Comparison of nicotine oral consumption and baseline anxiety measures in adolescent and adult C57BL/6J and C3H/Ibg mice

Approximately 80% of smokers initiate tobacco use during adolescence, suggesting that nicotine initiation and nicotine dependence have a substantial age component. There also is a substantial genetic influence on smoking behaviors such as age of initiation and the development of nicotine dependence. The goal of this study was to examine both genetic background and age dependent effects on oral nicotine self-administration and anxiety-like behaviors in mice. Two inbred mouse strains (C3H/Ibg and C57BL/6J) were assessed for oral nicotine preference during early adolescence (postnatal day 24-35), middle adolescence (postnatal day 36-47), late adolescence (postnatal day 48-59), adulthood (postnatal day 60+) and 2 months following their initial exposure to nicotine. Mice also were assessed for innate anxiety using an elevated zero maze to determine if age and/or genetic background influenced anxiety-like behaviors. Results indicated that initial nicotine preference and nicotine preference two months after an initial exposure are both strain and age dependent. Age also had an effect on some baseline anxiety measures but strain differences for most zero maze measures were present throughout all age groups. In general, early adolescent C3H mice exhibited greater nicotine preference while C57 mice displayed greater preference during middle adolescence and upon a second exposure to nicotine. In contrast, C57 mice exhibited reduced anxiety across all ages tested. These studies indicate that genetic background should be considered when evaluating age-dependent effects of drugs of abuse and baseline anxiety-like behaviors.     

Anxiety-like behavior during nicotine withdrawal predict subsequent nicotine consumption in adolescent C57BL/6 mice

We have previously demonstrated that anxiety-like behavior assessed in the elevated plus maze does not predict subsequent nicotine consumption in naïve adolescent mice. However, an association between anxiety and relapse to drug use has been suggested. In the present study, we investigated whether anxiety levels during nicotine withdrawal predict subsequent nicotine consumption in adolescent mice. C57BL/6 mice were either exposed to (−)-nicotine-free base (nicotine, 50 μg/ml) or tap water (water) from postnatal day 30 to 45 (PN30–PN45, priming period). By the end of PN48, all animals were submitted to the elevated plus maze and classified as either having high (HiAnx) or low (LoAnx) levels of anxiety. Immediately after finishing the test, all animals were returned to their home cages and were given a free choice (from PN49 to PN55, free-choice period) between two bottles, one containing a nicotine solution (10 μg/ml) and the other tap water. Nicotine consumption during the free-choice period was affected by the priming treatment (nicotine or water) in a way that was dependent on the anxiety level (HiAnx or LoAnx): the nicotine HiAnx group had lower nicotine consumption than the other groups. No differences were observed between the nicotine LoAnx, water HiAnx and water LoAnx groups. The present study provides experimental evidence for the role of anxiety on the regulation of drug consumption. Specifically, our results suggest that the anxiety-like behavior during nicotine withdrawal is associated with subsequent nicotine self-administration.     

Length of social deprivation differently affects free-choice morphine consumption in C57BL/6J mice.

The effects of social deprivation on morphine consumption in C57BL/6J mice were investigated. Social or isolated animals (length of isolation: 7, 12, 17 days) were submitted to a free-choice between water and morphine hydrochloride solution (0.5 mg/ml). Isolation affects morphine consumption, with solitary housed mice drinking more morphine solution than socially housed animals. Moreover the amount of morphine intake depends on the duration of the isolation period to which mice are subjected. The importance of environmental factors on opiate consumption may be considered in terms of the possible implications of drug addiction in humans.     

Topiramate reduces ethanol consumption by C57BL/6 mice

BACKGROUND: Previous reports indicate that topiramate (TPM) might be an effective treatment for alcohol dependence, perhaps due to a decrease alcohol's rewarding effects resulting from inhibition mesocorticolimbic dopamine (DA) release. Additional reports indicate that TPM antagonizes chronic changes induced by alcohol at the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate receptors. In the present study, a C57BL/6 (B6) murine model (n = 40) was used to evaluate the effect of TPM on the consumption of 12% alcohol over a 21-h period. METHODS: TPM (0, 10, 30, 90 mg/kg) injected subcutaneously into B6 mice 60 min prior to access to a 12% ethanol solution (v/v) over 8 days produced dose-responsive reduction in consumption during the first 2-h period after injection. RESULTS: Across the 8 days of treatment ethanol intake (g/kg) for SAL, T10, T30, and T90, respectively, was 1.34, 1.03, 0.72, and 0.67. This reduction appears to require systemically available TPM since it was not statistically supported when assessed over the entire 21-h period of ethanol availability. None of the TPM doses affected food consumption or body weight, and T90 dose did not reduce motor activity either by itself or in combination with ethanol. CONCLUSIONS: Unlike previous experiments using the same B6 mouse model to assess naltrexone or tiagabine, there was no evidence that mice developed tolerance to the TPM-induced reductions in ethanol consumption. Thus, in the B6 mouse, TPM reduced ethanol intake at doses with no readily apparent adverse side effects, an effect consistent with recent clinical reports. Additional study will be directed toward characterizing TPM as a treatment for alcohol dependence.     

