Shannon G. Matta

Guidelines on nicotine dose selection for in vivo research

RationaleThis review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure.ObjectivesThis review capitalizes on the authors’ collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models.ResultsSeven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses.ConclusionsThe selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose–response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.

Self-administration in rats allowed unlimited access to nicotine

Abstract The purpose of the present study was to develop an animal model of nicotine self-administration that more closely approximates the conditions of human nicotine use than do existing models. In most nicotine self-administration models, rats acquire self-administration during brief daily sessions in which rapid injections of a relatively high dose of the drug, 0.03 mg/kg, serve as the reinforcer. The present study examined nicotine self-administration in rats that acquired the behavior while having virtually unlimited access to injections of a relatively low dose of the drug; the rats did not have any prior operant training or shaping. Under these conditions, rats readily acquire nicotine self-administration at doses at least as low as 0.00375 mg/kg per injection, and they self-administer throughout the active portion of their light cycle. The daily nicotine intake of rats, which ranged from 0.18 to 1.38 mg/kg per day, appears to be comparable to that of human smokers.

Nicotine administration enhances NPY expression in the rat hypothalamus

Epidemiological studies have shown an inverse relationship between cigarette smoking and body weight. In rodents, a negative correlation between nicotine and body weight has been reported, but this observation was largely derived from studies where relatively high doses of nicotine ( approximately 12 mg/kg/day) were used. In the current study, we showed that a negative relationship also holds for low doses of nicotine that are comparable to that consumed by average human smokers (<6 mg/kg/day). We also demonstrated that 14 days of nicotine administration (4 mg/kg/day) reduced average daily food intake by 19.5% (P<0.01) in the free-feeding nicotine-treated group compared to saline controls. No significant differences in body weight were detected between the nicotine-treated and pair-fed groups. To determine whether the effects of nicotine on food intake and body weight were related to neuropeptide Y (NPY) expression, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and radioimmunoassay were utilized to measure NPY mRNA and peptide levels in various regions of the hypothalamus. Significantly higher levels of NPY mRNA (ca. 20-50%) and peptide (ca. 24-69%) were only detected in the nicotine-treated groups. In addition, significantly higher NPY contents were also obtained in two hypothalamic areas of pair-fed control animals. In summary, our data suggest that the pharmacological effects of nicotine on food intake and body weight may be mediated by changes in hypothalamic NPY levels, a neuropeptide that is pivotal to the hypothalamic regulation of food intake.

Acquisition of Nicotine Self-Administration in Adolescent Rats Given Prolonged Access to the Drug

As most human tobacco use begins during adolescence and ongoing development of the adolescent central nervous system could affect acquisition of nicotine self-administration (SA), our established rat SA procedure was modified to study adolescent acquisition of SA with prolonged access to nicotine (23 h/day). Postnatal age 43–45 female Lewis rats, without prior shaping, conditioning, or food deprivation, were housed in operant chambers equipped with two levers; pressing the active lever triggered an i.v. injection of nicotine. By the 10th day of SA, rats receiving 7.5, 15, 30, or 60 μg/kg/injection nicotine (free base) obtained 23±16, 50±8, 65±8, or 48±5 injections (mean±SE), respectively. In the 30 μg/kg/injection group, active : inactive ratio was greater than 2 after SA day 4; 92% of injections occurred during the 12-h active (dark) phase of the light cycle. Main effects (analysis of variance) were shown for day and lever (ie active vs inactive) (p<0.001). Adolescent males showed similar dose-dependent nicotine SA. With the increasing workload imposed by raising the fixed ratio (FR), less nicotine was self-administered at FR 5 and 7 compared to FR 1 and 3. In comparison to adult females self-administering 30 μg/kg/injection of nicotine at FR 1, adolescents acquired nicotine SA at an accelerated rate (p<0.05) and received a greater number of injections (p<0.05) by day 10. In conclusion, when given prolonged access to the drug, both female and male adolescent Lewis rats rapidly acquire nicotine SA within the dosage range and FR constraints previously observed in adult Lewis rats. However, adolescent females acquired the behavior more rapidly and attained higher levels of stable nicotine SA than adults.

Rat strain differences in nicotine self-administration using an unlimited access paradigm

An effective animal model for elucidating the neurobiological basis of human smoking should simulate important aspects of this behavior. Therefore, a 23 h unlimited access nicotine self-administration model was used to compare inbred Lewis rats, which have a propensity to self-administer drugs of abuse, to inbred Fisher 344 rats and to the outbred Holtzman strain. Using this unlimited access model, 88.8% of Lewis vs. 57.1% of Holtzman rats achieved maintenance self-administration at a fixed ratio 1 (FR 1) at 0.03 mg/kg IV nicotine (P<0.05). In contrast, Fisher rats did not acquire self-administration under these conditions. Of the Lewis and Holtzman rats that achieved maintenance self-administration on an FR 1 schedule, a greater percentage of Lewis rats acquired nicotine self-administration at FR 2 (P<0.05) and progressed to FR 4 (P<0.05). Using naïve cohorts in a progressive dose reduction study, 83.3% of Lewis rats achieved maintenance at 0.0075 mg/kg nicotine as compared to 31.8% of Holtzman rats (P<0.05). Furthermore, only Lewis rats showed differences in active vs. inactive bar presses during maintenance at sequential dose reductions (P<0.001). Thus, in this unlimited access model, inbred Lewis rats will more reliably acquire nicotine self-administration than outbred Holtzman rats. Moreover, Lewis rats showed a significantly higher likelihood of continuing to self-administer nicotine in face of both increasing work requirements and decreasing drug reinforcement. Therefore, it is likely that Lewis rats would be genetically susceptible to nicotine addiction.

