Balaji Krishnan

Circadian rhythms in olfactory responses of Drosophila melanogaster

The core mechanism of circadian timekeeping in arthropods and vertebrates consists of feedback loops involving several clock genes, including period (per) and timeless (tim),. In the fruitfly Drosophila, circadian oscillations in per expression occur in chemosensory cells of the antennae, even when the antennae are excised and maintained in isolated organ culture. Here we demonstrate a robust circadian rhythm in Drosophila in electrophysiological responses to two classes of olfactory stimuli. These rhythms are observed in wild-type flies during light–dark cycles and in constant darkness, but are abolished in per or tim null-mutant flies (per01 and tim01) which lack rhythms in adult emergence and locomotor behaviour. Olfactory rhythms are also abolished in the per 7.2:2 transgenic line in which per expression is restricted to the lateral neurons of the optic lobe. Because per 7.2:2 flies do not express per in peripheral oscillators, our results provide evidence that peripheral circadian oscillators are necessary for circadian rhythms in olfactory responses. As olfaction is essential for food acquisition, social interactions and predator avoidance in many animals, circadian regulation of olfactory systems could have profound effects on the behaviour of organisms that rely on this sensory modality.

A new role for cryptochrome in a Drosophila circadian oscillator

Cryptochromes are flavin/pterin-containing proteins that are involved in circadian clock function in Drosophila and mice. In mice, the cryptochromes Cry1 and Cry2 are integral components of the circadian oscillator within the brain and contribute to circadian photoreception in the retina. In Drosophila, cryptochrome (CRY) acts as a photoreceptor that mediates light input to circadian oscillators in both brain and peripheral tissue. A Drosophila cry mutant, cryb, leaves circadian oscillator function intact in central circadian pacemaker neurons but renders peripheral circadian oscillators largely arrhythmic. Although this arrhythmicity could be caused by a loss of light entrainment, it is also consistent with a role for CRY in the oscillator. A peripheral oscillator drives circadian olfactory responses in Drosophila antennae. Here we show that CRY contributes to oscillator function and physiological output rhythms in the antenna during and after entrainment to light–dark cycles and after photic input is eliminated by entraining flies to temperature cycles. These results demonstrate a photoreceptor-independent role for CRY in the periphery and imply fundamental differences between central and peripheral oscillator mechanisms in Drosophila.

Circadian Clocks in Antennal Neurons Are Necessary and Sufficient for Olfaction Rhythms in Drosophila

BACKGROUND The Drosophila circadian clock is controlled by interlocked transcriptional feedback loops that operate in many neuronal and nonneuronal tissues. These clocks are roughly divided into a central clock, which resides in the brain and is known to control rhythms in locomotor activity, and peripheral clocks, which comprise all other clock tissues and are thought to control other rhythmic outputs. We previously showed that peripheral oscillators are required to mediate rhythmic olfactory responses in the antenna, but the identity and relative autonomy of these peripheral oscillators has not been defined. RESULTS Targeted ablation of lateral neurons by using apoptosis-promoting factors and targeted clock disruption in antennal neurons with newly developed dominant-negative versions of CLOCK and CYCLE show that antennal neurons, but not central clock cells, are necessary for olfactory rhythms. Targeted rescue of antennal neuron oscillators in cyc(01) flies through wild-type CYCLE shows that these neurons are also sufficient for olfaction rhythms. CONCLUSIONS Antennal neurons are both necessary and sufficient for olfaction rhythms, which demonstrates for the first time that a peripheral tissue can function as an autonomous pacemaker in Drosophila. These results reveal fundamental differences in the function and organization of circadian oscillators in Drosophila and mammals and suggest that components of the olfactory signal transduction cascade could be targets of circadian regulation.

Dopamine receptor mechanisms mediate corticotropin‐releasing factor‐induced long‐term potentiation in the rat amygdala following cocaine withdrawal

Corticotropin‐releasing factor (CRF) in the amygdala is involved in stress responses. Moreover, dopaminergic neurotransmission in the brain reward system including the amygdala plays a significant role in the pathology of cocaine addiction. The present study analysed CRF‐induced synaptic plasticity, its pharmacological sensitivity and interactions with the dopamine (DA) system in the basolateral to lateral capsula central amygdala (lcCeA) pathway after a 2‐week withdrawal from repeated cocaine administration. A physiologically relevant CRF concentration (25 nm) induced long‐term potentiation (LTP) that was enhanced after cocaine withdrawal. In saline‐treated rats, CRF‐induced LTP was mediated through N‐methyl‐d‐aspartate (NMDA) receptors, L‐type voltage‐gated calcium channels (L‐VGCCs) and CRF1 receptors. However, in cocaine‐withdrawn animals, activation of CRF1 and CRF2 receptors was found to enhance LTP. This enhanced CRF‐induced LTP after cocaine withdrawal was mediated through endogenous activation of both D1‐ and D2‐like receptors. Furthermore, expression of the D1 receptor (D1R) but not the D2R, D3R, D4R or D5R was significantly increased after cocaine withdrawal. CRF1 but not CRF2 protein expression was increased, suggesting that elevated levels of these proteins contributed to the enhancement of CRF‐induced LTP during cocaine withdrawal. CRF interactions with the DA system in the amygdala may represent a fundamental neurochemical and cellular mechanism linking stress to cocaine‐induced neuronal plasticity.

