Consumption of a High-Fat Diet Alters Perineuronal Nets in the Prefrontal Cortex.

A key factor in the development of obesity is the overconsumption of fatty foods, which, in addition to facilitating weight gain, alters neuronal structures within brain reward circuitry. Our previous work demonstrates that sustained consumption of a high-fat diet (HFD) attenuates spine density in the prefrontal cortex (PFC). Whether HFD promotes structural adaptation among inhibitory cells of the PFC is presently unknown. One structure of interest is the perineuronal net (PNN), a specialized extracellular matrix surrounding, primarily, parvalbumin-containing GABAergic interneurons. PNNs contribute to synaptic stabilization, protect against oxidative stress, regulate the ionic microenvironment within cells, and modulate regional excitatory output. To examine diet-induced changes in PNNs, we maintained rats on one of three dietary conditions for 21 days: ad libitum chow, ad libitum 60% high fat (HF-AL), or limited-access calorically matched high fat (HF-CM), which produced no significant change in weight gain or adiposity with respect to chow controls. The PNN "number" and intensity were then quantified in the prelimbic (PL-PFC), infralimbic (IL-PFC), and ventral orbitofrontal cortex (OFC) using Wisteria floribunda agglutinin (WFA). Our results demonstrated that fat exposure, independent of weight gain, induced a robust decrease in the PNN intensity in the PL-PFC and OFC and a decrease in the PNN number in the OFC.

Sex and region-specific effects of high fat diet on PNNs in obesity susceptible rats.

Perineuronal nets (PNNs) are specialized extracellular matrix structures that primarily surround fast-spiking parvalbumin (PV)-containing interneurons within the PFC. They regulate PV neuron function and plasticity to maintain cortical excitatory/inhibitory balance. For example, reductions in PNN intensity are associated with reduced local inhibition and enhanced pyramidal neuron firing. We previously found that exposure to dietary high fat reduced PNN intensity within the PFC of male Sprague-Dawley (SD) rats. However, how high fat affects PNNs in the PFC of females or in obesity-vulnerable vs. -resistant models is unknown. Therefore, we gave male and female SD, selectively bred obesity-prone (OP), and obesity-resistant rats (OR) free access to standard lab chow or 60% high fat for 21 days. We then measured the number of PNN positive cells and PNN intensity (determined by Wisteria floribunda agglutinin [WFA] staining) as well as the number of PV positive neurons using immunohistochemistry. We found sex and region-specific effects of dietary high fat on PNN intensity, in the absence of robust changes in cell number. Effects were comparable in SD and OP but differed in OR rats. Specifically, high fat reduced PNN intensities in male SD and OP rats but increased PNN intensities in female SD and OP rats. In contrast, effects in ORs were opposite, with males showing increases in PNN intensity and females showing a reduction in intensity. Finally, these effects were also region specific, with diet-induced reductions in PNN intensity found in the prelimbic PFC (PL-PFC) and ventral medial orbital frontal cortex (vmOFC) of SD and OP males in the absence of changes in the infralimbic PFC (IL-PFC), and increases in PNN intensity in the IL-PFC of SD and OP females in the absence of changes in other regions. These results are discussed in light of roles PNNs may play in influencing PFC neuronal activity and the differential role of these sub-regions in food-seeking and motivation.

Tumor promotion by phorbol esters in skin: evidence for a memory effect.

By means of a two-stage promotion protocol in mouse epidermis with 12-O-tetradecanoylphorbol-13-acetate as first-stage promoter and 12-O-retinoylphorbol-13-acetate as second-stage promoter, the effects of the former that are critical and obligatory for tumor promotion were shown to be irreversible in nature for at least 8 weeks. The reversibility of tumor promotion was related to the second stage of promotion, reflecting the reversibility of epidermal hyperplasia induced by 12-O-tetradecanoylphorbol-13-acetate.

Characterization of extracellular dopamine clearance in the medial prefrontal cortex: role of monoamine uptake and monoamine oxidase inhibition.

