Neonatal Ethanol Exposure Causes Behavioral Deficits in Young Mice.

BACKGROUND: Fetal ethanol (EtOH) exposure can damage the developing central nervous system and lead to cognitive and behavioral deficits, known as fetal alcohol spectrum disorders (FASD). EtOH exposure to mouse pups during early neonatal development was used as a model of EtOH exposure that overlaps the human third-trimester "brain growth spurt"-a model that has been widely used to study FASD in rats. METHODS: C57BL/6 male and female mice were exposed to EtOH (4 g/kg/d) on postnatal days (PD) 4 to 10 by oral intubation. Intubated and nontreated controls were also included. Behavioral testing of the offspring, including open field, elevated plus maze, and Morris water maze, was performed on PD 20 to 45. RESULTS: EtOH exposure during PD 4 to 10 resulted in hyperactivity and deficits in learning and memory in young mice with no apparent sex differences. CONCLUSIONS: Based on these data, this neonatal intubation mouse model may be useful for future mechanistic and genetic studies of FASD and for screening of novel therapeutic agents.

A patient reported outcome measure in homeopathic clinical practice for long-term conditions.

BACKGROUND: This study was initiated as part of a quality improvement audit process to create standards around goal setting with our patients to understand and improve outcomes of homeopathic treatment. METHOD: We used the Measure Yourself Medical Outcome Profile (MYMOP2) as a tool to assist clinicians in setting the treatment goals across a wide range of diagnoses and other complaints in routine clinical practice at the Bristol Homeopathic Hospital. The data collected from the MYMOP2 is of significance in its own right and the results are now reported in this paper. RESULTS: A total of 198 patients with a wide range of complaints attended one to five consultations with 20 homeopathic doctors. Diagnostic categories were most commonly neoplasms (16.7%), psychological (13.9%) and genitourinary complaints (12.3%), with 66.7% suffering from these problems for at least one year. The three symptoms that bothered patients the most were pain, mental symptoms and tiredness/fatigue. A paired-samples t-test using an intention-to-treat analysis showed that the MYMOP2 profile score improved from 4.25 (IQR 3.50-5.00), with a mean change of 1.24 (95% CI 1.04, 1.44) from the first to the last consultation (p<0.001). Results were statistically significant both for completers (n=91) (p<0.001) and non-completers (n=107) (p<0.001) using last-observation-carried-forward, although completers did better than non-completers (p<0.001). The overall clinical significance of improvements was at least moderate. A repeated measures ANOVA test also showed statistically significant improvements (p<0.001). CONCLUSION: The MYMOP2 results add to a growing body of observational data which demonstrates that when patients with long term conditions come under homeopathic care their presenting symptoms and wellbeing often improve. Offering a low cost high impact intervention to extend the range of choice to patients and to support self-care could be an important part of the NHS.

The Dietary Flavonoid Rhamnetin Inhibits Both Inflammation and Excitotoxicity During Ethanol Withdrawal in Rat Organotypic Hippocampal Slice Cultures.

BACKGROUND: Ethanol (EtOH) causes neurotoxicity via several mechanisms including neuroinflammation (during EtOH exposure), and excitotoxicity (during EtOH withdrawal [EWD]). Alpha7 nicotinic acetylcholine receptor (nAChR) selective agonists have the potential to reduce both. The aim of this study was to evaluate the anti-inflammatory and neuroprotective potential of rhamnetin, a dietary flavonoid with alpha7 nAChR selective activity, in an in vitro model of EtOH-induced neurotoxicity. METHODS: The anti-inflammatory and neuroprotective properties of rhamnetin were assessed in neonatal organotypic hippocampal slice cultures undergoing EWD (or not) and challenged with N-methyl-D-aspartate (NMDA) and/or lipopolysaccharide (LPS). Neurotoxicity was determined using propidium iodide uptake, and the inflammatory response was evaluated by measuring the release of tumor necrosis factor (TNF)-alpha (NO; quantified by ELISA) and nitric oxide (quantified by the Griess reaction) into culture media. RESULTS: As predicted, rhamnetin reduced LPS-induced release of TNF-alpha and NO both under control conditions and during EWD. Additionally, rhamnetin had no effect on NMDA-induced neurotoxicity under control conditions, but significantly reduced NMDA toxicity during EWD. In contrast, rhamnetin had no effect on neurotoxicity induced by NMDA and LPS combined despite reducing TNF-alpha and NO levels under these conditions. CONCLUSIONS: Rhamnetin is anti-inflammatory and neuroprotective during EWD and therefore has potential value in treating neurotoxicity caused by EtOH.

