Caffeine has no effect on eyeblink conditioning in mice.

Caffeine is one of the most widely used drugs in the world. In the brain, caffeine acts as an antagonist for the adenosine A1 and A2B receptors. Since A1 receptors are highly concentrated in the cortex of the cerebellum, we hypothesized that caffeine could potentially affect learning tasks that require the cerebellar cortex, such as eyeblink conditioning. To test this hypothesis, we examined the effect of low (5mg/kg) and high (50mg/kg) doses of caffeine, injected intraperitoneally before training, on eyeblink conditioning in mice. The results show that, at the dosages we used, caffeine affects neither the rate of acquisition, nor the timing of the onset or peak of the conditioned blink responses. Therefore, we conclude that caffeine neither improves nor worsens performance on eyeblink conditioning.

Maternal iron deficiency worsens the associative learning deficits and hippocampal and cerebellar losses in a rat model of fetal alcohol spectrum disorders.

BACKGROUND: Gestational alcohol exposure causes lifelong physical and neurocognitive deficits collectively referred to as fetal alcohol spectrum disorders (FASDs). Micronutrient deficiencies are common in pregnancies of alcohol-abusing women. Here we show the most common micronutrient deficiency of pregnancy-iron deficiency without anemia-significantly worsens neurocognitive outcomes following perinatal alcohol exposure. METHODS: Pregnant rats were fed iron-deficient (ID) or iron-sufficient diets from gestational day 13 to postnatal day (P) 7. Pups received alcohol (0, 3.5, 5.0 g/kg) from P 4 to P 9, targeting the brain growth spurt. At P 32, learning was assessed using delay or trace eyeblink classical conditioning (ECC). Cerebellar interpositus nucleus (IPN) and hippocampal CA1 cellularity was quantified using unbiased stereology. RESULTS: Global analysis of variance revealed that ID and alcohol separately and significantly reduced ECC learning with respect to amplitude (ps ≤ 0.001) and conditioned response [CR] percentage (ps ≤ 0.001). Iron and alcohol interacted to reduce CR percentage in the trace ECC task (p = 0.013). Both ID and alcohol significantly reduced IPN (ps < 0.001) and CA1 cellularity (ps < 0.005). CR amplitude correlated with IPN cellularity (delay: r = 0.871, trace: r = 0.703, ps < 0.001) and CA1 cellularity (delay: r = 0.792, trace: r = 0.846, ps < 0.001) across both tasks. The learning impairments persisted even though the offsprings' iron status had normalized. CONCLUSIONS: Supporting our previous work, gestational ID exacerbates the associative learning deficits in this rat model of FASD. This is strongly associated with cellular reductions within the ECC neurocircuitry. Significant learning impairments in FASD could be the consequence, in part, of pregnancies in which the mother was also iron inadequate.

Effects of intravenous nicotine on prepulse inhibition in smokers and non-smokers: relationship with familial smoking.

RATIONALE: The reinforcing properties of nicotine may be, in part, derived from its ability to enhance certain forms of cognitive processing. Several animal and human studies have shown that nicotine increases prepulse inhibition (PPI) of the startle reflex. However, it remains unclear whether these effects are related to smoking susceptibility. OBJECTIVES: The current study examined the effects of intravenously delivered nicotine on PPI in smokers and non-smokers, as well as its association with a quantitative index of familial smoking. METHODS: The sample consisted of 30 non-smokers and 16 smokers, who completed an initial assessment, followed on a separate day by a laboratory assessment of PPI prior to and following each of two intravenous nicotine infusions. Separate doses were used in smoker and non-smoker samples. RESULTS: Analyses indicated that both nicotine infusions acutely enhanced PPI among non-smokers, and this enhancement was positively related to the degree of smoking among first and second-degree relatives. Smokers also displayed PPI enhancement after receiving the first infusion, but this effect was unrelated to familial smoking. CONCLUSIONS: These data suggest that the PPI paradigm may have utility as an endophenotype for cognitive processes which contribute to smoking risk.