Dopamine-Induced Plasticity, Phospholipase D (PLD) Activity and Cocaine-Cue Behavior Depend on PLD-Linked Metabotropic Glutamate Receptors in Amygdala

Cocaine-cue associations induce synaptic plasticity with long lasting molecular and cellular changes in the amygdala, a site crucial for cue-associated memory mechanisms. The underlying neuroadaptations can include marked alterations in signaling via dopamine (DA) receptors (DRs) and metabotropic glutamate (Glu) receptors (mGluRs). Previously, we reported that DR antagonists blocked forms of synaptic plasticity in amygdala slices of Sprague-Dawley rats withdrawn from repeated cocaine administration. In the present study, we investigated synaptic plasticity induced by exogenous DA and its dependence on mGluR signaling and a potential role for phospholipase D (PLD) as a downstream element linked to mGluR and DR signaling. Utilizing a modified conditioned place preference (CPP) paradigm as a functional behavioral measure, we studied the neurophysiological effects after two-weeks to the last cocaine conditioning. We recorded, electrophysiologically, a DR-induced synaptic potentiation in the basolateral to lateral capsula central amygdala (BLA-lcCeA) synaptic pathway that was blocked by antagonists of group I mGluRs, particularly, the PLD-linked mGluR. In addition, we observed 2–2.5 fold increase in PLD expression and 3.7-fold increase in basal PLD enzyme activity. The enhanced PLD activity could be further stimulated (9.3 fold) by a DA D1-like (D1/5R) receptor agonist, and decreased to control levels by mGluR1 and PLD-linked mGluR antagonists. Diminished CPP was observed by infusion of a PLD-linked mGluR antagonist, PCCG-13, in the amygdala 15 minutes prior to testing, two weeks after the last cocaine injection. These results imply a functional interaction between D1/5Rs, group I mGluRs via PLD in the amygdala synaptic plasticity associated with cocaine-cues.

Cocaine withdrawal reduces group I mGluR-mediated long-term potentiation via decreased GABAergic transmission in the amygdala

Cocaine relapse can occur when cocaine‐associated environmental cues induce craving. Conditioned place preference (CPP) is a behavioral paradigm modeling the association between cocaine exposure and environmental cues. The amygdala is involved in cocaine cue associations with the basolateral amygdala (BLA) and central amygdala (CeA) acting differentially in cue‐induced relapse. Activation of metabotropic glutamate receptors induces synaptic plasticity, the mechanism of which is thought to underlie learning, memory and drug–cue associations. The goal of this study was to examine the neural alterations in responses to group I metabotropic glutamate receptor (mGluR) agonists in the BLA to lateral capsula of CeA (BLA–CeLc) pathway in slices from rats exposed to cocaine‐CPP conditioning and withdrawn for 14 days. mGluR1, but not mGluR5, agonist‐induced long‐term potentiation (mGluR1‐LTP) in the BLA–CeLc pathway was reduced in rats withdrawal from cocaine for 2 and 14 days, and exhibited an altered concentration response to picrotoxin. Cocaine withdrawal also reduced γ‐aminobutyric acid (GABA)ergic synaptic inhibition in CeLc neurons. Blocking cannabinoid receptor 1 (CB1) reduced mGluR1‐LTP in the saline‐treated but not cocaine‐withdrawn group. Response to CB1 but not CB2 agonist was altered after cocaine. Additionally, increasing endocannabinoid (eCB) levels abolished mGluR1‐LTP in the saline but not cocaine‐withdrawn group. However, CB1 and CB2 protein levels were increased in the amygdala of cocaine‐withdrawn rats while mGluR1 and mGluR5 remained unchanged. These data suggested that the mechanisms underlying the diminished mGluR1‐LTP in cocaine‐withdrawn rats involve an altered GABAergic synaptic inhibition mediated by modulation of downstream eCB signaling. These changes may ultimately result in potentiated responses to environmental cues that would bias behavior toward drug‐seeking.

Near infrared light decreases synaptic vulnerability to amyloid beta oligomers

Synaptic dysfunction due to the disrupting binding of amyloid beta (Aβ) and tau oligomers is one of the earliest impairments in Alzheimer’s Disease (AD), driving initial cognitive deficits and clinical manifestation. Consequently, there is ample consensus that preventing early synaptic dysfunction would be an effective therapeutic strategy for AD. With this goal in mind, we investigated the effect of a treatment of mice with near infrared (NIR) light on synaptic vulnerability to Aβ oligomers. We found that Aβ oligomer binding to CNS synaptosomes isolated from wild type (wt) mice treated with NIR light was significantly reduced and the resulting suppression of long term potentiation (LTP) by Aβ oligomers was prevented. Similarly, APP transgenic mice treated with NIR showed a significant reduction of endogenous Aβ at CNS synapses. We further found that these phenomena were accompanied by increased synaptic mitochondrial membrane potential in both wt and Tg2576 mice. This study provides evidence that NIR light can effectively reduce synaptic vulnerability to damaging Aβ oligomers, thus furthering NIR light therapy as a viable treatment for AD.