A comparison of the behavior of C57BL/6 and C57BL/10 mice

Selection of an appropriate animal model is a crucial first step in many research programs. The C57BL/6 (B6) mouse is the most widely used inbred mouse strain in biomedical research; this is particularly so in behavioral studies. However, there are several C57BL substrains, all derived from common ancestors. C57BL/10 (B10) mice are superficially almost identical to B6 mice in appearance and behavior and widely used in inflammation and immunology research, yet rarely in behavioral studies. The present study assessed the comparability of behavioral results from these two strains, to determine whether they could be used interchangeably in future behavioral experiments. The results showed that the behavior of B6 mice clearly differed from that of B10 mice: in tests of cognition, species-typical behaviors, and motor coordination the B6 strain performed better. Consequently, B6 mice will probably remain the preferred choice for behavioral studies. Interpretation of results derived from the B10 strain should take into account its particular behavioral characteristics.     

Comparative hemostatic parameters in BALB/c, C57BL/6 and C3H/He mice

This study describes micro-methods to determine biological parameters in plasma of three strains of mice. Platelet count was significantly different among strains. C57BL/6 mice showed the highest values (988 × 10³/μL) and BALB/c the lowest (782 × 10³/μL). Fibrinogen levels were 2.55 (C57BL/6), 2.37 (BALB/c) and 2.28 g/L (C3H/He). Some inter-strain differences were observed in factor XIII (94, 118 and 114%) and plasminogen levels (142, 80 and 135%) in C57BL/6, BALB/c and C3H/He, respectively. Additionally, we observed individual mice factor XIII and plasminogen levels between 80 to 200% and 65 to 180%, respectively, in relation to pooled human plasma; and between 70 to 185% and 70 to 155%, respectively, against pooled mice plasma. To our knowledge, this is first report in the literature in diverse mice strains regarding hemostasis, mainly on factor XIII, plasminogen levels, and a very simple test that allows measurement of endogenous fibrinolytic activity present in the plasma. The different results are discussed in relationship with existing literature regarding if the animals in some studies were maintained under strict pathogen-free conditions, the collection of blood was from the heart or eye and if the analysis method was tested by counting manually or automatically. This work could contribute useful knowledge to the field of investigations regarding hemostatic disorders using mouse models, especially for laboratories that are not well equipped.     

Toll-like receptor 3 dynamics in female C57BL/6J mice: Regulation of alcohol intake

Although there are sex differences in the effects of alcohol on immune responses, it is unclear if sex differences in immune response can influence drinking behavior. Activation of toll-like receptor 3 (TLR3) by polyinosinic:polycytidylic acid (poly(I:C)) produced a rapid proinflammatory response in males that increased alcohol intake over time (Warden et al., 2019). Poly(I:C) produced a delayed and prolonged innate immune response in females. We hypothesized that the timecourse of innate immune activation could regulate drinking behavior in females. Therefore, we chose to test the effect of two time points in the innate immune activation timecourse on every-other-day two-bottle-choice drinking: (1) peak activation; (2) descending limb of activation. Poly(I:C) reduced ethanol consumption when alcohol access occurred during peak activation. Poly(I:C) did not change ethanol consumption when alcohol access occurred on the descending limb of activation. Decreased levels of MyD88-dependent pathway correlated with decreased alcohol intake and increased levels of TRIF-dependent pathway correlated with increased alcohol intake in females. To validate the effects of poly(I:C) were mediated through MyD88, we tested female mice lacking Myd88. Poly(I:C) did not change alcohol intake in Myd88 knockouts, indicating that poly(I:C)-induced changes in alcohol intake are dependent on MyD88 in females. We next determined if the innate immune timecourse also regulated drinking behavior in males. Poly(I:C) reduced ethanol consumption in males when alcohol was presented at peak activation. Therefore, the timecourse of innate immune activation regulates drinking behavior and sex-specific dynamics of innate immune response must be considered when designing therapeutics to treat excessive drinking.     