In vitro rotating disk electrode (RDE) voltammetry and in vivo microdialysis were used to characterize dopamine clearance in the rat medial prefrontal cortex (mPFC). RDE studies indicate that inhibition by cocaine, specific inhibitors of the dopamine transporter (DAT) and norepinephrine transporter (NET), and low Na(+) produced a 50-70% decrease in the velocity of dopamine clearance. Addition of the monoamine (MAO) inhibitors, l-deprenyl, clorgyline, pargyline, or in vivo nialamide produced 30-50% inhibition. Combined effects of uptake inhibitors with l-deprenyl on dopamine clearance were additive (up to 99% inhibition), suggesting that at least two mechanisms may contribute to dopamine clearance. Dopamine measured extracellularly 5 min after exogenous dopamine addition to incubation mixtures revealed that most conditions of DAT/NET inhibition did not produce elevated dopamine levels above controls. Inhibition of MAO produced elevated dopamine levels only after long-term, but not short-term, incubation in vitro. Short-term incubation of l-deprenyl combined with DAT and NET uptake inhibitors increased dopamine above control levels, consistent with more than one mechanism of dopamine clearance. Local infusion of pargyline (100 or 300 microm) into the mPFC or striatum via microdialysis produced more pronounced and immediate increases in mPFC dopamine levels compared with striatum. Furthermore, dopamine elevation in the mPFC was not accompanied by a decrease in the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, as found in the striatum. These findings may have revealed a unique mechanism of mPFC dopamine clearance and therefore contribute to the understanding of multiple behaviors that involve mPFC dopamine transmission, such as schizophrenia, drug abuse, and working memory function.

Repeated low-level formaldehyde exposure produces cross-sensitization to cocaine: possible relevance to chemical sensitivity in humans.

Sensitivity to chemicals in humans has been proposed to be an acquired disorder in which individuals become increasingly sensitive to chemicals in the environment. A possible link between the manifestation of psychiatric symptoms in individuals claiming sensitivity to chemicals was investigated based on a leading hypothesis put forth by Bell and co-workers (1992) to explain the amplification of symptoms after chemical exposure. The hypothesis is that chemical sensitivities may be akin to sensitization observed in rodents after repeated psychostimulants. Repeated exposure to psychostimulants enhances behavioral activity and the underlying neurochemical responses in specific limbic pathways; a similar sensitization of limbic pathways has been proposed to occur in individuals who become sensitive to chemicals. To test this hypothesis, female Sprague-Dawley rats were exposed to either air or formaldehyde (Form) for 1 h/day for 7 days or 20 days (5 days/week x 4 weeks). Two to 4 days after the last exposure, rats were given a cocaine challenge (= early withdrawal) followed by an additional cocaine challenge 4-6 weeks later (= late withdrawal). No differences in cocaine-induced locomotor activity were noted between groups after 7 days of exposure. However, after 20 days of exposure to Form, vertical activity was significantly elevated at both early and late withdrawal times. These studies demonstrate that behavioral sensitization occurs after long-term, but not short-term, low-level exposure to Form, and lends support to the limbic system sensitization hypothesis of sensitivity to chemicals in humans.

In vivo assessment of release and metabolism of dopamine in the ventrolateral striatum of awake rats following administration of dopamine D1 and D2 receptor agonists and antagonists.

The ability of specific dopamine (DA) receptor agonists and antagonists to modify the release and metabolism of DA in the ventrolateral striatum of awake rats was assessed using in vivo microdialysis. The specific DA D2 receptor antagonist, raclopride (0.1, 0.5 and 2.0 mg/kg, i.p.), dose-dependently increased release of DA and levels of the metabolites DOPAC and HVA, while the D2 receptor agonist, quinpirole (0.03, 0.1 and 0.3 mg/kg), decreased levels of DA, DOPAC and HVA. The DA D1 receptor antagonist, SCH23390 [(R + (+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl- 1H-3-benzazepin-7-ol) (0.01, 0.05 and 0.25 mg/kg), produced an increase in DA, DOPAC and HVA but of a lesser magnitude than raclopride. The D1 agonist SKF38393 (1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol) (1.0, 3.0 and 10.0 mg/kg) failed to affect the release of metabolism of DA at any dose. These results support previous findings that activation of D2 receptors has greater control over in vivo DA function, than drugs specifically affecting D1 receptors.