Differential effects of progesterone and medroxyprogesterone on delay eyeblink conditioning in ovariectomized rats.

Ovarian hormones modulate acquisition processes involved in classical conditioning. Although progesterone has been indirectly implicated, its role in classical conditioning of the eyeblink response has not been directly investigated. We assessed the effects of daily dosing of progesterone or medroxyprogesterone (MPA), a non-metabolized synthetic progestin, upon the acquisition of a classically conditioned eyeblink response in ovariectomized (OVX) female rats. Rats were dosed 4h prior to each training session with 0.1 or 1.5 mg/kg of either of these hormones or sesame oil. A delay conditioning paradigm was employed using a 500 ms conditioned stimulus coterminating with a 10 ms 10 V unconditioned stimulus. At the low dose, progesterone and MPA rats did differ from each other, with MPA-treated rats learning slower, but neither group differed from OVX-oil or Sham-oil controls. No group differences in acquisition were observed at the higher dose. During extinction trials, high-dose MPA-treatment and OVX-oil groups extinguished quicker than the high-dose progesterone-treated group. In addition, unconditional response (UR) amplitudes were lower in all OVX groups, regardless of hormone or oil treatment, compared to the sham-oil group. Since MPA did not affect extinction, it is likely the slower extinction in the progesterone-treated rats is due to a metabolite of progesterone. Corticosterone is discussed as a likely candidate for such a role. In addition, we found chronic absence of ovarian hormones decreased UR amplitudes, although differences in UR amplitudes were not associated with changes in the acquisition process. These results are discussed with respect to differences in the hormonal effects upon acquisition versus extinction processes and how these data may explain reports of learning differences in women based on oral contraceptive usage.

Neonatal corticosterone administration impairs adult eyeblink conditioning and decreases glucocorticoid receptor expression in the cerebellar interpositus nucleus.

Neonatal maternal separation alters adult learning and memory. Previously, we showed that neonatal separation impaired eyeblink conditioning in adult rats and increased glucocorticoid receptor (GR) expression in the cerebellar interpositus nucleus, a critical site of learning-related plasticity. Daily neonatal separation (1 h/day on postnatal days 2-14) increases neonatal plasma corticosterone levels. Therefore, effects of separation on GR expression in the interpositus and consequently adult eyeblink conditioning may be mediated by neonatal increases in corticosterone. As a first step in exploring a potential role for corticosterone in the neonatal separation effects we observed, we assessed whether systemic daily (postnatal days 2-14) corticosterone injections mimic neonatal separation effects on adult eyeblink conditioning and GR expression in the interpositus. Control uninjected animals were compared to animals receiving either daily corticosterone injections or daily injections of an equal volume of vehicle. Plasma corticosterone values were measured in a separate group of control, neonatally separated, vehicle injected, or corticosterone injected pups. In adulthood, rats underwent surgery for implantation of recording and stimulating electrodes. After recovery from surgery, rats underwent 10 daily sessions of eyeblink conditioning. Then, brains were processed for GR immunohistochemistry and GR expression in the interpositus nucleus was assessed. Vehicle and corticosterone injections both produced much larger increases in neonatal plasma corticosterone than did daily maternal separation, with the largest increases occurring in the corticosterone-injected group. Neonatal corticosterone injections impaired adult eyeblink conditioning and decreased GR expression in the interpositus nucleus, while the effects of vehicle injections were intermediate. Thus, while neonatal injections and maternal separation both produce adult impairments in learning and memory, these manipulations produce opposite changes in GR expression. This suggests an inverted U-shaped relationship may exist between both neonatal corticosterone levels and adult GR expression in the interpositus nucleus, and adult GR expression in the interpositus and eyeblink conditioning.

Variability in initial nicotine sensitivity due to sex, history of other drug use, and parental smoking.

