The development of olfactory preferences for artificial odors briefly experienced by the precocial spiny mouse young

Groups of pups, aged 2, 4, 6, 12, 16, and 18 days of the precocial murid rodent spiny mouse (Acomys cahirinus), were each exposed for 1.5 h to one of the experimental odors, cinnamon or cumin, present in the surrounding environment. The odors were not contingent upon home-cage odors or any other reinforcers. This simple exposure produced a strong preference for the exposed odor in multichoice preference tests carried out 48 h after the exposure in pups from groups aged 2 through 16 days. Older pups exposed to cinnamon or cumin at Day 18 did not show a significant preference for the familiar odor. Both experimental odors used were found to be neutral for control experimentally naive pups within a tested age span. These results point at a stimulus familiarity, rather than associative learning in the development of olfactory preferences in young spiny mouse pups.     

Unilateral olfactory conditioning in 6-day-old rat pups

The potential that early olfactory learning might be laterally organized in the brain was investigated in 6-day-old rats. This hypothesis is based on the finding that the commissural systems that subserve bilateral olfactory communication do not mature until the second week of postnatal life. Pups were trained with pairings of cedar odor and intraoral infusions of milk while one nostril was occluded. Animals expressed a conditioned orientation towards cedar if tested with the trained nostril open. No such conditioning was observed if the untrained nostril was open during testing. Further, when individual pups received cedar odor/milk pairings with one nostril open and orange odor/milk pairings with the other open, they expressed a conditioned preference for orange when tested with the orange-trained nostril open, and a preference for cedar when tested with the cedar-trained nostril open. Classically conditioned oral responses (mouthing) also appeared to be lateralized. However, no such unilateral conditioning occurred with respect to behavioral activation, which is also conditioned in this paradigm. Increases in activity to the odor CS were observed regardless of whether the trained or untrained nostril was open during testing. These results suggest that in developing rodents, olfactory memories may be partly represented in structures that can be unilaterally accessed during training and testing. They provide a starting point for isolation of neural substrates of the olfactory conditioning process.     

Behavioral modulation induced by food odor aversive conditioning and its influence on the olfactory responses of an oscillatory brain network in the slug Limax marginatus

We compared behaviorally and physiologically the olfactory responses of slugs (Limax marginatus) that had been subjected to aversive, appetitive, or unpaired training with food odors (carrot or cucumber). In the aversive training, the slugs were exposed to the food odor as a conditioned stimulus (CS), and then quinidine sulfate solution as an unconditioned stimulus (UCS) was immediately applied to the lip of the slugs. This training caused a decrease in preference level for the CS. The unpaired training, in which the CS and the UCS were presented to the slugs with a 5-min interval, induced no change in the preference level for the CS. In the appetitive training, the slugs were allowed to eat the CS odor source without UCS application. When we used nonstarved slugs, it was found that the preference level for the CS increased upon the appetitive training. These results indicate that each training changed the preference for the odors in a characteristic manner. In the physiological experiments, we used brain-inferior tentacular nose preparations isolated from slugs and investigated the olfactory responses of the oscillations in the local field potential (LFP) of the procerebral (PC) lobe. We found that odor presentation induced various types of changes in the LFP oscillation frequency, although the rate of occurrence of the frequency modulation differed between odors used in the aversive and the unpaired training (aversive-conditioned and unpaired odors). The aversive-conditioned odors induced a decrease in the oscillatory frequency. Unpaired odors did not change it. Moreover, odors used in the appetitive training (appetitive-conditioned odors) induced an increase in the frequency. Thus, it was considered that those modulations of PC lobe oscillatory activity were independent of odor and reflected learned preference for odors.     