A Central Mechanism Is Involved in the Secretion of ACTH in Response to IL-6 in Rats: Comparison to and Interaction with IL-1β

The cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha are known to be potent effectors of ACTH secretion. Some of the peripheral effects of IL-1 beta appear to be related to the secretion of IL-6 induced by IL-1 beta. Thus, we evaluated the effect of IL-6 on ACTH secretion and its interaction with IL-1 beta. Rats received recombinant human (rhIL-6) or murine (rmIL-6) IL-6 through indwelling jugular cannulae. rhIL-6 (200 ng or 2 micrograms/rat) produced peak plasma ACTH levels which were 3- to 4-fold greater than basal levels. rmIL-6 produced similar responses. Neither species of IL-6 affected plasma prolactin levels. Comparison of rhIL-1 beta (200 ng) to rhIL-6 (200, 100 or 50 ng) showed that IL-6 elevated ACTH in a dose-dependent manner and that IL-1 beta was significantly more effective. IL-1 beta was also administered concomitantly with or 10 min after IL-6. Delivered together, IL-1 beta (100, 30 or 10 ng) and IL-6 (100 ng) produced significantly higher ACTH levels than when given alone. This additivity was also evident when IL-6 was given 10 min prior to IL-1 beta. The coadministration of IL-6 (2 micrograms) with corticotropin-releasing factor (CRF, 1 micrograms/kg, b.w.) also had an additive effect on ACTH secretion (at 20 min: 300 +/- 40 pg/ml for CRF; 320 +/- 83 pg/ml for IL-6; and 540 +/- 44 pg/ml for CRF + IL-6), whereas a higher dose of CRF (10 micrograms/kg b.w.) yielded ACTH levels of 1,000 +/- 107 pg/ml at 20 min, with no further enhancement by IL-6. Incubation of pituitary cells with IL-6 alone (0.1, 1.0 or 3.0 nM) produced a slight but significant stimulation of ACTH secretion within 2 h in response to the higher doses of IL-6 only (p < 0.05), but did not modify the effect of CRF in vitro. To determine if the action of IL-6 was at a site(s) within the brain, IL-6 (30 or 100 ng/0.5 microliters) was injected into the third cerebroventricle of alert rats. 100 ng IL-6 elicited peak plasma ACTH levels (300 +/- 65 pg/ml) within 30 min; these were significantly higher than the buffer responses (90 +/- 25 pg/ml, p < 0.01), and lower than the responses to 30 ng IL-1 beta (530 +/- 50 pg/ml, p < 0.001). 30 ng IL-6 was ineffective.(ABSTRACT TRUNCATED AT 400 WORDS)

Constitutively active TRPC3 channels regulate basal ganglia output neurons.

A hallmark of the GABA projection neurons of the substantia nigra pars reticulata (SNr), a key basal ganglia output nucleus, is its depolarized membrane potential and rapid spontaneous spikes that encode the basal ganglia output. Parkinsonian movement disorders are often associated with abnormalities in SNr GABA neuron firing intensity and/or pattern. A fundamental question remains regarding the molecular identity of the ion channels that drive these neurons to a depolarized membrane potential. We show here that SNr GABA projection neurons selectively express type 3 canonical transient receptor potential (TRPC3) channels. These channels are tonically active and mediate an inward, Na+-dependent current, leading to a substantial depolarization in these neurons. Inhibition of TRPC3 channels induces hyperpolarization, decreases firing frequency, and increases firing irregularity. These data demonstrate that TRPC3 channels play important roles in ensuring the appropriate firing intensity and pattern in SNr GABA projection neurons that are crucial to movement control.