Effects of cocaine and footshock stress on extracellular dopamine levels in the medial prefrontal cortex.

The interaction between cocaine and footshock stress was evaluated by determining changes in extracellular dopamine and metabolite levels by in vivo microdialysis in the medial prefrontal cortex. In the first experiment, rats were given one of three treatments: no pretreatment (naive); or five daily injections of cocaine (15 mg/kg, i.p.); or daily saline. Six to seven days later, animals had a microdialysis probe implanted into the medial prefrontal cortex and an acute 20-min footshock stress (0.45 mA/200 ms per s) or sham shock was delivered. The results showed that acute footshock in daily saline pretreated rats increased medial prefrontal cortex extracellular dopamine concentrations to 203% of baseline levels. In rats pretreated with daily cocaine, this stress-induced response was completely abolished and extracellular dopamine was reduced 38% below baseline levels by 100 min post-shock. Naive animals showed a response to footshock that was intermediate between cocaine and saline pretreated rats (156% of baseline). In the second experiment, rats were given either no pretreatment (naive) or five daily 20-min footshock treatments (as above) or daily sham shock. Six to seven days later, an acute cocaine or saline injection was given. In daily sham-pretreated rats, extracellular dopamine levels were increased to 500% of baseline in response to acute cocaine. Pretreatment with daily footshock significantly reduced the response to acute cocaine (216% of baseline). Naive rats showed an intermediate increase that was not significantly different from footshock-pretreated animals (265% of baseline). Locomotor activity measured concurrently with dialysis showed a non-significant trend towards enhanced activity in daily footshock animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of cocaine sensitization: regulation by the medial prefrontal cortex.

Extracellular levels of dopamine are increased in response to systemic administration of cocaine in several brain areas including the nucleus accumbens and medial prefrontal cortex. While the cocaine-induced increase in extracellular dopamine levels in the nucleus accumbens is augmented after repeated daily cocaine, the response of extracellular dopamine levels in the medial prefrontal cortex is attenuated. Since dopamine in the medial prefrontal cortex has an inhibitory effect on nucleus accumbens dopamine levels and locomotor activity, the role of medial prefrontal cortex dopamine tolerance in the expression of sensitized locomotor behavior was further examined by injection of D-amphetamine sulfate into the prelimbic portion of the medial prefrontal cortex just prior to cocaine challenge in cocaine-sensitized rats. Male Sprague-Dawley rats were non-handled (naive) or injected with either saline (1 ml/kg, i.p.) or cocaine (15 mg/kg, i.p.) for five consecutive days. After a seven to 12 day withdrawal period, rats were microinjected with either saline or various doses of amphetamine into primarily the prelimbic region of the medial prefrontal cortex followed by systemic injection of saline or cocaine. In naive rats, intramedial prefrontal cortex amphetamine produced a trend toward decreased locomotor responding to cocaine challenge while no effect of amphetamine was evident in daily saline pretreated rats. Daily cocaine pretreated rats that received saline in the medial prefrontal cortex demonstrated a sensitized locomotor response compared to their daily saline pretreated counterparts. This sensitization was blocked by a low dose of amphetamine (0.175 microg/side) in the medial prefrontal cortex, an effect which disappeared in animals administered higher amphetamine doses. The results suggest that in rats sensitized to cocaine, decreased medial prefrontal cortex dopamine levels in response to cocaine challenge may contribute to behavioral sensitization. Furthermore, the data indicate the possibility that there is an optimal range at which medial prefrontal cortex amphetamine exerts maximal behavioral inhibition. These findings implicate a role for decreased cortical control in producing sensitized behavioral responding to cocaine.

Regulation of medial prefrontal cortex dopamine by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors.

