Mariusz Sacharczuk

Canine Olfactory Receptor Gene Polymorphism and Its Relation to Odor Detection Performance by Sniffer Dogs

The outstanding sensitivity of the canine olfactory system has been acknowledged by using sniffer dogs in military and civilian service for detection of a variety of odors. It is hypothesized that the canine olfactory ability is determined by polymorphisms in olfactory receptor (OR) genes. We investigated 5 OR genes for polymorphic sites which might affect the olfactory ability of service dogs in different fields of specific substance detection. All investigated OR DNA sequences proved to have allelic variants, the majority of which lead to protein sequence alteration. Homozygous individuals at 2 gene loci significantly differed in their detection skills from other genotypes. This suggests a role of specific alleles in odor detection and a linkage between single-nucleotide polymorphism and odor recognition efficiency.

Differences in ethanol drinking between mice selected for high and low swim stress-induced analgesia

Alcoholism is a complex disorder, still not fully understood, in which environmental and inherited risk factors play essential roles. Of particular importance may be chronic exposure to stress thought to increase preference for ethanol in genetically susceptible individuals. Animal and human data suggest that the opioid system may be involved in the development of alcohol dependence. We studied the effects of chronic mild stress (CMS) on the voluntary intake of 8% ethanol in the mouse lines displaying high (HA) or low (LA) swim stress-induced analgesia. These lines differ in the activity of the endogenous opioid system. Normally, 8% ethanol is aversive to rodents. We found that LA mice with the low opioid system activity exposed to CMS manifested greater ethanol intake than under no stress conditions. No such effect of CMS on ethanol consumption was observed in HA mice that display the enhanced opioid system activity. We conclude that CMS imposed on individuals with a genetically determined low opioid activity may favor the development of ethanol abuse.

A polymorphism in exon 2 of the δ-opioid receptor affects nociception in response to specific agonists and antagonists in mice selectively bred for high and low analgesia

&NA; This study searched for polymorphic sites in the murine &mgr;‐, &dgr;‐ and &kgr;‐opioid receptors that presumably influence pain perception. We employed two mouse lines divergently bred for high (HA, high analgesia line) and low (LA, low analgesia line) swim stress‐induced analgesia (SSIA). These mouse lines differ substantially in pain sensitivity, measured as hind paw withdrawal latency in the hot‐plate test. We found a novel C320T transition in exon 2 of the &dgr;‐opioid receptor gene, resulting in an A107V substitution in the first extracellular loop (EL1) of the peptide chain. Using hot‐plate and tail‐flick tests, we found a significant association between the genotype of this locus and basal nociception and SSIA magnitude. Moreover, this transition affects the pharmacological effects of two specific &dgr;‐opioid receptor ligands, the agonist SNC80 and the antagonist naltrindole. The impact of the C320T polymorphism on the magnitude of SSIA was partially mediated by endogenous opioids. The effectiveness of exogenous &dgr;‐opioid receptor ligands was greater in the HA than in the LA line, and was greater in C320C homozygotes than in C320T heterozygotes within each line. Our results indicate that the C320T polymorphism in the &dgr;‐opioid receptor gene is at least partly responsible for the divergent nociceptive thresholds in HA and LA mice under both basal and post‐stress conditions.

Association between the A107V substitution in the δ‐opioid receptors and ethanol drinking in mice selected for high and low analgesia

Experimental evidence suggests that endogenous opioids play an important role in the development of ethanol addiction. In this study, we employed two mouse lines divergently bred for opioid‐mediated stress‐induced analgesia. In comparison with HA (high analgesia line) mice, LA (low analgesia line) mice, having lower opioid receptor system activity, manifest enhanced basal as well as stress‐induced ethanol drinking. Here, we found that recently discovered C320T transition in exon 2 of the δ‐opioid receptor gene (EU446125.1), which results in an A107V substitution (ACA23171.1), leads to higher ethanol preference in CT mice compared with CC homozygotes. This genetic association is particularly evident under chronic mild stress (CMS) conditions. The interaction between stress and ethanol intake was significantly stronger in HA than in LA mice. Ethanol almost completely attenuated the pro‐depressive effect of CMS (assessed with the tail suspension test) in both the CC and CT genotypes in the HA line. In the LA mice, a lack of response to ethanol was observed in the CC genotype, whereas ethanol consumption strengthened depressive‐like behaviours in CT individuals. Our results suggest that constitutively active A107V substitution in δ‐opioid receptors may be involved in stress‐enhanced vulnerability to ethanol abuse and in the risk of ethanol dependence.