Odor-aversion learning and retention span in neonatal mouse pups

One hundred and sixty-four litters of Swiss CD-1 random-bred mice were used to assess learning and retention capacities during the first postnatal week. In Experiment 1, whole 7-day litters were exposed for 65 min to commercial extracts of either mint or lemon sprinkled over wood shavings. Five minutes after the beginning of the exposure, half of the litters were injected ip with the illness-inducing agent lithium chloride (LiCl; 0.20 M, 2% of body weight); the other half was treated with saline solution (8% NaCl). On Postnatal Day 10, the animals were singly introduced in a warmed arena for a 180-s preference test, and the time spent in the mint- and lemon-scented areas of the apparatus was recorded. When compared with saline-injected pups, mice that experienced lemon-LiCl pairings showed a significant aversion for the lemon-scented area, while the mint aversion in the mint-LiCl group just missed statistical significance. Three additional control groups (unhandled on Day 7, or only LiCl- or saline-injected) did not show significant preferences for either the mint or the lemon odor. In Experiment 2, litters of 3, 5, or 7 days were similarly exposed to lemon-scented shavings for either 5 or 20 min, injected with LiCl or saline, and then exposed for an additional 60 min to the shavings. On Postnatal Day 10, tests like those of Experiment 1 showed a significant odor-aversion in animals conditioned on Day 7, but not in those conditioned on Day 3 or 5. In Experiment 3, 3- and 5-day old pups were exposed to lemon odor-LiCl or -NaCl pairings, and tested for aversion after 3 or 7 days (CS duration 5 min before injection and either 30 or 60 min after injection). Only when the conditioning-testing interval was limited to 3 days did LiCl-injected groups show a significant aversion, which did not depend on duration of CS exposure.     

pCREB in the neonate rat olfactory bulb is selectively and transiently increased by odor preference-conditioned training

Early olfactory preference learning in rat pups occurs when novel odors are paired with tactile stimulation, for example stroking. cAMP-triggered phosphorylation of cAMP response element binding protein (pCREB) has been implicated as a mediator of learning and memory changes in various animals (Frank and Greenberg 1994). In the present study we investigate whether CREB is phosphorylated in response to conditioned olfactory training as might be predicted given the proposed role of the phosphorylated protein in learning. On postnatal day 6, pups were trained for 10 min using a standard conditioned olfactory learning paradigm in which a conditioned stimulus, Odor, was either used alone or paired with an unconditioned stimulus, Stroking (using a fine brush to stroke the pup). In some instances stroking only was used. The pups were sacrificed at 0, 10, 30, or 60 min after the training. Using Western blot analysis, we observed that the majority of olfactory bulbs in conditioned pups (Odor + Stroking) had a greater increase in pCREB activation at 10 min after training than pups given nonlearning training (Odor only or Stroking only). The phosphorylated protein levels were low at 0 min and at 60 min after training. This is in keeping with the slightly delayed and short-lived activation period for this protein. The localization of pCREB increases within the olfactory bulb as seen by immunocytochemistry. Naive pups were not exposed to odor or training. There was a significantly higher level of label in mitral cell nuclei within the dorsolateral quadrant of the bulb of pups undergoing odor-stroke pairing. No significant differences were observed among nonlearning groups (Naive, Odor only, or Stroking only) or among any training groups in the granule or periglomerular cells of the dorsolateral region. The localized changes in the nuclear protein are consistent with studies showing localized changes in the bulb in response to a learned familiar odor. The present study demonstrates that selective increases in pCREB occur as an early step following pairing procedures that normally lead to the development of long-term olfactory memories in rat pups. These results support the hypothesized link between pCREB and memory formation.     

Second-order conditioning in Drosophila

Associative conditioning in Drosophila melanogaster has been well documented for several decades. However, most studies report only simple associations of conditioned stimuli (CS, e.g., odor) with unconditioned stimuli (US, e.g., electric shock) to measure learning or establish memory. Here we describe a straightforward second-order conditioning (SOC) protocol that further demonstrates the flexibility of fly behavior. In SOC, a previously conditioned stimulus (CS1) is used as reinforcement for a second conditioned stimulus (CS2) in associative learning. This higher-order context presents an opportunity for reassessing the roles of known learning and memory genes and neuronal networks in a new behavioral paradigm.     

