Pradeep K. Shukla

Gene expression patterns in the hippocampus and amygdala of endogenous depression and chronic stress models

The etiology of depression is still poorly understood, but two major causative hypotheses have been put forth: the monoamine deficiency and the stress hypotheses of depression. We evaluate these hypotheses using animal models of endogenous depression and chronic stress. The endogenously depressed rat and its control strain were developed by bidirectional selective breeding from the Wistar–Kyoto (WKY) rat, an accepted model of major depressive disorder (MDD). The WKY More Immobile (WMI) substrain shows high immobility/despair-like behavior in the forced swim test (FST), while the control substrain, WKY Less Immobile (WLI), shows no depressive behavior in the FST. Chronic stress responses were investigated by using Brown Norway, Fischer 344, Lewis and WKY, genetically and behaviorally distinct strains of rats. Animals were either not stressed (NS) or exposed to chronic restraint stress (CRS). Genome-wide microarray analyses identified differentially expressed genes in hippocampi and amygdalae of the endogenous depression and the chronic stress models. No significant difference was observed in the expression of monoaminergic transmission-related genes in either model. Furthermore, very few genes showed overlapping changes in the WMI vs WLI and CRS vs NS comparisons, strongly suggesting divergence between endogenous depressive behavior- and chronic stress-related molecular mechanisms. Taken together, these results posit that although chronic stress may induce depressive behavior, its molecular underpinnings differ from those of endogenous depression in animals and possibly in humans, suggesting the need for different treatments. The identification of novel endogenous depression-related and chronic stress response genes suggests that unexplored molecular mechanisms could be targeted for the development of novel therapeutic agents.

Protective effect of curcumin against lead neurotoxicity in rat.

Curcumin (diferuloylmethane), an active ingredient of turmeric, is known to have multiple activities, including an antioxidant property, and has been suggested to be of use in treatment of several diseases. The present study has been undertaken to investigate the protective effect of curcumin against lead-induced neurotoxicity in rats. Exposure of rats to lead (50 mg/kg po) for 45 days caused an increase in lipid peroxidation (LPO) and a decrease in reduced glutathione (GSH) levels in cerebellum, corpus striatum, hippocampus and frontal cortex as compared with controls. Lead levels were significantly increased in these rats. Activity of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) decreased in all the brain regions following lead exposure. Interestingly, cotreatment with curcumin (100 mg/kg po) and lead (50 mg/kg po) for 45 days caused a significant decrease in LPO with concomitant decrease in lead levels in all the brain regions as compared with those treated with lead alone. A significant increase in reduced glutathione (GSH) levels, SOD and CAT activities was also observed in all the four brain regions in rats simultaneously treated with curcumin and lead. The results suggest that curcumin may prevent lead-induced neurotoxicity.

Protein kinase a in postmortem brain of depressed suicide victims: Altered expression of specific regulatory and catalytic subunits

BACKGROUND We recently reported reduced [3H]cyclic adenosine monophosphate binding and catalytic activity of protein kinase A in prefrontal cortex of depressed suicide victims. Here we examined the molecular basis of these alterations and whether these findings can be replicated in another cohort. METHODS Prefrontal cortex from depressed suicide victims and nonpsychiatric controls were obtained from the Lenhossek Human Brain Program, Budapest and the Maryland Brain Collection Program. [3H]cyclic adenosine monophosphate binding and protein kinase A activity were determined by radioligand binding and enzymatic assay, respectively. Expression of catalytic and regulatory subunits was determined by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. RESULTS [3H]cyclic adenosine monophosphate binding and total and endogenous protein kinase A activity were significantly decreased in membrane and cytosol fractions of prefrontal cortex of depressed suicide victims from the Budapest cohort, with a similar magnitude (33%-40% reduction) as reported for the Maryland cohort. In both cohorts, selective reduction (36%-41%) in mRNA and protein expression of the regulatory RIIbeta and the catalytic Cbeta was observed. CONCLUSIONS Our results suggest abnormalities in [3H]cyclic adenosine monophosphate binding and catalytic activity kinase A in brain of depressed suicide victims, which could be due to reduced expression of RIIbeta and Cbeta. These abnormalities in PKA may be critical in the pathophysiology of depression.

Neuroprotective efficacy of curcumin in arsenic induced cholinergic dysfunctions in rats