In the medial prefrontal cortex, repeated cocaine produces tolerance of the extracellular dopamine response to subsequent cocaine injection. These studies characterized the influence of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors on the medial prefrontal cortex dopamine response to acute cocaine, amphetamine and potassium chloride as a first step to assess whether these receptor subtypes may be candidates for mediating dopamine tolerance after repeated cocaine. Local infusion of 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) produced an approximate 40% increase in dopamine levels in the medial prefrontal cortex, while a 30 microM dose did not alter basal levels infused over a 3-h period. Thus, 30 microM CNQX was chosen for the remaining experiments, and was infused for 1 h prior to and during all in vivo treatments. Local medial prefrontal cortex infusion of the 30 microM dose blocked the small increase in dopamine levels elicited by systemic saline injection (maximum of 26%), as well as the much larger increase in response to acute cocaine injection (maximum of 340%). Local infusion of D-amphetamine (3 and 30 microM) through the probe increased dopamine to 300 and 600% of basal levels, respectively. Co-infusion of CNQX partially blocked the response for the first 40 min, but dopamine levels recovered by 60 min later. Local infusion of 100 mM potassium chloride elicited a 600% increase in dopamine levels, which was attenuated approximately 50% by CNQX co-infusion. Potassium-stimulated release of dopamine was also measured in vitro in medial prefrontal cortical and striatal tissue. By 30 s after potassium addition, dopamine levels increased to 800% above baseline in the medial prefrontal cortex, and this increase was blocked by the presence of 30 microM CNQX. In contrast, potassium-stimulated dopamine release in striatal tissue was approximately 250% above basal levels, with no effect of CNQX on dopamine release. Locomotor behavior collected during dialysis experiments demonstrated that increased activity induced by local infusion of potassium chloride was severely attenuated by co-infusion of 30 microM CNQX, while no effects of this drug were found for cocaine-elicited behavior. These results suggest a potent influence of glutamate via alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptors on extracellular dopamine in the medial prefrontal cortex, and these receptors may regulate dopamine release through a presynaptic mechanism. The findings may help elucidate the role of medial prefrontal cortex dopamine-glutamate interactions in drug abuse and stress- and drug-precipitated psychosis.

Activation of dopamine D1 receptors in the medial prefrontal cortex produces bidirectional effects on cocaine-induced locomotor activity in rats: effects of repeated stress.

We examined the effects of repeated stress and D1 receptor activation in the medial prefrontal cortex (mPFC) on acute-cocaine-induced locomotor activity in rats. Male rats were given 7 days of either handling (Controls) or a variety of stressors. After 8-17 days' withdrawal, rats received an intra-mPFC microinjection of the full D1 agonist, SKF 81297: 0, 0.03, 0.1 or 0.3 microg/side followed by an i.p. saline or cocaine injection (15 mg/kg, i.p.). The target sites were either the dorsal or ventral mPFC. We also divided rats into either high or low responders based on their locomotor response to an acute cocaine injection. In the dorsal PFC, low responder Control and Stress groups demonstrated an augmentation of cocaine-induced increases in activity after SKF 81297, compared with vehicle, microinjection. In contrast, high responder rats demonstrated a suppression of cocaine-induced increases in activity after intra-mPFC SFK 81297 infusion, with an apparent 10 times higher sensitivity in the Stress group. In the ventral PFC, low responder Controls showed no changes after SKF 81297 infusion, while the Stress group showed an increase in cocaine-induced activity in response to SKF 81297. In high responders given SFK 81297 into the ventral mPFC, cocaine-induced activity was suppressed in Controls, while stress pretreatment rendered animals resistant to SKF 81297 effects. These results indicate that D1 receptor activation effects in the mPFC are bidirectional depending on whether rats have a high or low locomotor response to cocaine. Further, daily stress alters the sensitivity of the mPFC to SKF 81297, which is dependent on whether the dorsal or ventral mPFC is targeted.

Subchronic administration of clozapine, but not haloperidol or metoclopramide, decreases dopamine D2 receptor messenger RNA levels in the nucleus accumbens and caudate-putamen in rats.