Neonatal ventral hippocampus lesions disrupt extra-dimensional shift and alter dendritic spine density in the medial prefrontal cortex of juvenile rats

Recent data showed that neonatal ventral hippocampus (VH) lesions, an approach used to model schizophrenia symptoms in rodents, produce premature deficits of working memory believed to be associated with early medial prefrontal cortex (mPFC) maldevelopment. This experiment expands the investigation of mPFC integrity in juvenile rats with neonatal VH lesions by assessing behavioral flexibility and dendritic spine density. Sixteen Sprague-Dawley male pups received bilateral microinjections of ibotenic acid in the VH or SHAM surgery on postnatal day (PND) 6. On PND 29 and 30, rats were subjected to a spatial shift task in a cross-maze; an attentional set-shifting task was then administered on two consecutive days, between PND 33 and PND 35. Rats were sacrificed at PND 36 and dendritic spine density in the mPFC was assessed using Golgi-Cox staining procedure. Results revealed impaired extra-dimensional shift in VH-lesioned rats and inconsistent reversal discrimination outcomes. Although lesioned animals displayed intact performance in the spatial shift, rates of perseverative responses were higher than normal in this task. Neonatal VH damage resulted in lower dendritic spine density in the mPFC than measured in control brains; however, no significant correlation was found between this outcome and behavioral data. Juvenile morphological and cognitive perturbations are consistent with the early emergence of mPFC anomalies following neonatal VH lesions. Results are discussed in relation with potential common mechanisms linking pre- and post-pubertal onsets of behavioral dysfunction.     

Olfactory stimulation induces filial preferences for huddling in rat pups.

Rat pups of all ages huddle with conspecifics, but the senosory control of contact behavior changes ontogenetically. Thermal cues control huddling until about Day 15, at which time species' odors become the dominant stimulus. The present experiments indicate that the filial response to conspecifics is dependent on olfactory experience. A synthetic chemical scent was added to the smells of the dam from Day 1 to Day 20 postpartum. Standardized videographic tests were used to assess the development of huddling preference. Preferences for nest-typical smells emerged by Day 15 in pups from both scented and nonscented litters. Pups from scented nests preferred to huddle with a scented stimulus rat, whereas control pups preferred a nonadulterated rat stimulus. Additional testing indicated that the affiliative preferences were specific to rearing odor and were not based on decreased aversion to test scents, or on disrupted olfactory discrimination. The ontogeny of species-typical contact behavior is discussed in terms of the induction of a perceptual preference that is based on early odor stimulation.     

Interactions between perinatal and neonatal associative learning defined by contiguous olfactory and tactile stimulation

Tactile stimulation of the neonate, as performed by the mother during and after delivery, has been described as an effective unconditioned stimulus during early ontogeny (Leon, 1987; Ronca & Alberts, 1994). The present experiments examined the interaction between perinatal and neonatal learning determined by the explicit association between alcohol odor and vigorous body stimulation of the perinatal organism. In Experiment 1, rat fetuses were exposed to either alcohol or saline 10 min prior to cesarean delivery. The alcohol administration procedure here employed was sufficient to provide sensory contamination of the amniotic fluid but avoid fetal alcohol intoxication. Pups in the two prenatal treatments later experienced the smell of alcohol, tactile stimulation, or both stimuli explicitly paired or unpaired. Other postnatal groups were composed of pups that had no explicit experience with either experimental stimulus. Pups subjected to alcohol odor in utero displayed more overall motor activity in response to that odor than saline controls. The increased motor responses were further potentiated in pups that experienced additional postnatal alcohol odor paired with tactile stimulation. In Experiment 2, pups were exposed to alcohol in the amniotic fluid 10 or 30 min prior to birth. As previously demonstrated the memory acquired in utero appears highly dependent upon contingency between exposure to this particular scent and delivery procedures. Pups in both prenatal treatment groups were then exposed to alcohol odor paired or unpaired with tactile stimulation. Some control animals received no further experience with either stimuli. Those pups exposed to alcohol odor paired with tactile stimulation both pre- and postnatally later showed maximum motor activity elicited by the odor of alcohol. The results support the notion of fetal associative learning comprising alcohol's chemosensory cues and behaviorally activating stimuli. Furthermore, the conditioned response under analysis is potentiated whenever neonates are reexposed to contingent presentations of the elements that defined the original associative memory.     