Our recent studies have shown that curcumin protects arsenic induced neurotoxicity by modulating oxidative stress, neurotransmitter levels and dopaminergic system in rats. As chronic exposure to arsenic has been associated with cognitive deficits in humans, the present study has been carried out to implore the neuroprotective potential of curcumin in arsenic induced cholinergic dysfunctions in rats. Rats treated with arsenic (sodium arsenite, 20mg/kg body weight, p.o., 28 days) exhibited a significant decrease in the learning activity, assessed by passive avoidance response associated with decreased binding of (3)H-QNB, known to label muscarinic-cholinergic receptors in hippocampus (54%) and frontal cortex (27%) as compared to controls. Decrease in the activity of acetylcholinesterase in hippocampus (46%) and frontal cortex (33%), staining of Nissl body, immunoreactivity of choline acetyltransferase (ChAT) and expression of ChAT protein in hippocampal region was also observed in arsenic treated rats as compared to controls. Simultaneous treatment with arsenic and curcumin (100mg/kg body weight, p.o., 28 days) increased learning and memory performance associated with increased binding of (3)H-QNB in hippocampus (54%), frontal cortex (25%) and activity of acetylcholinesterase in hippocampus (41%) and frontal cortex (29%) as compared to arsenic treated rats. Increase in the expression of ChAT protein, immunoreactivity of ChAT and staining of Nissl body in hippocampal region was also observed in rats simultaneously treated with arsenic and curcumin as compared to those treated with arsenic alone. The results of the present study suggest that curcumin significantly modulates arsenic induced cholinergic dysfunctions in brain and also exhibits neuroprotective efficacy of curcumin.

Reversal of chronic inflammatory pain by acute inhibition of Ca 2+/calmodulin-dependent protein kinase II

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major protein kinase that is capable of regulating the activities of many ion channels and receptors. In the present study, the role of CaMKII in the complete Freunds adjuvant (CFA)-induced inflammatory pain was investigated. Intraplantarly injected CFA was found to induce spinal activity of CaMKII (phosphorylated CaMKII), which was blocked by KN93 [[2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)], a CaMKII inhibitor. Pretreatment with KN93 (i.t.) dose-dependently prevented the development of CFA-induced thermal hyperalgesia and mechanical allodynia. Acute treatment with KN93 (i.t.) also dose-dependently reversed CFA-induced thermal hyperalgesia and mechanical allodynia. The action of KN93 started in 30 min and lasted for at least 2 to 4 h. KN92 (45 nmol i.t.) [2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], an inactive analog of KN93, showed no effect on CFA-induced CaMKII activation, allodynia, or hyperalgesia. Furthermore, our previous studies identified trifluoperazine, a clinically used antipsychotic drug, to be a potent CaMKII inhibitor. Inhibition of CaMKII activity by trifluoperazine was confirmed in the study. In addition, trifluoperazine (i.p.) dose-dependently reversed CFA-induced mechanical allodynia and thermal hyperalgesia. The drug was also effectively when given orally. In conclusion, our findings support a critical role of CaMKII in inflammatory pain. Blocking CaMKII or CaMKII-mediated signaling may offer a novel therapeutic target for the treatment of chronic pain.

Reversal of Morphine Antinociceptive Tolerance and Dependence by the Acute Supraspinal Inhibition of Ca2+/Calmodulin-Dependent Protein Kinase II

Previous studies have suggested that Ca2+/calmodulin-dependent protein kinase II (CaMKII) can modulate opioid tolerance and dependence via its action on learning and memory. In this study, we examined whether CaMKII could directly regulate opioid tolerance and dependence. CaMKII activity was increased after the treatment with morphine (100 mg/kg s.c. or 75 mg s.c. of morphine/pellet/mouse); the effect exhibited a temporal correction with the development of opioid tolerance and dependence. In mice treated with morphine (100 mg/kg s.c.), morphine tolerance and dependence developed in 2 to 6 h. An acute supraspinal administration of KN93 [2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)], a CaMKII inhibitor, was able to dose-dependently reverse the already-established antinociceptive tolerance to morphine (p < 0.001 for 15-30 nmol; not significant for 5 nmol). KN92 [2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine] (30 nmol i.c.v.), a kinase-inactive analog of KN93, did not affect opioid tolerance. Neither KN92 nor KN93 affected basal nociception or acute morphine antinociception (1-10 nmol i.c.v.). Likewise, dependence on morphine was abolished by the acute administration of KN93, but not KN92, in a dose-dependent manner. Pretreatment of mice with KN93 also prevented the development of morphine tolerance and dependence. The effect of acute CaMKII inhibition was not limited to the particular experimental model, because KN93 also acutely reversed the established opioid tolerance and dependence in mice treated with morphine (75 mg/pellet/mouse s.c.) for 6 days. Taken together, these data strongly support the hypothesis that CaMKII can act as a key and direct factor in promoting opioid tolerance and dependence. Identifying such a direct mechanism may be useful for designing pharmacological treatments for these conditions.

Neuroprotective effect of Acorus calamus against middle cerebral artery occlusion-induced ischaemia in rat.

The neuroprotective potential of ethanol:water (1:1) extract of rhizomes of Acorus calamus (AC–002) has been investigated in middle cerebral artery occlusion (MCAO)–induced ischaemia in rats. A significant behavioural impairment in Rota–Rod performance and grid walking was observed in rats, 72 hours after MCAO as compared to sham–operated animals. These rats also exhibited an increase in lipid peroxidation (cortex / 157%, corpus striatum – 58%) and a decrease in glutathione levels (cortex – 59%, corpus striatum – 34%) and superoxide dismutase (SOD) activity (cortex – 64%, corpus striatum – 32%) as compared to sham–operated animals. Ischaemic rats treated with AC–002 (25 mg/kg, p.o.) exhibited a significant improvement in neurobehavioural performance viz. Rota–Rod performance and grid walking as compared to the MCAO group. Interestingly, treatment with AC–002 in MCAO rats significantly decreased malonaldialdehyde levels in cortex as compared to ischaemic rats. A significant increase in reduced glutathione levels and SOD activity was also observed both in cortex and corpus striatum in MCAO rats treated with AC–002 in comparison to MCAO rats. Treatment with AC–002 in MCAO rats also reduced the contralateral cortical infarct area (19%) as compared to MCAO rats (33%). Neurological function score was improved in the AC–002–treated rats as compared to the MCAO group. The results of the present study indicate the neuroprotective efficacy of A. calamus in the rat model of ischaemia.