The effects of unique profile antipsychotic drugs on dopamine D2 receptors and D2 receptor messenger RNA were assessed following subchronic administration in rats. Male, Sprague-Dawley rats were administered oral haloperidol, clozapine, metoclopramide or no drug for three weeks via their drinking water. Tissue from the medial nucleus accumbens and dorsolateral caudate-putamen was dissected and analyzed by Northern blot analysis for levels of dopamine D2 receptor messenger RNA and binding assays conducted with [3H]spiperone for dopamine D2 receptors. Haloperidol and metoclopramide, but not clozapine, significantly increased [3H]spiperone in the caudate-putamen, but not the nucleus accumbens. Clozapine significantly decreased D2 messenger RNA levels in the caudate-putamen and the nucleus accumbens, while metoclopramide and haloperidol had no significant effect in either brain region. The finding of decreased D2 receptor messenger RNA levels produced by subchronic clozapine may account for the lack of striatal D2 receptor up-regulation, which was robustly observed after subchronic haloperidol and metoclopramide. Furthermore, since haloperidol and metoclopramide have a high liability for motor side effects, the current results relate favorably to the low motor side effect profile of clozapine.

Manipulation of dopamine d1-like receptor activation in the rat medial prefrontal cortex alters stress- and cocaine-induced reinstatement of conditioned place preference behavior.

These studies examined the ability of the dopamine D1-like agonist SKF 81297 and D1-like antagonist SCH 23390 in the medial prefrontal cortex to alter the reinstatement of cocaine-induced conditioned place preference behavior. Male Sprague-Dawley rats were fitted with bilateral cannulae over the medial prefrontal cortex and subsequently trained in a conditioned place preference task. Animals were trained in this task using four pairings of cocaine (12 mg/kg, i.p.). Conditioned place preference was demonstrated in all animals, and this behavior was then extinguished over a 5-10-day period before testing for reinstatement. Just prior to reinstatement by immobilization stress or a cocaine priming injection (5 mg/kg, i.p.), a microinjection of the D1-like receptor antagonist SCH 23390 (0.01, 0.1 or 1.0 microg/side), or the D1-like receptor agonist SKF 81297 (0.1, 0.3 or 1.0 microg/side) was given into the medial prefrontal cortex. SCH 23390 blocked both stress- and cocaine-induced reinstatement of conditioned place preference after the two higher doses were administered into the medial prefrontal cortex. The highest dose of SKF 81297 (1.0 microg/side) prevented immobilization stress- but not cocaine-induced reinstatement. The highest dose of these drugs given in the absence of stress or cocaine did not produce reinstatement. The results indicate that immobilization stress given within the place-preference chamber is capable of producing reinstatement of cocaine-seeking behavior. The microinjection studies suggest that D1-like receptor antagonism within the prefrontal cortex is sufficient to block reinstatement by stress and cocaine. Furthermore, the results from D1-like receptor activation in the medial prefrontal cortex point to utilization of different neural pathways for stress- and cocaine-induced reinstatement.

Multiple chemical sensitivity: potential role for neural sensitization.

An emerging issue in environmental health is the phenomenon of multiple chemical sensitivity (MCS). Multiple chemical sensitivity is a controversial disorder characterized by multiorgan symptoms in response to low-level chemical exposures that are considered safe for the general population. The onset of MCS is often attributed to prior repeated chemical exposures in the home and/or workplace, and, once initiated, symptoms are triggered by extremely low levels of many chemicals/foods. No single case definition exists for MCS due to several issues that call into question its validity as a distinct illness induced by prior chemical exposure. Hypotheses regarding the etiological basis for MCS range from direct toxicological effects of chemicals to the notion that MCS is purely a psychological "belief system". One leading hypothesis suggests that MCS represents a neural sensitization phenomenon, wherein susceptible individuals demonstrate extreme sensitivity to chemicals and odor intolerance due to central nervous system (CNS) sensitization processes. The recent development of an animal model for MCS provides some support for the sensitization hypothesis and may offer evidence for behavioral changes observed in at least a subset of those reporting MCS.

Phospholipids involved in specific binding of 12-O-(5-azido-2-nitrobenzoyl)phorbol-13-acetate to epidermal microsomes, a photolabeling study.

The preparation of 12-O-(5-azido-2-nitro)benzoylphorbol-13-acetate (NABPA) is described. It is used as a photoaffinity probe to study the biochemical components involved in the specific binding of phorbol esters to an epidermal particulate fraction (microsomes) from NMRI mice: without irradiation NABPA binds in a saturable and high affinity manner (KD = 12 nM; Rt = 2.6 pmol/mg protein) to microsomes; after irradiation (at 350 nm) specific photolabeling (representing specific binding of NABPA) is found of phospholipids (phosphatidyl-serine (PS) and -ethanolamine(PE)), but not of protein. The results are discussed in the context of protein kinase C being a receptor for phorbol esters.