Acute alcohol intoxication paired with aversive reinforcement: ethanol odor as a conditioned reinforcer in rat pups

Previous research has suggested that infant rats process ethanol sensory properties during acute alcohol intoxication. The present study was designed in order to examine if alcohol odor could act as an aversive conditioned stimulus after the organism experiences the state of intoxication paired with nociceptive stimulation (footshock). In a first experiment 11-day-old pups received intragastric alcohol administration (1.5 g/kg). At different postabsorptive intervals footshock was presented (0-30, 30-60, 60-90, or 90-120 min). An explicitly unpaired control group which experienced footshock prior to the state of intoxication was also employed. All animals were subsequently tested in terms of alcohol intake and ethanol locational odor preferences. Both assessments indicated that pups which were exposed to the unconditioned peripheral stimulus 30-60 min after receiving ethanol expressed strong alcohol aversions. In a second experiment pups were exposed to footshock during this postabsorptive interval. Twenty four hours later, pups experienced ambient ethanol odor paired with soft or rough texture surfaces. Differential texture aversions were registered in experimental animals when compared with controls which suffered the state of intoxication explicitly unpaired with footshock, or unpaired presentations of ethanol odor and the tactile stimuli under consideration. These results appear to support the hypothesis concerning orosensory processing during an acute state of intoxication. Additionally it seems that the hedonic value of sensory attributes of this drug varies as a function of associative processes occurring during such a state.     

Odor preference learning and memory modify GluA1 phosphorylation and GluA1 distribution in the neonate rat olfactory bulb: testing the AMPA receptor hypothesis in an appetitive learning model

An increase in synaptic AMPA receptors is hypothesized to mediate learning and memory. AMPA receptor increases have been reported in aversive learning models, although it is not clear if they are seen with memory maintenance. Here we examine AMPA receptor changes in a cAMP/PKA/CREB-dependent appetitive learning model: odor preference learning in the neonate rat. Rat pups were given a single pairing of peppermint and 2 mg/kg isoproterenol, which produces a 24-h, but not a 48-h, peppermint preference in the 7-d-old rat pup. GluA1 PKA-dependent phosphorylation peaked 10 min after the 10-min training trial and returned to baseline within 90 min. At 24 h, GluA1 subunits did not change overall but were significantly increased in synaptoneurosomes, consistent with increased membrane insertion. Immunohistochemistry revealed a significant increase in GluA1 subunits in olfactory bulb glomeruli, the targets of olfactory nerve axons. Glomerular increases were seen at 3 and 24 h after odor exposure in trained pups, but not in control pups. GluA1 increases were not seen as early as 10 min after training and were no longer observed 48 h after training when odor preference is no longer expressed behaviorally. Thus, the pattern of increased GluA1 membrane expression closely follows the memory timeline. Further, blocking GluA1 insertion using an interference peptide derived from the carboxyl tail of the GluA1 subunit inhibited 24 h odor preference memory providing causative support for our hypothesis. PKA-mediated GluA1 phosphorylation and later GluA1 insertion could, conjointly, provide increased AMPA function to support both short-term and long-term appetitive memory.     