Brain plasticity and cognitive functions after ethanol consumption in C57BL/6J mice

Acute or chronic administrations of high doses of ethanol in mice are known to produce severe cognitive deficits linked to hippocampal damage. However, we recently reported that chronic and moderate ethanol intake in C57BL/6J mice induced chromatin remodeling within the Bdnf promoters, leading to both enhanced brain-derived neurotrophic factor (BDNF) expression and hippocampal neurogenesis under free-choice protocol. We performed here a series of cellular and behavioral studies to analyze the consequences of these modifications. We showed that a 3-week chronic free-choice ethanol consumption in C57BL/6J mice led to a decrease in DNA methylation of the Bdnf gene within the CA1 and CA3 subfields of the hippocampus, and upregulated hippocampal BDNF signaling pathways mediated by ERK, AKT and CREB. However, this activation did not affect long-term potentiation in the CA1. Conversely, ethanol intake impaired learning and memory capacities analyzed in the contextual fear conditioning test and the novel object recognition task. In addition, ethanol increased behavioral perseveration in the Barnes maze test but did not alter the mouse overall spatial capacities. These data suggested that in conditions of chronic and moderate ethanol intake, the chromatin remodeling leading to BDNF signaling upregulation is probably an adaptive process, engaged via epigenetic regulations, to counteract the cognitive deficits induced by ethanol.     

Observational learning in C57BL/6j mice

The ability of mice to solve a complex task by observational learning was investigated with C57BL/6j mice. Four female demonstrators were trained to reliably perform a sequence that consisted in pushing a piece of food into a tube attached to the side of a puzzle box, and recovering it by opening a drawer in front of the box. They then performed this sequence in front of naive mice assigned to individual cubicles in a box with a wire mesh front arranged in a row facing the demonstrators. A total of 25 naive mice (13 males and 12 females) were used. Fifteen mice observed 14 demonstrations a day for 5 days; 10 control mice were placed in similar cubicles, but behind a plastic screen which prevented them from observing the demonstrators. The mice were post-tested in the demonstrator situation, and 6 of 15 observers immediately reproduced the complete task successfully, but none of the naive or control mice were able to solve the task. The observers and controls were then subjected to a five level individual learning schedule. Observers learned the individual task significantly faster than the controls. No sex difference was found. These results suggest that observational learning processes at work were based on stimulus enhancement and observational conditioning.     

H MRS identifies lactate rise in the striatum of MPTP-treated C57BL/6 mice

Mitochondrial dysfunction has been implicated in the death of nigrostriatal dopaminergic neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated experimental models of Parkinson's disease (PD). Here we utilized proton magnetic resonance spectroscopy ((1)H MRS) to identify changes in energy metabolism in the striatum of MPTP-treated C57BL/6 mice. Remarkable increases in lactate/creatine (Lac/Cr) ratio were observed at 2 h and then quickly returned to about the basal level by 7 h after injection of MPTP. Neurochemical and Western blot analyses revealed that dopamine contents and protein levels of tyrosine hydroxylase and dopamine transporter in the striatum were profoundly decreased at 3 days after MPTP treatment. Pretreatment with deprenyl, a monoamine oxidase B inhibitor, or GBR-12909, a dopamine uptake inhibitor, almost completely attenuated both the increases in striatal Lac/Cr ratio and the subsequent loss of dopaminergic nerve terminals in MPTP-treated mice. The present study indicates that (1)H MRS is a sensitive measure of biochemical alterations of the brain in a mouse model of PD, and further shows that the increases in striatal Lac/Cr ratio induced by MPTP may be associated with mitochondrial energy crisis, followed by dopaminergic neurotoxicity.     

Characterization of cortical spindles in DBA/2 and C57BL/6 inbred mice

Continuous twenty-four hour EEG recordings were conducted on freely-moving DBA/2 and C57BL/6 inbred mice. No brief spindle episodes (BSEs: 6-7 cps, 1-5 sec duration, high amplitude spindle bursts) were seen in the waking EEG of C57BL/6 mice. BSEs were a conspicuous element of the EEG during active waking (AW) and quiet waking (QW) in DBA/2 mice. BSEs occurred at a 10X faster rate in QW than in AW and had a longer duration. Sleep spindle bursts resembling BSEs were seen in both C57BL/6 and DBA/2 mice, and occasionally were observed to follow a K-complex. Rostropontine, but not midpontine, brainstem transection released spindles in both strains. Pentobarbital produced spindles in both strains. The waveforms of the waves comprising BSEs, sleep spindles, transection-induced spindles and barbiturate spindles were quite similar, though differing in frequencies and amplitude. Genetic factors may be critical for the lack of BSEs during AW and QW in C57BL/6 mice and for the occurrence of BSEs during AW in DBA/2 mice. In contrast, most other rodents whow a third pattern: BSEs only during QW. Since C57BL/6 mice can generate spindles under some circumstances, the absence of spindles during waking reflects some alteration in the mechanisms that control the initiation of BSEs rather than a lack of the circuits required to generate a BSE. These mechanisms are distinct from those processes of arousal that produce the background EEG desynchronization of waking. Following both rostropontine and midpontine transection, the background EEG is desynchronized, yet after rostropontine, but not midpontine transection, BSEs occur freely, at a rate over 200 per hour.     