Chromosomal NOR Activity in Mice Selected for High and Low Swim Stress-Induced Analgesia

Metaphase nucleolar organizer region (NOR) activity was studied in mouse lines selectively bred for high analgesia (HA) and for low analgesia (LA) induced by 3-min swimming in 20°C water. Apart from pain-related traits, HA mice also manifest, as compared to the LA line, higher emotionality in certain behavioral tests and are less capable of coping with the hypothermic challenge of swimming in cold water, in addition to being more susceptible to the mutagenic effect of whole-body γ-radiation and mitomycin C injection. We here compared NOR activity in HA and LA mice. A statistically significant difference (p ≤ .01) was detected in mean silver-stained nucleolar number/cell between the HA and LA lines, being lower for HA mice. We propose that the breeding strategy, along with the differentiation of stress-related phenomena, has altered the activity of genes coding rRNA and that this activity is important in controlling DNA repair in each line. It is concluded that quantitative studies of nucleolar changes may be useful in evaluating the biological reactivity to stress stimuli.

Opposite effects of alcohol in regulating stress-induced changes in body weight between the two mouse lines with enhanced or low opioid system activity.

Considering the involvement of the opioid system in alcoholism, depression and metabolism - known risk factors in human obesity, we studied the effects of chronic mild stress (CMS) and alcohol intake on body weight in two mouse lines selected for high (HA-high analgesia) or low (LA-low analgesia) swim stress-induced analgesia. In comparison to LA mice, HA mice exhibit an upregulation of opioid receptor system function, different depression-like behavior and reduced energy expenditure in stress. LA animals showed enhanced basal and CMS-induced alcohol drinking versus HA. Now we report different effects of alcohol under no stress (control) and CMS conditions on food intake and body weight between the lines. CMS in animals with no access to alcohol increased body weight in both HA and LA mice, with no effect of CMS on food intake in either line and without differences between the lines. In LA mice alcohol reduced body weight under both conditions although significantly more under the control than CMS conditions. In contrast, in HA mice alcohol increased body weight more under the CMS than under control conditions. The results suggest that opioid system may modulate effects of alcohol on stress -induced changes in body weight.

Biphalin protects against cognitive deficits in a mouse model of mild traumatic brain injury (mTBI)

Traumatic brain injury (TBI) is often a result of traffic accidents, contact sports or battlefield explosions. A mild form of traumatic brain injury (mTBI) is frequently underestimated, as the immediate physical symptoms decrease rapidly and conventional neuroimaging studies often do not show visible evidence of brain lesions. However, cognitive impairments persist for weeks, months or even years after the incident. Endogenous opioids were documented to play a role in thmodulation of mTBI pathology, whereas exogenous opioids were shown to possess neuroprotective properties. In the present study, biphalin, a dimeric enkephalin analog, improved cognitive performance in the Morris Water Maze and Novel Object Recognition tests in a mouse weight-drop model of mTBI. The effect of a single systemic injection of 10 mg/kg biphalin immediately after trauma was reversed by naltrexone, suggesting an opioid receptor-mediated mechanism. Biphalin also reduced cortical and hippocampal neurodegeneration, as shown by silver staining. Our data indicates that opioid receptor activation by biphalin may provide neuroprotection of post-traumatic neurodegeneration processes and may protect against memory impairments.

Chronic mild stress facilitates melanoma tumor growth in mouse lines selected for high and low stress-induced analgesia.