啮齿动物主动母性行为动态改变的神经机制

主动母性行为是雌性哺乳动物在哺乳期内有效照料幼崽的一种动机行为, 对幼崽的生存和行为发展有重要影响。证据显示, 啮齿动物的主动母性行为会经历从产后早期的发动和维持到晚期衰退的动态改变, 反映了雌鼠对幼崽奖赏价值阶段性变化的适应; 这一过程不仅涉及分娩激素事件开启下丘脑内侧视前区(MPOA)-中脑腹侧被盖(VTA)-伏隔核(NA)-腹侧苍白球(VP)通路, 还需要杏仁核基底外侧核(BLA)和内侧前额皮层(MPFC)等脑区对上述通路进行实时调节。哺乳期主动母性行为动态改变及其神经机制的研究, 可以加深对行为进化和早期发展的认识, 也对人类母亲产后抑郁等临床问题的干预有借鉴意义。本文首先利用条件化位置偏好(CPP)任务的行为学证据分析幼崽奖赏价值与主动母性行为动态改变的关系; 然后系统阐述调控这一动态改变的神经机制; 最后对未来需要研究的一些重要问题或方向进行探讨。     

味觉厌恶性条件反射与条件反射性免疫抑制的研究

以环磷酰胺的注射为非条件刺激（ＵＣＳ）,糖精水的摄入为条件刺激（ＣＳ）,并以血白细胞和淋巴细胞数量及脾淋巴细胞的转化反应为免疫指标,糖精水的饮用量为行为指标,通过改变条件刺激与非条件刺激结合的次数来观察条件反射性厌恶行为与条件反射性免疫抑制的反应。结果表明不管是一次性的还是两次性的ＣＳ－ＵＣＳ结合训练都能使动物明显地建立起味觉厌恶性条件反射,然而条件反射性免疫抑制只在两次性ＣＳ－ＵＣＳ结合训练后才     

Simultaneous conditioning in honeybees (Apis mellifera).

Honeybees (Apis mellifera) were classically conditioned with odor as conditioned stimulus (CS), sucrose as unconditioned stimulus (US), and proboscis extension as response. The purpose of Experiment 1 (Ns = 26 and 27) was to look for facilitation of forward conditioning by CS-US overlap, but rapid conditioning without overlap left little room for improvement. In 2 further experiments, CS and US were simultaneous, and response to odor alone was measured in subsequent tests. In Experiment 2, a Simultaneous group (N = 25) responded more to the training odor than did an Unpaired control group (N = 25). In Experiment 3, a differentially conditioned Simultaneous group (N = 29) responded more to an odor paired with sucrose in training (S+) than to an odor presented alone (S-). The implications of the results for the problem of the role of amount of reward in honeybee learning are considered.     

以兔抗鼠淋巴细胞血清为非条件刺激的条件性免疫抑制

采用一种生物类免疫抑制剂-兔抗鼠淋巴血清（rabbit anti-rat lymphocyte serum,ALS）为非条件刺激（UCS）,糖精水为条件刺激（CS）,以双瓶给水法置于鼠笼前端饮用偏好侧。在一次性CS-UCS结合训练后,单独再次给予CS,使卵清蛋白（OVA）免疫过的大鼠表现出脾淋巴细胞对有丝分裂原PWM的增殖反应降低,血抗OVA抗体的总量及脾内抗OVA抗体生成细胞的减少,但动物未表现出条件性味觉厌恶的行为反应。这些结果表明条件性免疫抑制与味觉厌恶行为条件反射没有必然联系,并非是厌恶行为反应或情绪应激的伴随产物。UCS也并非必需具有感觉的毒副作用,条件性免疫抑制是脑高级神经活动调节免疫功能的结果。     

Effects of Early Hippocampal Lesions on Trace, Delay, and Long-Delay Eyeblink Conditioning in Developing Rats

The effects of bilateral hippocampal aspiration lesions on later acquisition of eyeblink conditioning were examined in developing Long-Evans rat pups. Lesions on postnatal day (PND) 10 were followed by evaluation of trace eyeblink conditioning (Experiment 1) and delay eyeblink conditioning (Experiment 2) on PND 25. Pairings of a tone conditioned stimulus (CS) and periocular shock unconditioned stimulus (US, 100 ms) were presented in one of three conditioning paradigms: trace (380 ms CS, 500 ms trace interval, 880 ms interstimulus interval [ISI]), standard delay (380 ms CS, 280 ms ISI), or long delay (980 ms CS, 880 ms ISI). The results of two experiments indicated that hippocampal lesions impaired trace eyeblink conditioning more than either type of delay conditioning. In light of our previous work on the ontogeny of trace, delay, and long-delay eyeblink conditioning (Ivkovich, Paczkowski, & Stanton, 2000) showing that trace and long-delay eyeblink conditioning had similar ontogenetic profiles, the current data suggest that during ontogeny hippocampal maturation may be more important for the short-term memory component than for the long-ISI component of trace eyeblink conditioning. The late development of conditioning over long ISIs may depend on a separate process such as protracted development of cerebellar cortex.     