Altered relation between lipopolysaccharide-induced inflammatory response and excitotoxicity in rat organotypic hippocampal slice cultures during ethanol withdrawal.

BACKGROUND: Ethanol (EtOH) causes neurotoxicity by several mechanisms including excitotoxicity and neuroinflammation, but little is known about the interaction between these mechanisms. Because neuroinflammation is known to enhance excitotoxicity, we hypothesized that neuroinflammation contributes to the enhanced excitotoxicity, which is associated with EtOH withdrawal (EWD). The aim of this study was to evaluate the lipopolysaccharide (LPS)-induced inflammatory response of cultured hippocampal tissue during EWD and its effects on the enhanced N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, which occurs at this time. METHODS: Using a neonatal organotypic hippocampal slice culture (OHSC) model, we assessed the effects of NMDA and LPS (separately or combined) during EWD after 10 days of EtOH exposure. Neurotoxicity was assessed using propidium iodide uptake, and the inflammatory response was evaluated by measuring the release of tumor necrosis factor (TNF)-alpha (quantified by enzyme-linked immunosorbent assay) and nitric oxide (NO; quantified by the Griess reaction) into culture media. Furthermore, we explored the potential role of the microglial cell type using immortalized BV2 microglia treated with EtOH for 10 days and challenged with LPS during EWD. RESULTS: As predicted, NMDA-induced toxicity was potentiated by LPS under control conditions. However, during EWD, the reverse was observed and LPS inhibited peak NMDA-induced toxicity. Additionally, LPS-induced release of TNF-alpha and NO during EWD was reduced compared to control conditions. In BV2 microglia, following EtOH exposure, LPS-induced release of NO was reduced, whereas TNF-alpha release was potentiated. CONCLUSIONS: During EWD following chronic EtOH exposure, OHSC exhibited a desensitized inflammatory response to LPS and the effects of LPS on NMDA toxicity were reversed. This might be explained by a change in microglia to an anti-inflammatory and neuroprotective phenotype. In support, studies on BV2 microglia indicate that EtOH exposure and EWD do alter the response of these cells to LPS, but this cannot fully explain the changes observed in the OHSC. The data suggest that neuroinflammation and excitotoxicity do interact during EWD. However, the interaction is not as simple as we originally proposed. This in turn illustrates the need to assess the extent, importance, and relation of these mechanisms in models of EtOH exposure producing neurotoxicity.

Polyaminergic agents modulate contextual fear extinction in rats.

Polyamines, such as spermidine and spermine, have been reported to improve memory retention through the activation of N-methyl-d-aspartate receptors (NMDAr). However whether polyamine agonists and antagonists alter extinction remains unclear. In the current study, we investigated whether spermidine and polyamine antagonists that selectively block the NR2B subunit at the NMDAr alter the extinction of contextual conditioned fear in male Wistar rats. The bilateral intra-hippocampal administration of exogenous spermidine (2 nmol/site) immediately after, but not 6h after extinction training, facilitated the extinction of fear conditioning. The injection of the NMDAr antagonists arcaine (0.2 nmol/site), ifenprodil (20 nmol/site) and traxoprodil (0.2 nmol/site), disrupted fear extinction and, at doses that had no effect per se, reversed the facilitatory effect of spermidine on fear extinction. These results suggest that exogenous and endogenous polyamines facilitate the extinction of contextual conditioned fear through activation of NR2B subunit-containing NMDAr in the hippocampus. Since extinction-based exposure therapy is widely used as treatment for a number of anxiety-related disorders, including phobias and post-traumatic stress, the currently reported facilitation of extinction by polyaminergic agents suggest these compounds as putative candidates for drug development.