Initial sensitivity to nicotine's effects during early exposure to tobacco may relate to dependence vulnerability. We examined the association of initial nicotine sensitivity with individual difference factors of sex, other drug use history (i.e. cross-tolerance or cross-sensitization), and parental smoking status in young adult nonsmokers (N=131). Participants engaged in 4 sessions, the first 3 to assess the dose-response effects of nasal spray nicotine (0, 5, 10 microg/kg) on rewarding, mood, physiological, sensory processing, and performance effects, and the fourth to assess nicotine reinforcement using a choice procedure. Men had greater initial sensitivity than women to some self-reported effects of nicotine related to reward and incentive salience and to impairment in sensory processing, but men and women did not differ on most other effects. Prior marijuana use was associated with greater nicotine reward, nicotine reinforcement was greater in men versus women among those with prior marijuana use, and having parents who smoked was related to increased incentive salience. However, history of other drug use and parental smoking were not otherwise associated with initial nicotine sensitivity. These findings warrant replication with other methods of nicotine administration, especially cigarette smoking, and in more diverse samples of subjects naïve to nicotine. Yet, they suggest that sex differences in initial sensitivity to nicotine reward occur before the onset of dependence. They also suggest that parental smoking may not increase risk of nicotine dependence in offspring by altering initial nicotine sensitivity, and that cross-tolerance between other drugs and nicotine may not be robust in humans.

Inhibition of cortisol production by metyrapone enhances trace, but not delay, eyeblink conditioning.

RATIONALE: Hypercortisolism [ corrected] impairs trace classical conditioning of the eyeblink response to an air puff but does not affect delay conditioning. OBJECTIVES: The opposite neurohormonal condition, hypocortisolism, may facilitate trace classical conditioning, which might be informative in understanding the role of classical conditioning in stress-sensitive syndromes such as fibromyalgia. MATERIALS AND METHODS: Volunteers (n = 82) were randomized to receive either an inhibitor of cortisol production (metyrapone, 1500 mg) or placebo and to complete a delay or a trace eyeblink conditioning protocol (unconditioned stimulus: corneal air puff, 10 psi, 50 ms; conditioned stimulus: binaural pure tone, 75 dB, 1000 Hz, 400 ms; empty interval in trace conditioning: 600 ms), where conditioned eyeblink response probability was assessed electromyographically. RESULTS: Metyrapone induced hypocortisolism, reflected by a 30% decrease of salivary cortisol levels (p < 0.01), and facilitated trace eyeblink conditioning (p < 0.001), while delay eyeblink conditioning remained unaffected. Moreover, extinction of delay-conditioned eyeblink responses was impaired (p = 0.023), but extinction of trace-conditioned responses remained unaffected. CONCLUSIONS: We conclude that acute mild metyrapone-induced hypocortisolism facilitates hippocampus-mediated classical trace eyeblink conditioning but suppresses the extinction of cerebellum-based delay-conditioned responses. Both results may be of theoretical and clinical significance for the generation and persistence of psychosomatic symptoms in patient groups characterized by relative hypocortisolism (e.g., fibromyalgia and chronic fatigue).

The nicotinic agonist RJR-2403 compensates the impairment of eyeblink conditioning produced by the noncompetitive NMDA-receptor antagonist MK-801.

The classical conditioning of eyelid responses using trace paradigms is a hippocampal-related model of associative learning, involving the activation of N-methyl-D-aspartate (NMDA) receptors. We have evaluated here the effects of NMDA-receptor blockage with the selective noncompetitive antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (dizocilpine, MK-801). Mice were implanted with stimulating electrodes on the supraorbitary nerve and with recording electrodes in the ipsilateral orbicularis oculi muscle. Animals were conditioned with a trace shock-SHOCK paradigm. MK-801-injected animals (0.02 mg/kg) seemed unable to acquire this type of associative learning task, but the latency and amplitude of their unconditioned eyelid responses was not affected by drug administration. The administration of the nicotinic agonist (E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403; 2 mg/kg) was able to restore completely the acquisition of the conditioned response when administered both before and after MK-801. In vitro recordings of field excitatory postsynaptic potentials (fEPSPs) evoked in the hippocampal CA1 area by the electrical stimulation of the Schaffer collateral pathway indicates that RJR-2403 application to the bath enhance the release of glutamate by a presynaptic mechanism. These findings reveal that nicotinic acetylcholine receptors enhance glutamatergic transmission in hippocampal circuits involved in the acquisition of associative learning.