早期剥夺所致大鼠抑郁样行为与海马BDNF mRNA表达变化

为探讨早期剥夺对大鼠行为与海马脑源性神经营养因子(BDNF)mRNA表达的影响,将新生的SD大鼠随机分为正常对照组与早期剥夺组,早期剥夺组在出生后1～14d每天孤养4h。监测体重,在出生后36?64天进行液体消耗试验,每周一次。12w龄时,进行穿梭箱试验,酶联免疫法检测海马BDNF含量,原位杂交法观察海马BDNF mRNA的表达。早期剥夺组大鼠体重显著低于正常对照组(p0.01),糖水摄入量与糖水偏爱显著低于正常对照组(p0.05或p0.01)。穿梭箱试验中,早期剥夺组大鼠在训练时的穿梭反应次数显著少于正常对照组(p0.05),在测试时,两组大鼠的被动逃避行为、主动逃避行为与逃避错误次数无显著差异。早期剥夺组大鼠海马BDNF含量、CA1区和CA3区BDNF mRNA表达显著低于正常对照组(p=0.05或p0.05)。提示早期剥夺可导致大鼠生长减慢,表现出快感缺失的抑郁样行为,但未诱导成年大鼠表现出明显的习得性无助倾向,早期剥夺能下调成年大鼠海马BDNFmRNA的表达。     

Context-dependent olfactory learning in an insect

We studied the capability of the cricket Gryllus bimaculatus to select one of a pair of odors and to avoid the other in one context and to do the opposite in another context. One group of crickets was trained to associate one of a pair of odors (conditioned stimulus, CS1) with water reward (appetitive unconditioned stimulus, US+) and another odor (CS2) with saline solution (aversive US, US-) under illumination and to associate CS1 with US- and CS2 with US+ in the dark. Another group of crickets received training of the opposite stimulus arrangement. At 1 d after the training for 3 d, the former group significantly preferred CS1 over CS2 under illumination but preferred CS2 over CS1 in the dark, and the latter group exhibited the opposite odor preference. The results of control experiments showed that the background light condition had no significant effects on memory formation or retrieval unless it was explicitly associated with US during training. Thus, the visual context affected learning performance only when crickets were requested to use it to disambiguate the meaning of CSs and to predict USs.     

Development switch in neural circuitry underlying odor-malaise learning

Fetal and infant rats can learn to avoid odors paired with illness before development of brain areas supporting this learning in adults, suggesting an alternate learning circuit. Here we begin to document the transition from the infant to adult neural circuit underlying odor-malaise avoidance learning using LiCl (0.3 M; 1% of body weight, ip) and a 30-min peppermint-odor exposure. Conditioning groups included: Paired odor-LiCl, Paired odor-LiCl-Nursing, LiCl, and odor-saline. Results showed that Paired LiCl-odor conditioning induced a learned odor aversion in postnatal day (PN) 7, 12, and 23 pups. Odor-LiCl Paired Nursing induced a learned odor preference in PN7 and PN12 pups but blocked learning in PN23 pups. 14C 2-deoxyglucose (2-DG) autoradiography indicated enhanced olfactory bulb activity in PN7 and PN12 pups with odor preference and avoidance learning. The odor aversion in weanling aged (PN23) pups resulted in enhanced amygdala activity in Paired odor-LiCl pups, but not if they were nursing. Thus, the neural circuit supporting malaise-induced aversions changes over development, indicating that similar infant and adult-learned behaviors may have distinct neural circuits.