MBP-PLP fusion protein-induced EAE in C57BL/6 mice

Gene knock-out and knock-in mice are becoming increasingly indispensable for mechanism-oriented studies of EAE. Most gene-modified mice are on the C57BL/6 background, for which presently there are only two EAE models available, the MOG peptide 35-55 and the PLP 178-191 peptide induced disease. However, because MS is not a single pathogenic entity, different EAE models are required to reproduce and study its various features. Here we are introducing MBP-PLP fusion protein (MP4)-induced EAE for C57BL/6 mice. B cell- and CD8+ T cell-dependence, as well as multi-determinant recognition are among the unique features of this demyelinating EAE.     

Lesions of the Edinger-Westphal nucleus in C57BL/6J mice disrupt ethanol-induced hypothermia and ethanol consumption

The Edinger-Westphal nucleus (EW) is a brain region that has recently been implicated as an important novel neural target for ethanol. Thus, the EW is the only brain region consistently showing elevated c-Fos expression following both voluntary and involuntary ethanol administration. Ethanol-induced c-Fos expression in the EW has been shown to occur in urocortin I-positive neurons. Moreover, previous reports using several genetic models have demonstrated that differences in the EW urocortin I system are correlated with ethanol-mediated behaviours such as ethanol-induced hypothermia and ethanol consumption. The aim of this study was to confirm these relationships using a more direct strategy. Thus, ethanol responses were measured following electrolytic lesions of the EW in male C57BL/6J mice. Both EW-lesioned and sham-operated animals were tested for several ethanol sensitivity measures and ethanol consumption in a two-bottle choice test. The results show that lesions of the EW significantly disrupted ethanol-induced hypothermia, while having no effect on pupillary dilation, locomotor activity or ethanol-induced sedation. In addition, EW-lesioned animals showed significantly lower ethanol preference and total ethanol dose consumed in the two-bottle choice test. EW-lesioned animals also consumed less sucrose than sham-operated animals, but did not have altered preferences for sucrose or quinine in a two-bottle choice test. These data support previously observed genetic correlations between EW urocortin I expression and both ethanol-induced hypothermia and ethanol consumption. Taken together, the findings suggest that the EW may function as a sensor for ethanol, which can influence ethanol consumption and preference.     

The effects of galantamine on nicotine withdrawal-induced deficits in contextual fear conditioning in C57BL/6 mice

Current smoking cessation aids are relatively ineffective at maintaining abstinence during withdrawal. Nicotine withdrawal is associated with a variety of symptoms including cognitive deficits and targeting these deficits may be a useful strategy for maintaining abstinence. Galantamine is an acetylcholinesterase inhibitor and allosteric modulator of nicotinic acetylcholine receptors (nAChRs) with cognitive enhancing effects that may alleviate cognitive deficits associated with nicotine withdrawal. The effects of galantamine on nicotine withdrawal-induced deficits in contextual fear conditioning in C57BL/6 mice were examined. An initial acute dose-response experiment revealed that 0.5 and 1 mg/kg galantamine had no effect on fear conditioning. To determine if galantamine would reverse nicotine withdrawal-related deficits in contextual fear conditioning, mice were implanted with osmotic mini-pumps that delivered chronic saline or 6.3 mg/kg/d nicotine for 12 days and then pumps were removed. Training and testing of fear conditioning occurred 24 and 48 h later, respectively. Nicotine withdrawal disrupted contextual fear conditioning, which was reversed with 1 but not 0.5 mg/kg galantamine. Across all conditions in both studies 2 mg/kg galantamine led to high levels of freezing that were likely due to nonspecific effects. The ability of galantamine to reverse nicotine withdrawal-deficits in contextual conditioning is likely mediated through enhanced levels of acetylcholine via inhibition of acetylcholinesterase, potentiation of hippocampal α4β2* nAChRs, or both. The present study suggests that acetylcholinesterase inhibitors and/or drugs that act as allosteric modulators of nAChRs might be targets for smoking cessation aids because they may alleviate withdrawal symptoms such as cognitive deficits that can lead to relapse.     