Potential role of stress and sensitization in the development and expression of multiple chemical sensitivity.

Chemical sensitivity in humans may be an acquired disorder in which individuals become increasingly sensitive to chemicals in the environment. It is hypothesized that in individuals with multiple chemical sensitivity (MCS), a sensitization process has occurred that is akin to behavioral sensitization and kindling observed in rodents. In the rodent sensitization model, repeated exposure to stress or drugs of abuse enhances behavioral and neurochemical responses to subsequent stimuli (stress or drugs of abuse). Kindling is a form of sensitization in which repeated application of electrical stimuli applied to the brain at low levels culminates in the induction of full-blown seizures when the same stimulus is applied at a later time. A similar sensitization of specific limbic pathways in the brain may occur in individuals with MCS. The time-dependent nature of sensitization and kindling and the role of stress in the development of sensitization are discussed in the context of rodent models, with an emphasis on application of these models to human studies of MCS.

Testing the neural sensitization and kindling hypothesis for illness from low levels of environmental chemicals.

Sensitization in the neuroscience and pharmacology literatures is defined as progressive increase in the size of a response over repeated presentations of a stimulus. Types of sensitization include stimulant drug-induced time-dependent sensitization (TDS), an animal model related to substance abuse, and limbic kindling, an animal model for temporal lobe epilepsy. Neural sensitization (primarily nonconvulsive or subconvulsive) to the adverse properties of substances has been hypothesized to underlie the initiation and subsequent elicitation of heightened sensitivity to low levels of environmental chemicals. A corollary of the sensitization model is that individuals with illness from low-level chemicals are among the more sensitizable members of the population. The Working Group on Sensitization and Kindling identified two primary goals for a research approach to this problem: to perform controlled experiments to determine whether or not sensitization to low-level chemical exposures occurs in multiple chemical sensitivity (MCS) patients; and to use animal preparations for kindling and TDS as nonhomologous models for the initiation and elicitation of MCS.

Cocaine alters glutamic acid decarboxylase differentially in the nucleus accumbens core and shell.

The effects of acute and repeated daily cocaine on the levels of mRNA coding for glutamic acid decarboxylase (GAD), preproenkephalin (PPE), preprotachykinin (PPT), and the dopamine D2 receptor were determined in the striatum, nucleus accumbens core and shell areas (NAcore, NAshell), and medial prefrontal cortex. Rats were given repeated saline or cocaine for 6 days. A cocaine challenge administered 24 h later resulted in an augmented locomotor response in daily cocaine-pretreated rats. Six h after the challenge, rats were sacrificed and Northern blot analysis revealed that acute cocaine increased GAD mRNA levels by 44% in the NAshell, while repeated cocaine prevented the acute cocaine-induced increase. These data suggest that cocaine may differentially regulate GABA release at NA core and shell projection fields.

Systemic promoting action and leukemogenesis in SWR mice by phorbol and structurally related polyfunctional diterpenes.

Phorbol and six structurally related compounds representing the polyfunctional diterpenes of the tigliane, ingenane, and lathyrane types were tested for systemic promoting and leukemogenic activity in SWR mice. For systemic initiation soon after birth, 15 microgram dimethylnitrosamine (DMN) was injected s.c. The diterpenes were administered i.p. either with or without prior systemic initiation with DMN. Systemic promotion was expressed for liver by induction of adenomas with all the diterpenes tested, some of them being more potent than phorbol. The relatively high dose of DMN used as initiator prevented an evaluation of promoting action in relation to lung carcinogenesis. The leukemogenic effect of phorbol in SWR mice was confirmed at three different dose levels. The other diterpenes tested had no significant leukemogenic activity. The leukemogenic action of phorbol was totally inhibited by prior DMN injection. The lack of correlation between promoting action in skin, systemic promoting action in liver and leukemogenic action, among the diterpenes tested, is discussed.