Topiramate attenuates the stress-induced increase in alcohol consumption and preference in male C57BL/6J mice.

Stress increases the risk for alcohol abuse and relapse behaviors. However, there are hardly any medications to counteract stress-induced alcoholism and relapse behaviors. The present study examined the effects of topiramate (intraperitoneal injections of 10, 20, and 30 mg/kg) in its ability to attenuate alcohol consumption on exposure to restraint stress in C57BL/6J mice on a 2-choice test procedure. Mice were either restrained for 1h/day for 5 successive days or left unrestrained. Subsequently, the effects of topiramate were studied in post-restraint days. Results showed that restrained animals increased alcohol consumption and alcohol preference significantly compared to control group on day 5. On post-restraint days, topiramate reduced alcohol consumption and alcohol preference on days 2-5 compared to saline. This experiment suggests that one mechanism of topiramate in reducing alcohol consumption and alcohol preference may involve an interaction with stress.

Difluoromethylornithine (DFMO) reduces deficits in isolation-induced ultrasonic vocalizations and balance following neonatal ethanol exposure in rats.

Neonatal ethanol (EtOH) exposure is associated with central nervous system dysfunction and neurotoxicity in rats. Increases in polyamine levels have been implicated as one underlying mechanism for some of EtOH's effects on the developing brain. In this study we addressed whether the inhibition of polyamine biosynthesis by alpha-difluoromethylornithine (DFMO) could reduce behavioral deficits induced by early EtOH exposure. Male and female rat pups received ethanol (6 g/kg/day EtOH i.g.), or isocaloric maltose (control) from postnatal days (PND) 1-8. On PND 8, animals were injected with either saline or DFMO (500 mg/kg, s.c.) immediately following the final neonatal treatment. Subjects were tested for isolation-induced ultrasonic vocalizations (USV) on PND 16; spontaneous activity in an open field apparatus on PND 20 and 21; and balance on PND 31. Animals exposed to EtOH neonatally displayed an increased latency to the first USV and reduced frequencies of USV, hyperactivity and preference for the center of the open field and poorer balance relative to controls. DFMO minimized these deficits in latency to the first USV and balance. These data provide further support that polyamines play a role in some of the functional deficits associated with EtOH exposure during early development and that reducing polyamine activity can improve outcome.

Effects of mecamylamine on alcohol consumption and preference in male C57BL/6J mice.

Alcohol and nicotine (in the form of tobacco) are 2 commonly used recreational drugs and studies show a high correlation between tobacco use and alcohol consumption. In the present study, using C57BL/6J mice, we investigated the ability of mecamylamine (a nicotinic antagonist) to reduce alcohol consumption and alcohol preference with free 24-hour access using a 2-bottle choice test drinking procedure. Male C57BL/6J mice were individually housed and acclimatized to 10% alcohol. Immediately following the last day of alcohol acclimatization, the mice (n = 5/group) received subcutaneous injections of mecamylamine (0.5, 1 and 2 mg/kg) or saline consisting of either intermittent (3 injections given every other day) or daily (injections on all 5 days) exposures. Fluid consumption (alcohol and water) was recorded daily. The results showed that mecamylamine significantly reduced alcohol consumption and alcohol preference in both phases of intermittent and daily drug exposures, while the total fluid consumption was unchanged. These results provide further support that mecamylamine is effective in reducing alcohol consumption and preference, and nicotinic-receptor-based drugs could further be explored as potential treatments for alcoholism.

Age and gender differences in response to neonatal ethanol withdrawal and polyamine challenge in organotypic hippocampal cultures.