Genetic analysis of cerebellar folial pattern in crosses of C57BL/6J and DBA/2J inbred mice

Variation in the cerebellar folial pattern of mice is influenced by genetic elements [Inouye, M. and Oda, S.,J. Comp. Neurol., 190 (1980) 357–362]. In crosses of C57BL/6J and DBA/2J inbred mice, the presence or absence of a specific fissure, the intraculminate fissure, is largely determined by a single genetic locus (Cfp-1), which is located on distal Chromosome 4 [Neumann et al.,Brain Res., 524 (1990) 85–89]. In the present study, the mid-sagittal cerebellar folial pattern has been examined in crosses of C57BL/6J and DBA/2J mice and in BXD recombinant inbred strains. At least three loci, includingCfp-1, are involved in variation in vermian pattern formation. Genetic variation in thyroid hormone function may be involved in the inheritance of folial pattern. A locus (Cfp-2) that appears to be partially responsible for this negative genetic correlation in mice may be linked toAfp on Chromosome 5. This hypothesis was suggested by the negative correlation between neonatal serum T₄ level and the number of folia in rats given neonatal injections of thyroxine or propylthiouracil [Lauder, J.M. et al.,Brain Res., 76 (1974) 33–40].     

Tripchlorolide protects against MPTP-induced neurotoxicity in C57BL/6 mice

Many current studies of Parkinson's disease (PD) suggest that inflammation is involved in the neurodegenerative process. Tripchlorolide (TW397), a traditional Chinese herbal compound with anti-inflammatory and immunosuppressive properties, has been shown to protect dopaminergic neurons against, and restore their function after, the neurotoxicity induced by 1-methyl-4-phenylpyridinium ions in vitro. This study was designed to investigate the effect of TW397 in vivo in the PD model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned C57BL/6 mice. In the animals that received vehicle-only (i.e., no TW397) treatment with MPTP i.p. injection, the survival ratios of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the substantia nigra pars compacta and TH-IR fibres in the striatum were only 59 and 13%, respectively, compared with the normal controls. Intriguingly, in conjunction with MPTP, treatment with TW397, 1 microg/kg for 16 days, once per day, dramatically improved the survival rate of the TH-IR neurons and TH-IR fibres to 80 and 43% of the control. The treatment with TW397 also significantly improved the level of dopamine in the substantia nigra and striatum to 157 and 191%, respectively, of the MPTP- plus vehicle-treated group. In addition, in MPTP-treated animals the rota-rod performances of those treated with 0.5 or 1 microg/kg TW397 were significantly improved, by approximately 2- and 3-fold, respectively, relative to vehicle-treated animals. The neuroprotective effect of TW397 was coincident with an attenuated astroglial response within the striatum. These data demonstrate a neuroprotective action of TW397 in vivo against MPTP toxicity, with important implications for the treatment of PD.     

Acetylcholinesterase molecular forms in C57BL/6J dystrophic mice

Acetylcholinesterase (AChE) was extracted from normal and dystrophic C57BL/6J mouse hindlimb muscles and its molecular forms fractionated by sucrose density gradient ultracentrifugation. In the soleus muscles from 6- to 7-week-old mice an increase in the 3 Svedberg unit (S) and a decrease in the 16S AChE molecular forms was observed in dystrophic animals compared to controls. At 12-13 weeks of age, no major significant differences in the relative proportions of AChE molecular forms were noted. In the extensor digitorum longus (EDL) muscles of 6- to 7-week-old dystrophic mice a significant decrease in the proportion of the 10S AChE molecular form and an increase in the 3S and 5S forms was observed. At 12-13 weeks, the dystrophic EDL muscles again displayed a decrease in the 10S form; however, the increase in the 3S and 5S AChE forms, while still apparent, was not significant. These results provide evidence for a biochemical abnormality in the distribution of specific AChE molecular forms, and a differential expression of this abnormality in the soleus and EDL muscles.     

Lesion-induced DA supersensitivity in aging C57BL/6J mice

Lesion-induced dopaminergic supersensitivity was investigated in 4-, 10-, and 28-month-old C57BL/6J mice. Apomorphine-induced rotational behavior was examined 5, 10, and 20 days after destruction of the dopamine-containing nigro-striatal pathway by intrastriatal infusion of 6-OHDA. No major differences between ages were observed in the extent or rate of development of contralateral rotation. It is concluded that age-differences in dopaminergic supersensitization are dependent upon the nature and/or severity of the sensitizing stimulus.