2)). We apply it to assess cocaines effects in rat cortical brain and show an immediate decrease (3.5±0.9 min, phase 1) in the oxygen content of hemoglobin and the cerebral blood flow followed by an overshoot (7.1±0.2 min, phase 2) lasting over 20 min whereas Ca(2+) increased immediately (peaked at t=4.1±0.4 min) and remained elevated. This enabled us to identify a delay (2.9±0.5 min) between peak neuronal and vascular responses in phase 2. The ability of this multimodality optical approach for simultaneous imaging at high spatiotemporal resolutions permits us to distinguish the vascular versus cellular changes of the brain, thus complimenting other neuroimaging modalities for brain functional studies (e. g., PET, fMRI).

The effect of intravenous lidocaine on brain activation during non-noxious and acute noxious stimulation of the forepaw: a functional magnetic resonance imaging study in the rat.

BACKGROUND: Lidocaine can alleviate acute as well as chronic neuropathic pain at very low plasma concentrations in humans and laboratory animals. The mechanism(s) underlying lidocaine’s analgesic effect when administered systemically is poorly understood but clearly not related to interruption of peripheral nerve conduction. Other targets for lidocaine’s analgesic action(s) have been suggested, including sodium channels and other receptor sites in the central rather than peripheral nervous system. To our knowledge, the effect of lidocaine on the brain’s functional response to pain has never been investigated. Here, we therefore characterized the effect of systemic lidocaine on the brain’s response to innocuous and acute noxious stimulation in the rat using functional magnetic resonance imaging (fMRI). METHODS: Alpha-chloralose anesthetized rats underwent fMRI to quantify brain activation patterns in response to innocuous and noxious forepaw stimulation before and after IV administration of lidocaine. RESULTS: Innocuous forepaw stimulation elicited brain activation only in the contralateral primary somatosensory (S1) cortex. Acute noxious forepaw stimulation induced activation in additional brain areas associated with pain perception, including the secondary somatosensory cortex (S2), thalamus, insula and limbic regions. Lidocaine administered at IV doses of either 1 mg/kg, 4 mg/kg or 10 mg/kg did not abolish or diminish brain activation in response to innocuous or noxious stimulation. In fact, IV doses of 4 mg/kg and 10 mg/kg lidocaine enhanced S1 and S2 responses to acute nociceptive stimulation, increasing the activated cortical volume by 50%–60%. CONCLUSION: The analgesic action of systemic lidocaine in acute pain is not reflected in a straightforward interruption of pain-induced fMRI brain activation as has been observed with opioids. The enhancement of cortical fMRI responses to acute pain by lidocaine observed here has also been reported for cocaine. We recently showed that both lidocaine and cocaine increased intracellular calcium concentrations in cortex, suggesting that this pharmacological effect could account for the enhanced sensitivity to somatosensory stimulation. As our model only measured physiological acute pain, it will be important to also test the response of these same pathways to lidocaine in a model of neuropathic pain to further investigate lidocaine’s analgesic mechanism of action.

Differential effects of anesthetics on cocaine's pharmacokinetic and pharmacodynamic effects in brain

Most studies of the effect of cocaine on brain activity in laboratory animals are preformed under anesthesia, which could potentially affect the physiological responses to cocaine. Here we assessed the effects of two commonly used anesthetics [α‐chloralose (α‐CHLOR) and isofluorane (ISO)] on the effects of acute cocaine (1 mg/kg i.v.) on cerebral blood flow (CBF), cerebral blood volume (CBV), and tissue hemoglobin oxygenation (StO2) using optical techniques and cocaine’s pharmacokinetics (PK) and binding in the rat brain using (PET) and [11C]cocaine. We showed that acute cocaine at a dose abused by cocaine abusers decreased CBF, CBV and StO2 in rats anesthetized with ISO, whereas it increased these parameters in rats anesthetized with α‐CHLOR. Importantly, in ISO‐anesthetized animals cocaine‐induced changes in CBF and StO2 were coupled, whereas for α‐CHLOR these measures were uncoupled. Moreover, the clearance of [11C]cocaine from the brain was faster for ISO (peak half‐clearance 15.8 ± 2.8 min) than for α‐CHLOR (27.5 ± 0.6 min), and the ratio of specific to non‐specific binding of [11C]cocaine in the brain was higher for ISO‐ (3.37 ± 0.32) than for α‐CHLOR‐anesthetized rats (2.24 ± 0.4). For both anesthetics, cocaine‐induced changes in CBF followed the fast uptake of [11C]cocaine in the brain (peaking at ∼2.5–4 min), but only for ISO did the duration of the CBV and StO2 changes correspond to the rate of [11C]cocaine’s clearance from the brain. These results demonstrate that anesthetics influence cocaine’s hemodynamic and metabolic changes in the brain, and its binding and PK, which highlights the need to better understand the interactions between anesthetics and pharmacological challenges in brain functional imaging studies.

Quantification of cocaine-induced cortical blood flow changes using laser speckle contrast imaging and Doppler optical coherence tomography.

We present a dual-imaging technique combining laser speckle contrast imaging and spectral-domain Doppler optical coherence tomography to enable quantitative characterization of local cerebral blood flow (CBF) changes in rat cortex in response to drug stimulus (e.g., cocaine) at high spatiotemporal resolutions. To examine the utility of this new technique, animal experiments were performed to study the influences of anesthetic regimes (e.g., isoflurane, alpha-chloralose) on the pharmadynamic effects of acute cocaine challenge. The results showed that cocaine-evoked CBF patterns (e.g., increases in alpha-chloralose and decreases in isoflurane regimes) were quantitatively characterized, thus rendering it a potentially useful tool for imaging studies of brain functions.