Brain Region Specific Alterations in the Protein and mRNA Levels of Protein Kinase A Subunits in the Post-Mortem Brain of Teenage Suicide Victims

Protein kinase A (PKA), a critical component of the adenylyl cyclase signaling system, phosphorylates crucial proteins and has been implicated in the pathophysiology of depression and suicide. The objective of the study was to examine if changes in PKA activity or in the protein and messenger RNA (mRNA) expression of any of its subunits are related to the pathophysiology of teenage suicide. We determined PKA activity and the protein and mRNA expression of different subunits of PKA in cytosol and membrane fractions obtained from the prefrontal cortex, (PFC) hippocampus, and nucleus accumbens (NA) of post-mortem brain from 17 teenage suicide victims and 17 nonpsychiatric control subjects. PKA activity was significantly decreased in the PFC but not the hippocampus of teenage suicide victims as compared with controls. However, the protein and mRNA expression of only two PKA subunits, that is, PKA RIα and PKA RIβ, but not any other subunits were significantly decreased in both membrane and cytosol fractions of the PFC and protein expression of RIα and RIβ in the NA of teenage suicide victims as compared to controls. A decrease in protein and mRNA expression of two specific PKA subunits may be associated with the pathogenesis of teenage suicide, and this decrease may be brain region specific, which may be related to the specific behavioral functions associated with these brain areas. Whether these changes in PKA subunits are related to suicidal behavior or are a result of suicide or are specific to suicide is not clear at this point.

Altered protein kinase a in brain of learned helpless rats: effects of acute and repeated stress

BACKGROUND Stress-induced learned helplessness (LH) in animals serves as a model of behavioral depression and some aspects of posttraumatic stress disorder. We examined whether LH behavior is associated with alterations in protein kinase A (PKA), a critical phosphorylating enzyme, how long these alterations persist after inescapable shock (IS), and whether repetition of IS prolongs the duration of LH behavior and changes in PKA. METHODS Rats were exposed to IS either on day 1 or twice, on day 1 and day 7. Rats were tested for escape latency on days 2 and 4 after day 1 IS or days 2, 8, and 14 after day 1 and day 7 IS. [(3)H]cAMP (cyclic adenosine monophosphate) binding, catalytic activity and expression of PKA subunits were determined in frontal cortex and hippocampus. RESULTS Higher escape latencies were observed in rats tested on day 2 after single IS and on day 14 after repeated IS. Concurrently, reduced [(3)H]cAMP binding, PKA activity, and expression of selective PKA RIIbeta and Calpha and Cbeta subunits were observed in the brains of these rats. CONCLUSIONS Repeated IS prolongs the duration of LH behavior, and LH behavior is associated with reductions in apparent activity and expression of PKA. These reductions in PKA may be critical in the pathophysiology of depression and other stress-related disorders.

Single and Repeated Stress-Induced Modulation of Phospholipase C Catalytic Activity and Expression: Role in LH Behavior

PI-PLC, a critical enzyme of the phosphoinositide (PI) signaling pathway, mediates many physiological functions in the brain, including cellular plasticity. Stress-induced learned helplessness (LH) in animals serves as a model of behavioral depression. Recently, we observed that repeated stress prolongs the duration of LH behavior in rats, enabling us to compare neurobiologic abnormalities in acute and chronic depression. Here we examine whether LH behavior is associated with alterations in phospholipase C (PLC), and whether repetition of inescapable shock has similar or dissimilar effects on PLC to those of the single-stress paradigm. Rats were exposed to inescapable shock either once on day 1, or twice, on days 1 and 7. Rats were tested for escape latency on days 2 and 4 after day 1 inescapable shock or on days 2, 8, and 14 after day 1 and 7 inescapable shock. PI-PLC activity and mRNA and protein expression of three different PLC isozymes were determined in the frontal cortex and hippocampus. Higher escape latencies were observed in LH rats tested on day 2 after single inescapable shock and on day 14 after repeated inescapable shock. Single inescapable shock reduced PI-PLC activity in the frontal cortex and hippocampus of LH rats. On the other hand, repeated inescapable shock not only reduced PI-PLC activity in these brain areas of LH rats but also selectively decreased the expression of PLC β1 and PLC γ1 isozymes. Our results suggest different responsiveness at the level of PI-PLC after single vs repeated stress, and that reductions in PLC may be critical in the pathophysiology of depression and other stress-related disorders.