Dietary cancer risk from conditional cancerogens (tumor promoters) in produce of livestock fed on species of spurge (Euphorbiaceae). V. Skin irriitant and tumor-promoting diterpene ester toxins of the tigliane and ingenane type in the herbs Euphorbia nubica and Euphorbia helioscopia contaminating fodder of livestock.

Irritant diterpene ester toxins were isolated from Euphorbia nubica and E. helioscopia, which are contaminants of the green fodder of livestock in Egypt. Fractionations of methanol extracts of aerial parts of both plants were monitored by the irritation unit on the mouse ear. Plant extracts were subjected to multiplicative distribution methods, yielding irritant hydrophilic fractions that were further purified by column chromatography. Final purification of the materials was achieved by TLC (silica gel) followed by HPLC, or by TLC alone. In this way, from E. nubica, five Euphorbia factors (Nu1-Nu5) were isolated and characterized as short-chain polyfunctional diterpene esters of tigliane-type parent alcohols. The two weak irritants Nul and Nu3 were triesters of 4-deoxy(4alpha)phorbol. Nu2 was shown to be a triester of the stereoisomeric tigliane-type parent alcohol 4-deoxyphorbol. Weak irritant Nu4 probably is a positional isomer of Nu2. Nu5 was characterized as a short-chain triester of 4,20-dideoxy-5xi-hydroxyphorbol. From E. helioscopia, six short- to medium-chain polyfunctional diterpene esters of the ingenane type, generally containing unsaturated acids were obtained, i.e., four irritant esters of ingenol (Euphorbia factors H1, H2, H5, and H6) and two esters of 20-deoxyingenol (non-irritant Euphorbia substance HS4, and irritant Euphorbia factor H8). All irritant Euphorbia factors of the tigliane and ingenane diterpene ester type described in this investigation are considered to be more or less active tumor promoters, i.e., conditional (non-genotoxic) cancerogens. The Euphorbia factors assayed exhibited moderate (H1) to low (H8) relative tumor-promoting potency in comparison to the ingenane prototype DTE tumor promoter 3-TI.

Stress-induced cross-sensitization to cocaine: effect of adrenalectomy and corticosterone after short- and long-term withdrawal.

We have recently shown that adrenalectomy (ADX) in rats blocks the appearance of cocaine-induced sensitization when this behavioral response is tested at early withdrawal times (1-2 days), but not after later withdrawal from cocaine (12 days). To determine if a similar phenomenon occurred with stress-induced sensitization, male Sprague-Dawley rats were given a sham ADX, ADX surgery, or ADX plus s.c. implanted corticosterone (CORT) pellets (CORT 12.5% pellets or CORT 50% pellets). A fifth group was given ADX surgery, but CORT 50% pellets were implanted after repeated stress treatment. One week after surgery, each group was divided into two additional groups, naive and stress. Naive animals remained unhandled, while stress rats were given a variety of daily stressors administered twice per day for 6 consecutive days. One day after the last stress, rats were given a saline injection followed by a cocaine injection (15 mg/kg, i.p.) the next day, and locomotor activity was monitored (early withdrawal). Two weeks after the last stress, the locomotor responses to an additional saline and cocaine injection were monitored (late withdrawal). At early withdrawal, no significant sensitization occurred for horizontal activity, but cross-sensitization was demonstrated for vertical activity. At late withdrawal, sham controls showed a stress-induced elevation in horizontal activity, with only a trend toward increased vertical activity. Animals given ADX surgery or ADX and CORT 12.5% pellets did not demonstrate sensitization to repeated stress, while CORT 50% pellets in ADX rats restored the sensitized horizontal response to cocaine challenge at late withdrawal. In contrast, stress-pretreated rats which were given CORT 50% pellets during the 2-week withdrawal period after the stress showed a marked decrease in horizontal activity in response to cocaine challenge at late withdrawal. The results provide evidence for a necessary role for adrenal hormones in long term, but not short-term, stress-induced cross-sensitization. Together with our previous study on the role of CORT in cocaine-induced sensitization, the results indicate that CORT is not the common factor mediating the long-term sensitization to cocaine and stress.