An Ultra-Short Dopamine Pathway Regulates Basal Ganglia Output

Substantia nigra pars reticulata (SNr) is a key basal ganglia output nucleus critical for movement control. Its GABA-containing projection neurons intermingle with nigral dopamine (DA) neuron dendrites. Here we show that SNr GABA neurons coexpress dopamine D1 and D5 receptor mRNAs and also mRNA for TRPC3 channels. Dopamine induced an inward current in these neurons and increased their firing frequency. These effects were mimicked by D1-like agonists, blocked by a D1-like antagonist. D1-like receptor blockade reduced SNr GABA neuron firing frequency and increased their firing irregularity. These D1-like effects were absent in D1 or D5 receptor knock-out mice and inhibited by intracellularly applied D1 or D5 receptor antibody. These D1-like effects were also inhibited when the tonically active TRPC3 channels were inhibited by intracellularly applied TRPC3 channel antibody. Furthermore, stimulation of DA neurons induced a direct inward current in SNr GABA neurons that was sensitive to D1-like blockade. Manipulation of DA neuron activity and DA release and inhibition of dopamine reuptake affected SNr GABA neuron activity in a D1-like receptor-dependent manner. Together, our findings indicate that dendritically released dopamine tonically excites SNr GABA neurons via D1–D5 receptor coactivation that enhances constitutively active TRPC3 channels, forming an ultra-short substantia nigra pars compacta → SNr dopamine pathway that regulates the firing intensity and pattern of these basal ganglia output neurons.

Gestational nicotine exposure reduces nicotinic cholinergic receptor (nAChR) expression in dopaminergic brain regions of adolescent rats

Children of women who smoked during pregnancy are at increased risk of dependence when smoking is initiated during adolescence. We previously reported that gestational nicotine exposure attenuated dopamine release induced by nicotine delivered during adolescence. In this study, we determined the effects of gestational nicotine exposure on nicotinic cholinergic receptor (nAChR) expression. Timed pregnant rats received nicotine (2 mg/kg/day) or vehicle via mini‐osmotic pumps during gestation. Treatments continued in pups via maternal nursing during postnatal days (PN) 2–14 (equivalent to the human in utero third trimester). On PN35, 125I‐epibatidine binding to nAChR was measured. The Bmax values (fmol/mg) in prefrontal cortex (PFC), nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) were reduced by 26.6% (P < 0.05), 32.6% (P < 0.01), 23.0% (P < 0.01) and 27.6% (P < 0.05), respectively. In addition, gender differences were found in vehicle‐treated groups; in SN and VTA, females were 79.3% (P < 0.005) and 82.9% (P = 0.08) of males, respectively. The expression of nAChR subunit mRNAs was measured using real‐time RT‐PCR on laser‐capture microdissected tissues. In adolescent VTA, gestational nicotine exposure reduced (P < 0.05) nAChR subunit mRNAs encoding α3 (53.0%), α4 (23.9%), α5 (46.7%) and β4 (61.4%). In NAcc core, the treatment increased α3 mRNA (75.8%). In addition, the number of neurons in VTA was reduced by 15.0% (P < 0.001). These studies indicate that gestational exposure to nicotine induces long‐lasting changes in nAChR expression that may underlie the vulnerability of adolescents to dependence on nicotine.

Local α-bungarotoxin-sensitive nicotinic receptors in the nucleus accumbens modulate nicotine-stimulated dopamine secretion in vivo

Nicotinic cholinergic receptors in the ventral tegmental area are required for the accumbal dopamine response to systemic nicotine. In contrast, the role of nicotinic receptors located within the nucleus accumbens itself has not been clarified for systemically administered nicotine. In the present study, in vivo microdialysis of accumbal dopamine secretion and receptor antagonist blockade in both the ventral striatal nucleus accumbens and the midbrain ventral tegmental area were used to evaluate this question. The nicotinic receptor antagonists methyllycaconitine or mecamylamine were delivered through the accumbal dialysis probe, followed by 0.09mg/kg nicotine (i.v.). The alpha7 subunit antagonist methyllycaconitine inhibited 71% of the dopamine response (P<0.01), whereas mecamylamine was completely ineffective. In addition, the classical alpha7 subunit antagonist alpha-bungarotoxin infused into the nucleus accumbens adjacent to the microdialysis probe, significantly reduced dopamine release by 0.065 or 0.09mg/kg nicotine (i.v.; P<0. 05). Combined, these data indicate the involvement of alpha7 subunit-containing nicotinic receptors in the nucleus accumbens. In contrast, local infusion of mecamylamine into the ventral tegmental area effectively blocked nicotine-induced accumbal dopamine release. Simultaneous infusions of methyllycaconitine into the accumbens and mecamylamine into the ventral tegmental area induced greater blockade of nicotine-stimulated dopamine secretion than methyllycaconitine or mecamylamine alone. In conclusion, the present study demonstrates that different types of nicotinic cholinergic receptors, located in the ventral striatal nucleus accumbens (alpha-bungarotoxin sensitive and mecamylamine insensitive) and the midbrain ventral tegmental area (mecamylamine sensitive), may be required for the full effects of nicotine on the mesostriatal dopaminergic pathway. While activation of nicotinic cholinergic receptors in the ventral tegmentum is required for the accumbal dopamine response to systemic nicotine, accumbal nicotinic receptors themselves act as modulators of this response. This fine tuning of the dopamine reward pathway through alpha7 nicotinic cholinergic receptors in the nucleus accumbens may amplify the secretion of dopamine, allowing a subthreshold brain concentration of nicotine to become an effective stimulus for dopamine secretion.