BACKGROUND: Polyamines are synthesized and released in high concentrations during CNS development. These agents can potentiate N-methyl-D-aspartate receptor (NMDAR) function and appear to play an important role in CNS development. Previous work has shown that polyamine release is increased during ethanol withdrawal (EWD). This likely promotes NMDAR overactivity and contributes to neurotoxicity during EWD, however, little is known regarding such effects in early neonatal brain. The present study compared the effects of EWD and polyamine exposure on toxicity in hippocampal slice cultures derived from postnatal day 2 (PND 2) or postnatal day 8 (PND 8) day-old rats. Due to changes in NMDAR subtypes and response to polyamines, we predicted that slices taken from PND 2 pups would be more sensitive to EWD and polyamine challenge. METHODS: Organotypic hippocampal slice cultures were obtained from neonatal rats either 2 or 8 days of age (PND 2 or PND 8). Five days after explantation, cultures were exposed to ETOH (50 mM- typically subthreshold for EWD induced cell death) for 10 days and then withdrawn from ETOH for 24-hour in the presence of 100 microM of the polyamine spermidine and/or 100 microM ifenprodil, an NMDAR antagonist that blocks the NMDAR that is the most sensitive to polyamine modulation. Cytotoxicity was measured after 24-hour by visualization of propidium iodide (PI) fluorescence. RESULTS: There were clear age and gender-dependent differences in response to EWD and to polyamines. EWD produced significant increases in PI uptake in all subregions (CA1, CA3 and DG) of cultures derived from PND 2 pups, but not PND 8 pups. Exposure of cultures to spermidine for 24-hour also produced significant increases in cytotoxicity in all 3 regions of PND 2 cultures with no gender differences. In contrast, there were both gender and region-specific differences in response to spermidine in cultures from PND 8. While the CA1 region of both sexes displayed increased cytotoxicity following spermidine exposure, only females showed increased cytotoxicity in the CA3 region while the DG appeared relatively insensitive to spermidine. Exposure to spermidine during EWD produced enhanced toxicity in all 3 hippocampal subregions in tissue from both PND 2 and PND 8 rats and this was reduced or prevented by co-exposure to ifenprodil. Of interest, the PND 2 hippocampus was significantly more sensitive than the PND 8 hippocampus to the toxic effects of EWD and to spermidine during EWD in the DG and CA3 regions. CONCLUSIONS: Hippocampal slice cultures derived from PND 2 rats were more sensitive to the toxic effects of both EWD and EWD + spermidine exposure than were those derived from PND 8 rats. These findings are similar to recent behavioral data collected from our lab showing greater sensitivity to ETOH's behavioral teratogenic effects when ETOH exposure in vivo occurred during the first postnatal week relative to the second postnatal week. Ifenprodil's ability to block the toxic effects of spermidine during EWD suggests that excess activity of NR2B subunits of the NMDAR contributed to the excitatory and cytotoxic effects of EWD plus spermidine. While no sex differences in toxicity were observed in cultures taken from pups during the first postnatal week, these data do suggest that later in neonatal life (i.e., the second postnatal week), the female hippocampus may be more sensitive to polyamine-induced neurotoxicity than males.

Alcohol withdrawal-induced hippocampal neurotoxicity in vitro and seizures in vivo are both reduced by memantine.

BACKGROUND: The ethanol withdrawal (EWD) syndrome is typically treated using benzodiazepines such as diazepam. However there is concern that benzodiazepines may not prevent neurotoxicity associated with EWD. Antagonists of glutamate/N-Methyl-D-Aspartate receptors (NMDARs) such as MK801 have been shown to be effective against both EWD-induced neurotoxicity in vitro and seizures in vivo. However, most of these agents have adverse side effects. An exception is the moderate affinity NMDAR channel blocker memantine, used in Alzheimer's dementia. The present studies examined the ability of memantine to protect against EWD-related toxicity in vitro and seizures in vivo. METHODS: Organotypic hippocampal slice cultures from neonatal rat pups were treated starting at 15 days in vitro with 100 mM ethanol for 10 days followed by a 24-hour EWD period. During the 24-hour EWD period cultures were treated with memantine (15 or 30 microM). MK801 (10 microM) was utilized as a positive control. For the in vivo studies, the ability of memantine (2, 5, 10, and 15 mg/kg) to reduce convulsions was analyzed in Swiss-Webster mice using the handling induced convulsion test paradigm. RESULTS: In vitro studies demonstrated that memantine is effective at blocking EWD-induced neurotoxicity. In vivo experiments showed that memantine also significantly reduced convulsions induced by EWD in mice. CONCLUSIONS: Memantine may be of therapeutic value during alcohol detoxification by virtue of its having neuroprotective effects in addition to anti-seizure activity. The potential role of memantine in treatment of alcoholism is deserving of further study.