Cerebellar norepinephrine modulates learning of delay classical eyeblink conditioning: evidence for post-synaptic signaling via PKA.

The neurotransmitter norepinephrine (NE) has been shown to modulate cerebellar-dependent learning and memory. Lesions of the nucleus locus coeruleus or systemic blockade of noradrenergic receptors has been shown to delay the acquisition of several cerebellar-dependent learning tasks. To date, no studies have shown a direct involvement of cerebellar noradrenergic activity nor localized the post-synaptic response to cerebellar beta-noradrenergic receptor signaling. Using ipsilateral, localized infusions into cerebellar lobule HVI and interpositus (IP), we have established that blocking beta-noradrenergic receptors with propranolol significantly impairs acquisition of conditioned responses. Furthermore, interrupting activation of cAMP-dependent PKA in the cerebellum using Rp-cAMPS completely prevents acquisition. However, neither blocking beta-adrenergic receptors nor blocking PKA activation significantly interferes with performance of established conditioned responses when administered after the learned response is formed.

Mecamylamine reversal by nicotine and by a partial alpha7 nicotinic acetylcholine receptor agonist (GTS-21) in rabbits tested with delay eyeblink classical conditioning.

The aim of this experiment was to investigate the effects of nicotinic acetylcholine receptor (nAChR) agonism and antagonism on learning. Eyeblink classical conditioning (750ms delay procedure) was tested for 15 daily sessions in a total of 82 young rabbits: 58 rabbits were tested in the paired procedure when the conditioned stimulus (CS) was always followed by the unconditioned stimulus (US), and 24 rabbits were tested in the explicitly unpaired procedure in which CS and US presentations were independent. We used the nAChR agonists nicotine and GTS-21 (a selective alpha7 nAChR partial agonist that antagonizes alpha4beta2 nAChRs) and the relatively nonselective nAChR antagonist, mecamylamine. Groups of young rabbits were injected with 0.5mg/kg mecamylamine alone and in combination with two doses of nicotine or GTS-21 and compared to vehicle-treated rabbits. Explicitly unpaired control groups received vehicle, mecamylamine plus the highest nicotine dose, or mecamylamine plus the highest GTS-21 dose. Both GTS-21 and nicotine reversed the deleterious effect of mecamylamine on the acquisition of conditioned responses. Combinations of GTS-21 or nicotine and mecamylamine did not cause sensitization or habituation in the unpaired condition. Reversal of mecamylamine-induced learning deficits by nicotine and GTS-21 suggests that nAChR agonists may have efficacy in ameliorating deficits caused by the loss of some types of nAChRs in diseases such as AD.

Disrupted retention of the classically conditioned nictitating membrane response in the aluminum-intoxicated rabbit using electrical stimulation of the brain as a conditioned stimulus.

Rabbits were classically conditioned to emit an eyeblink conditioned response (CR) to electrical stimulation (ESB) of the medial geniculate nucleus (MGN) paired with a corneal air puff until they attained a criterion of two consecutive days of greater than 90% CRs. They then received intraventricular injections of 1% AlCl3, HCL, or normal saline. Ten days postinjection, each animal underwent a retention test consisting of 50 ESB alone presentations. Whereas all saline and HCL animals gave at least 90% CRs during retention, no aluminum rabbit emitted more than 30% CRs. Considered with the results of previous work, these data suggest that aluminum-induced neurofibrillary degeneration disrupts retention of the CR by affecting central associative processes.