Abstract Both chronic stress conditions and hyperergic reaction to environmental stress are known to enhance cancer susceptibility. We described two mouse lines that displayed high (HA) and low (LA) swim stress-induced analgesia (SSIA) to investigate the relationship between inherited differences in sensitivity to stress and proneness to an increased growth rate of subcutaneously inoculated melanoma. These lines display several genetic and physiological differences, among which distinct sensitivity to mutagens and susceptibility to cancer are especially noticeable. High analgesic mice display high proneness both to stress and a rapid local spread of B16F0 melanoma. However, stress-resistant LA mice do not develop melanoma tumors after inoculation, or if so, tumors regress spontaneously. We found that the chronic mild stress (CMS) procedure leads to enhanced interlinear differences in melanoma susceptibility. Tumors developed faster in stress conditions in both lines. However, LA mice still displayed a tendency for spontaneous regression, and 50% of LA mice did not develop a tumor, even under stressed conditions. Moreover, we showed that chronic stress, but not tumor progression, induces depressive behavior, which may be an important clue in cancer therapy. Our results clearly indicate how the interaction between genetic susceptibility to stress and environmental stress determine the risk and progression of melanoma. To our knowledge, HA/LA mouse lines are the first animal models of distinct melanoma progression mediated by inherited differences in stress reactivity.

Naloxone exacerbates memory impairments and depressive-like behavior after mild traumatic brain injury (mTBI) in mice with upregulated opioid system activity

HighlightsSeverity of post‐mTBI deficits correlates with endogenous opioid system activity.Increased opioid tone protects against mTBI‐induced memory deficits.Naloxone impairs memory and exacerbates depressive‐like behavior only in HA mice.mTBI strongly induces depressive‐like behavior in LA mice Abstract The neuroprotective role of the endogenous opioid system in the pathophysiological sequelae of brain injury remains largely ambiguous. Noteworthy, almost no data is available on how its genetically determined activity influences the outcome of mild traumatic brain injury. Thus, the aim of our study was to examine the effect of opioid receptor blockage on cognitive impairments produced by mild traumatic brain injury in mice selectively bred for high (HA) and low (LA) swim‐stress induced analgesia that show innate divergence in opioid system activity. Mild traumatic brain injury was induced with a weight‐drop device on anaesthetized mice. Naloxone (5 mg/kg) was intraperitoneally delivered twice a day for 7 days to non‐selectively block opioid receptors. Spatial memory performance and manifestations of depressive‐like behavior were assessed using the Morris Water Maze and tail suspension tests, respectively. Mild traumatic brain injury resulted in a significant deterioration of spatial memory performance and severity of depressive‐like behavior in the LA mouse line as opposed to HA mice. Opioid receptor blockage with naloxone unmasked cognitive deficits in HA mice but was without effect in the LA line. The results suggest a protective role of genetically predetermined enhanced opioid system activity in suppression of mild brain trauma‐induced cognitive impairments. Mice selected for high and low swim stress‐induced analgesia might therefore be a useful model to study the involvement of the opioid system in the pathophysiology and neurological outcome of traumatic brain injury.

Biphalin preferentially recruits peripheral opioid receptors to facilitate analgesia in a mouse model of cancer pain - A comparison with morphine

The search for new drugs for cancer pain management has been a long-standing goal in basic and clinical research. Classical opioid drugs exert their primary antinociceptive effect upon activating opioid receptors located in the central nervous system. A substantial body of evidence points to the relevance of peripheral opioid receptors as potential targets for cancer pain treatment. Peptides showing limited blood-brain-barrier permeability promote peripheral analgesia in many pain models. In the present study we examined the peripheral and central analgesic effect of intravenously administered biphalin - a dimeric opioid peptide in a mouse skin cancer pain model, developed by an intraplantar inoculation of B16F0 melanoma cells. The effect of biphalin was compared with morphine - a golden standard in cancer pain management. Biphalin produced profound, dose-dependent and naloxone sensitive spinal analgesia. Additionally, the effect in the tumor-bearing paw was largely mediated by peripheral opioid receptors, as it was readily attenuated by the blood-brain-barrier-restricted opioid receptor antagonist - naloxone methiodide. On the contrary, morphine facilitated its analgesic effect primarily by activating spinal opioid receptors. Both drugs induced tolerance in B16F0 - implanted paws after chronic treatment, however biphalin as opposed to morphine, showed little decrease in its activity at the spinal level. Our results indicate that biphalin may be considered a future alternative drug in cancer pain treatment due to an enhanced local analgesic activity as well as lower tolerance liability compared with morphine.