Acamprosate attenuates the handling induced convulsions during alcohol withdrawal in Swiss Webster mice.

In the present study, we examined the effects of acamprosate for its ability to reduce handling induced convulsions (HICs) during alcohol withdrawal. Diazepam was used as a positive control. Swiss Webster male mice received three daily IP injections of alcohol (2.5 g/kg) or alcohol (2.5 g/kg)+methylpyrazole (4-MP) (9 mg/kg). (4-MP, being an alcohol dehydrogenase inhibitor slows down the breakdown of alcohol. 4-MP in combination with alcohol exhibits a dramatic increase in blood alcohol level compared to alcohol alone). Ten hours following the last alcohol injection, the mice were picked up by the tail and examined for their seizure susceptibility (HICs). Diazepam, a benzodiazepine known to reduce seizures during alcohol withdrawal, significantly reduced these HICs at doses of 0.25, 0.5 and 1 mg/kg (p's<0.001). Acamprosate, an anti-relapse compound used clinically in newly abstinent alcoholics, also reduced these HICs at doses of 100, 200 and 300 mg/kg (p's<0.05). This study supports the use of acamprosate during periods of alcohol withdrawal as well as during abstinence.

Towards standard setting for patient-reported outcomes in the NHS homeopathic hospitals.

INTRODUCTION: We report findings from a pilot data collection study within a programme of quality assurance, improvement and development across all five homeopathic hospitals in the UK National Health Service (NHS). AIMS: (1) To pilot the collection of clinical data in the homeopathic hospital outpatient setting, recording patient-reported outcome since first appointment; (2) to sample the range of medical complaints that secondary-care doctors treat using homeopathy, and thus identify the nature and complexity of complaints most frequently treated nationally; (3) to present a cross section of outcome scores by appointment number, including that for the most frequently treated medical complaints; (4) to explore approaches to standard setting for homeopathic practice outcome in patients treated at the homeopathic hospitals. METHODS: A total of 51 medical practitioners took part in data collection over a 4-week period. Consecutive patient appointments were recorded under the headings: (1) date of first appointment in the current series; (2) appointment number; (3) age of patient; (4) sex of patient; (5) main medical complaint being treated; (6) whether other main medical complaint(s); (7) patient-reported change in health, using Outcome Related to Impact on Daily Living (ORIDL) and its derivative, the ORIDL Profile Score (ORIDL-PS; range, -4 to +4, where a score or=+2 indicates an effect on the quality of a patient's daily life); (8) receipt of other complementary medicine for their main medical complaint. RESULTS: The distribution of patient age was bimodal: main peak, 49 years; secondary peak, 6 years. Male:female ratio was 1:3.5. Data were recorded on a total of 1797 individual patients: 195 first appointments, 1602 follow-ups (FUs). Size of clinical service and proportion of patients who attended more than six visits varied between hospitals. A total of 235 different medical complaints were reported. The 30 most commonly treated complaints were (in decreasing order of frequency): eczema; chronic fatigue syndrome (CFS); menopausal disorder; osteoarthritis; depression; breast cancer; rheumatoid arthritis; asthma; anxiety; irritable bowel syndrome; multiple sclerosis; psoriasis; allergy (unspecified); fibromyalgia; migraine; premenstrual syndrome; chronic rhinitis; headache; vitiligo; seasonal allergic rhinitis; chronic intractable pain; insomnia; ulcerative colitis; acne; psoriatic arthropathy; urticaria; ovarian cancer; attention-deficit hyperactivity disorder (ADHD); epilepsy; sinusitis. The proportion of patients with important co-morbidity was higher in those seen after visit 6 (56.9%) compared with those seen up to and including that point (40.7%; P<0.001). The proportion of FU patients reporting ORIDL-PS>or=+2 (improvement affecting daily living) increased overall with appointment number: 34.5% of patients at visit 2 and 59.3% of patients at visit 6, for example. Amongst the four most frequently treated complaints, the proportion of patients that reported ORIDL-PS>or=+2 at visit numbers greater than 6 varied between 59.3% (CFS) and 73.3% (menopausal disorder). CONCLUSIONS: We have successfully piloted a process of national clinical data collection using patient-reported outcome in homeopathic hospital outpatients, identifying a wide range and complexity of medical complaints treated in that setting. After a series of homeopathy appointments, a high proportion of patients, often representing "effectiveness gaps" for conventional medical treatment, reported improvement in health affecting their daily living. These pilot findings are informing our developing programme of standard setting for homeopathic care in the hospital outpatient context.