Amygdala-Hippocampal Phospholipase D (PLD) Signaling As Novel Mechanism of Cocaine- Environment Maladaptive Conditioned Responses

Background: Drug-environment associative memory mechanisms and the resulting conditioned behaviors are key contributors in relapse to cocaine dependence. Recently, we reported rat amygdala phospholipase D as a key convergent downstream signaling partner in the expression of cocaine-conditioned behaviors mediated by glutamatergic and dopaminergic pathways. In the present study, 1 of the 2 known upstream serotonergic targets of phospholipase D, the serotonin (5-hydroxytryptamine) 2C receptor, was investigated for its role in recruiting phospholipase D signaling in cocaine-conditioned behaviors altered in the rat amygdala and dorsal hippocampus. Methods: Using Western-blot analysis, amygdala phospholipase D phosphorylation and total expression of phospholipase D/5-hydroxytryptamine 2C receptor were observed in early (Day-1) and late (Day-14) withdrawal (cocaine-free) states among male Sprague-Dawley rats subjected to 7-day cocaine-conditioned hyperactivity training. Functional studies were conducted using Chinese Hamster Ovary cells with stably transfected human unedited isoform of 5-hydroxytryptamine 2C receptor. Results: Phosphorylation of phospholipase D isoforms was altered in the Day-1 group of cocaine-conditioned animals, while increased amygdala and decreased dorsal hippocampus phospholipase D/5-hydroxytryptamine 2C receptor protein expression were observed in the Day-14 cocaine-conditioned rats. Functional cellular studies established that increased p phospholipase D is a mechanistic response to 5-HT2CR activation and provided the first evidence of a biased agonism by specific 5-hydroxytryptamine 2C receptor agonist, WAY163909 in phospholipase D phosphorylation 2, but not phospholipase D phosphorylation 1 activation. Conclusions: Phospholipase D signaling, activated by dopaminergic, glutamatergic, and serotonergic signaling, can be a common downstream element recruited in associative memory mechanisms altered by cocaine, where increased expression in amygdala and decreased expression in dorsal hippocampus may result in altered anxiety states and increased locomotor responses, respectively.

Fear potentiated startle increases phospholipase D (PLD) expression/activity and PLD-linked metabotropic glutamate receptor mediated post-tetanic potentiation in rat amygdala

Long-term memory (LTM) of fear stores activity dependent modifications that include changes in amygdala signaling. Previously, we identified an enhanced probability of release of glutamate mediated signaling to be important in rat fear potentiated startle (FPS), a well-established translational behavioral measure of fear. Here, we investigated short- and long-term synaptic plasticity in FPS involving metabotropic glutamate receptors (mGluRs) and associated downstream proteomic changes in the thalamic-lateral amygdala pathway (Th-LA). Aldolase A, an inhibitor of phospholipase D (PLD), expression was reduced, concurrent with significantly elevated PLD protein expression. Blocking the PLD-mGluR signaling significantly reduced PLD activity. While transmitter release probability increased in FPS, PLD-mGluR agonist and antagonist actions were occluded. In the unpaired group (UNP), blocking the PLD-mGluR increased while activating the receptor decreased transmitter release probability, consistent with decreased synaptic potentials during tetanic stimulation. FPS Post-tetanic potentiation (PTP) immediately following long-term potentiation (LTP) induction was significantly increased. Blocking PLD-mGluR signaling prevented PTP and reduced cumulative PTP probability but not LTP maintenance in both groups. These effects are similar to those mediated through mGluR7, which is co-immunoprecipitated with PLD in FPS. Lastly, blocking mGluR-PLD in the rat amygdala was sufficient to prevent behavioral expression of fear memory. Thus, our study in the Th-LA pathway provides the first evidence for PLD as an important target of mGluR signaling in amygdala fear-associated memory. Importantly, the PLD-mGluR provides a novel therapeutic target for treating maladaptive fear memories in posttraumatic stress and anxiety disorders.

INCREASED SYNAPTIC SENSITIVITY TO Aβ AND TAU OLIGOMERS IN THE AGING CNS AS A FUNCTION OF DECREASING NEURAL STEM CELLS

investigate correlation between synaptic activity and behavior. Quantal analysis of light-evoked IPSCs differentiate preand postsynaptic mechanisms of action. Baclofen (30mM) was used to illustrate a clear pre-synaptic action while Memantine (3-30mM) had mixed preand postsynaptic actions. Action of these drugs in calcium buffering is also being investigated. Continuing experiments with glutamate modulators are allowing us to uncover a novel mechanism of action for memantine. Conclusions:Our data so far has illustrated the potential of using quantal analysis in an optogenetic model in order to study preand post-synaptic actions of glutamate and calcium modulators in the neuron. Ultimately, we will be able to determine the mechanism of action of memantine in the Basal forebrain during aging.