DETECTION OF Ca2+-DEPENDENT NEURONAL ACTIVITY SIMULTANEOUSLY WITH DYNAMIC CHANGES IN CEREBRAL BLOOD VOLUME AND TISSUE OXYGENATION FROM THE LIVE RAT BRAIN

We present a catheter-based optical diffusion and fluorescence (ODF) probe to study the functional changes of the brain in vivo. This ODF probe enables the simultaneous detection of the multi-wavelength absorbance and fluorescence emission from the living rat brain. Our previous studies, including a transient stroke experiment of the rat brain as well as the brain response to cocaine, have established the feasibility of simultaneously determining changes in cerebral blood volume (CBV), tissue oxygenation (StO2) and intracellular calcium ([Ca2+]i, using the fluorescence indicator Rhod2). Here, we present our preliminary results of somatosensory response to electrical forepaw stimulation obtained from the rat cortical brain by using the ODF probe, which indicate that the probe could track brain activation by directly detecting [Ca2+]i along with separately distinguishing CBV and StO2 in real time. The changes of CBV, StO2 and [Ca2+]i are comparable with the blood-oxygen-level-dependent (BOLD) response to the stimulation obtained using functional magnetic resonance imaging (fMRI). However, the high temporal resolution of the optical methodology is advanced, thus providing a new modality for brain functional studies to understand the hemodynamic changes that underlie the neuronal activity.

Multichannel optical brain imaging to separate cerebral vascular, tissue metabolic, and neuronal effects of cocaine

Characterization of cerebral hemodynamic and oxygenation metabolic changes, as well neuronal function is of great importance to study of brain functions and the relevant brain disorders such as drug addiction. Compared with other neuroimaging modalities, optical imaging techniques have the potential for high spatiotemporal resolution and dissection of the changes in cerebral blood flow (CBF), blood volume (CBV), and hemoglobing oxygenation and intracellular Ca ([Ca2+]i), which serves as markers of vascular function, tissue metabolism and neuronal activity, respectively. Recently, we developed a multiwavelength imaging system and integrated it into a surgical microscope. Three LEDs of λ1=530nm, λ2=570nm and λ3=630nm were used for exciting [Ca2+]i fluorescence labeled by Rhod2 (AM) and sensitizing total hemoglobin (i.e., CBV), and deoxygenated-hemoglobin, whereas one LD of λ1=830nm was used for laser speckle imaging to form a CBF mapping of the brain. These light sources were time-sharing for illumination on the brain and synchronized with the exposure of CCD camera for multichannel images of the brain. Our animal studies indicated that this optical approach enabled simultaneous mapping of cocaine-induced changes in CBF, CBV and oxygenated- and deoxygenated hemoglobin as well as [Ca2+]i in the cortical brain. Its high spatiotemporal resolution (30μm, 10Hz) and large field of view (4x5 mm2) are advanced as a neuroimaging tool for brain functional study.

Local Cerebral Blood Flow Responses to Direct Stimulation in Somatosensory Cortex: Laser Doppler Flowmetric and Autoradiographic Analysis

We present the experiment results of in-vivo laser Doppler flowmetry (LDF) and FDG (18Fluorodeoxyglucose) labeled in-vitro autoradiography to study the local cerebral blood flow (LCBF) evoked by direct stimulation in the rat somatosensory cortex. Key Words: Local cerebral blood flow (LCBF), Activation-flow coupling (AFC), laser Doppler flowmetry (LDF), auto-radiography, somatosensory cortext.

In vivo optical microprobe imaging for intracellular Ca2+ dynamics in response to dopaminergic signaling in deep brain evoked by cocaine

Ca2+ plays a vital role as second messenger in signal transduction and the intracellular Ca2+ ([Ca2+]i) change is an important indicator of neuronal activity in the brain, including both cortical and subcortical brain regions. Due to the highly scattering and absorption of brain tissue, it is challenging to optically access the deep brain regions (e.g., striatum at >3mm under the brain surface) and image [Ca2+]i changes with cellular resolutions. Here, we present two micro-probe approaches (i.e., microlens, and micro-prism) integrated with a fluorescence microscope modified to permit imaging of neuronal [Ca2+]i signaling in the striatum using a calcium indicator Rhod2(AM). While a micro-prism probe provides a larger field of view to image neuronal network from cortex to striatum, a microlens probe enables us to track [Ca2+]i dynamic change in individual neurons within the brain. Both techniques are validated by imaging neuronal [Ca2+]i changes in transgenic mice with dopamine receptors (D1R, D2R) expressing EGFP. Our results show that micro-prism images can map the distribution of D1R- and D2R-expressing neurons in various brain regions and characterize their different mean [Ca2+]i changes induced by an intervention (e.g., cocaine administration, 8mg/kg., i.p). In addition, microlens images can characterize the different [Ca2+]i dynamics of D1 and D2 neurons in response to cocaine, including new mechanisms of these two types of neurons in striatum. These findings highlight the power of the optical micro-probe imaging for dissecting the complex cellular and molecular insights of cocaine in vivo.

Simultaneous assessing the intracellular potassium and calcium concentrations noninvasively in vivo with high resolution fluorescence imaging

Intracellular potassium and calcium plays a crucial role in maintaining the membrane potential to the functioning cells, and the irregular changes of their concentrations can induce severe clinical problems (e.g., cardiac arrhythmias, nerve dysfunction, kidney failure). Therefore, simultaneous assessing the intra-cellular potassium and calcium concentrations is clinically desired. Such assessments would benefit from tools which can perform high resolution imaging measurements noninvasively in vivo. Here we developed a high resolution fluorescence imaging technique to simultaneously image intra-cellular potassium and calcium concentrations from tissue level down to cellular resolution, and more importantly, with time-lapse imaging technique, we can track the dynamic concentration changes in response to functional or pharmacological stimuli in vivo.