The Effects of Perinatal Oxycodone Exposure on Behavioral Outcome in a Rodent Model.

Opiate addiction is now a major public health problem. Perinatal insults and exposure to opiates such as morphine in utero are well known to affect development of the hypothalamic-pituitary-adrenal axis of the offspring adversely and are associated with a higher risk of developing neurobehavioral problems. Oxycodone is now one of the most frequently abused pain killers during pregnancy; however, limited data are available regarding whether and how perinatal oxycodone exposure (POE) alters neurobehavioral outcomes of the offspring. We demonstrated that exposure to 0.5 mg/kg/day oxycodone in utero was associated with hyperactivity in adult rats in an open field. No significant effects of POE were detected on isolation-induced ultrasonic vocalizations in the early postnatal period or on learning and memory in the water maze in adult offspring. Our findings are consistent with hyperactivity problems identified in children exposed to opiates in utero.

Neonatal ethanol and nicotine exposure causes locomotor activity changes in preweanling animals.

Sprague-Dawley rats were used to investigate the effects of neonatal ethanol (ETOH) and nicotine (NIC) exposure on activity levels in preweanling offspring. Male and female pups received daily oral intubations of ethanol ((ETOH) 5 g/kg/day), nicotine ((NIC) 12 mg/kg/day), ethanol and nicotine ((ETOH+NIC) 5 g/kg/day+12 mg/kg/day) or isocaloric maltose (control) on either postnatal days (PND) 1-7 or PND 8-14. A non-treated control group was also included. Peak blood ethanol concentrations (BECs) measured in a separate subset of animals ranged from 167 and 344 mg/dl depending upon neonatal treatment and period of exposure. Subjects were tested in an open field apparatus on PND 19-21. Animals exposed to ETOH or ETOH+NIC on PND 1-7 were hyperactive relative to the other treatment groups. In contrast, animals exposed to NIC or ETOH+NIC during PND 8-14 were hypoactive relative to other treatment groups. Males appeared more sensitive than females on measures of anxiety (distance traveled in the center of the open field) but this also varied dependent on neonatal treatment and period of exposure. These findings suggest that the third trimester is a critical period for ETOH and NIC effects on offspring activity although the pattern of effects on activity are different depending on when drug exposure occurred during the neonatal period.

Effects of neonatal alcohol and/or cocaine exposure on stress in juvenile and adult female rats.

The effects of neonatal exposure to alcohol and/or cocaine on two measures of stress were studied in juvenile and young adult female rats. After implantation with an intragastric cannula, subjects were artificially reared from postnatal days 4-10. This "brain growth spurt" period is roughly equivalent to CNS development during the third trimester of human pregnancy. There were five treatment groups: alcohol (6 g/kg/day), cocaine (60 mg/kg/day), alcohol/cocaine (6 g/kg/day alcohol and 60 mg/kg/day cocaine), stock (an artificially reared control), and sham (a suckled control). Subjects were tested in open field and forced swim tests beginning at 21 or 60-70 days of age, respectively. Compared to controls, alcohol-exposed females displayed longer latencies to become immobile in the forced swim test as juveniles and cocaine-exposed females showed increased immobility as adults. Increased immobility can be interpreted as hyporesponsiveness to stress. In contrast, very few differences were observed in the open field. Furthermore, the group exposed to alcohol and cocaine in combination did not differ from controls in either paradigm. These findings suggest that the forced swim test may be more sensitive to neonatal drug effects than open field, although these effects may not be consistent across age.

Neonatal ethanol exposure produces a hyperalgesia that extends into adolescence, and is associated with increased analgesic and rewarding properties of nicotine in rats.

RATIONALE: Drug exposure during CNS development may alter subsequent dependence liability. We postulated that early alcohol exposure might produce persistent alterations in responses to noxious stimuli. Because relief of physical discomfort may be negatively reinforcing, changes in responses to noxious stimuli produced by early alcohol exposure may increase the rewarding properties of nicotine, a potent analgesic. Such factors may contribute to the high level of alcohol and nicotine co-abuse in humans. OBJECTIVES: The purpose of this study was to determine whether neonatal ethanol exposure in rats altered responses to noxious stimuli, and whether nicotine would then be more rewarding to the alcohol-exposed offspring, perhaps via its analgesic actions. METHODS: Neonatal rats received ethanol by gavage (5.0 or 6.5 g/kg) on postnatal days (PND) 9-13. An iso-caloric control group was also included. Rats were then tested to assess responsiveness to a mild noxious heat stimulus, as measured in the tail-flick assay (PND 14 and PND 28), for their response to acute analgesic injections of either nicotine or ethanol (PND 28), and for nicotine induced conditioned place preference (CPP) (PND 36). RESULTS: Neonatal ethanol exposure produced hyperalgesia during the first 24 h after alcohol withdrawal (PND 14) that continued through PND 28. The analgesic effects of 12.5 microg/kg nicotine were enhanced approximately 2-fold in adolescent rats with previous ethanol histories, relative to controls. These ethanol-exposed rats also showed a significant CPP to nicotine, whereas controls showed no CPP. CONCLUSIONS: Persistent decreases in tail-flick response latencies suggestive of hyperalgesia were observed following neonatal ethanol exposure in the rat. These changes were accompanied by increases in the analgesic and place-conditioning effects of nicotine in adolescence. If similar effects occur in humans, prenatal alcohol exposure may play a role in an increased risk for the rewarding effects and dependence liability of nicotine later in life.

Polyamine modulation of NMDARs as a mechanism to reduce effects of alcohol dependence.

Relapse and neurodegeneration are two of the major therapeutic targets in alcoholism. Fortuitously, the roles of glutamate/NMDA receptors (NMDARs) in withdrawal, conditioning and neurotoxicity mean that NMDAR inhibitors are potentially valuable for both targets. Preclinical studies further suggest that inhibitory modulators that specifically reduce the co-agonist effects of polyamines on NMDARs are potential non-toxic medications. Using agmatine as a lead compound, over 1000 novel compounds based loosely on this structure were synthesized using feedback from a molecular screen. A novel series of aryliminoguanidines with appropriate NMDAR activity in the molecular screen were discovered (US patent application filed 2007). The most potent and selective aryliminoguanidine, JR 220 [4- (chlorobenzylidenamino)- guanidine hydrochloride], has now been tested in a screening hierarchy for anti-relapse and neuroprotective activity, ranging from cell-based assay, through tissue culture to animal behavior. This hierarchy has been validated using drugs with known, or potential, clinical value at these targets (acamprosate (N-acetyl homotaurine), memantine and topiramate). JR220 was non-toxic and showed excellent activity in every screen with a potency 5-200x that of the FDA-approved anti-relapse agent, acamprosate. This chapter will present a review of the background and rationale for this approach and some of the findings garnered from this approach as well as patents targeting the glutamatergic system especially the NMDAR.

Traxoprodil decreases pentylenetetrazol-induced seizures.

Polyamines, including spermidine, facilitate seizures by positively modulating N-methyl-d-aspartate receptors (NMDAr). Although NMDAr antagonists decrease seizures, it remains to be determined whether traxoprodil, a selective antagonist at the NR2B subunit of the NMDAr, decreases seizures and whether spermidine facilitates pentylenetetrazol (PTZ)-induced seizures. Adult male Wistar rats were injected in the lateral ventricle with 0.9% NaCl (1µl, i.c.v.), spermidine (0.02, 0.2 or 2nmol/site, i.c.v.) or traxoprodil (0.2, 2 or 20nmol, i.c.v.) and with PTZ (35 or 70mg/kg, i.p.). The effect of orally administered traxoprodil (60mg/kg, p.o.) on seizures was also investigated. Latencies to clonic and generalized seizures, as well the total time spent in seizures were recorded by behavioral and electrographic methods (EEG). Spermidine (2nmol/site; i.c.v.) facilitated the seizures induced by a sub-threshold dose of PTZ (35mg/kg; i.p.), but did not alter seizure activity induced by a convulsant dose of PTZ (70mg/kg; i.p.). Traxoprodil (20nmol i.c.v.) increased the latency to generalized tonic-clonic seizures induced by PTZ (70mg/kg; i.p.). Traxoprodil (60mg/kg, p.o.) increased the latency to clonic and generalized seizures, and decreased the total time spent in seizures. These results support the role for the NR2B subunit in PTZ-induced seizures.

Agmatine reduces ultrasonic vocalization deficits in female rat pups exposed neonatally to ethanol.

Rat pups, in isolation, produce ultrasonic vocalizations (USVs). These USVs have been used as a diagnostic tool for developmental toxicity. We have shown that neonatal ethanol (ETOH) exposure produces deficits in this behavior. The current study was designed to examine whether agmatine (AG), which binds to imidazoline receptors and modulates n-methyl-d-aspartate receptors (NMDAR), could reduce these deficits. In addition, this study examined critical periods for ETOH's effects on USVs by administering ETOH during either the 1st or 2nd postnatal week. Neonatal rats received intragastric intubations of either ETOH (6g/kg/day), ETOH and AG (6g/kg/day and 20mg/kg/day), AG (20mg/kg/day), or maltose on postnatal days (PND) 1-7 or 8-14. A non-intubated control was also included. Subjects were tested on PND 15. Neonatal ETOH exposure significantly increased the latency to vocalize for females and reduced the rate of USVs in both males and females exposed to ETOH on PND 1-7. Agmatine reduced these deficits, in female but not male pups. Subjects exposed to ETOH on PND 8-14 showed no evidence of abnormal USVs. These findings suggest that there may be gender differences in response to AG following neonatal ETOH exposure and also provide further support that the first neonatal week is a particularly sensitive time for the developmentally toxic effects of ETOH in rodents.

Lobeline, a nicotinic partial agonist attenuates alcohol consumption and preference in male C57BL/6J mice.

Lobeline is a partial nicotinic agonist and is currently being investigated as a therapeutic drug for several addictive disorders particularly for smoking cessation. The present study evaluated the effects of repeated (continuous and recurring) administration of lobeline on alcohol consumption (10% alcohol vs. water) and alcohol preference using a 2-bottle choice test procedure. Male C57BL/6J mice were individually housed and acclimatized to 10% alcohol. Immediately following the last day of alcohol acclimatization and attainment of consistent drinking pattern, mice (n=5/group) received subcutaneous injections of lobeline (3, 5, or 10 mg/kg) or saline. Groups received either repeated-recurring (3 injections, given every other day) or repeated-continuous (daily injections for 5 days) subcutaneous injections of lobeline. Fluid consumption (alcohol and water) was recorded daily. Results showed that lobeline significantly reduced alcohol consumption and alcohol preference during the repeated (recurring and continuous) administration phases, while total fluid consumption remained unchanged. These results provide support that nicotinic receptor based drugs may be useful as potential treatments for alcoholism.