The effect of medical clowning on reducing pain, crying, and anxiety in children aged 2-10 years old undergoing venous blood drawing--a randomized controlled study.

UNLABELLED: Recently, the utilization of medical clowns to reduce anxiety, stress, and even pain associated with hospitalization has become popular. However, the scientific basis of this benefit and outcome is scant. Venipuncture and IV cannulation are very common sources of pain in ill children. To reduce pain, one common approach is to apply a local anesthetic prior to the procedure. In the current study, we sought to compare the utilization of medical clowning in this process with two control groups: (1) local anesthetic cream (EMLA®, Astrazeneca, London, UK) applied prior to the procedure (active control) and (2) the procedure performed with neither clown nor EMLA (control group). We hypothesized that a medical clown will reduce pain, crying, and anxiety in children undergoing this procedure.Children aged 2-10 years who required either venous blood sampling or intravenous cannulation were recruited and randomly assigned to one of the three groups. Outcome measures consisted of the duration of the whole procedure (measured objectively by an independent observer), the duration of crying (measured objectively by an independent observer), subjective assessment of pain level (a commonly used validated scale), and anxiety level regarding future blood exams (by questionnaire). Analysis of variance (ANOVA) was used to compare between the groups. p < 0.05 was considered statistically significant.One hundred children participated. Mean age was 5.3 ± 2.5 years (range 2-10 years). The duration of crying was significantly lower with clown than in the control group (1.3 ± 2.0 vs 3.8 ± 5.4 min, p = 0.01). With EMLA, this duration was 2.4 ± 2.9 min. The pain magnitude as assessed by the child was significantly lower with EMLA than in the control group (2.9 ± 3.3 vs 5.3 ± 3.8, p = 0.04), while with clown it was 4.1 ± 3.5, not significant when compared with the other two modalities. Hence, duration of crying was shortest with clown while pain assessment was lowest with EMLA. Furthermore, with clown duration of cry was significantly shorter than in controls, but pain perception did not significantly differ between these groups. As expected, the duration of the entire process was shortest in the control group (5.0 ± 3.8 min), moderate with clown (19.3 ± 5.8 min), and longest with EMLA (63.2 ± 11.4 min, p < 0.0001 between all). Parental reporting of a beneficial effect was greater with clown than with EMLA (3.6 ± 0.8 vs 3.0 ± 1.1, p = 0.02). Parental assessment of child's anxiety related to future blood tests as evaluated by telephone the following day revealed that it was significantly lower with clown than in the control group or EMLA (2.6 ± 1.2 vs 3.7 ± 1.3 or 3.8 ± 1.6, p < 0.01 for both). CONCLUSIONS: Distraction by a medical clown is helpful in children undergoing blood tests or line insertion. Although pain reduction was better with EMLA, both duration of cry and anxiety were lower with a medical clown. These results strongly encourage and support the utilization of medical clowns while drawing blood in children.

Epigenetic-Imprinting Changes Caused by Neonatal Fasting Stress Protect From Future Fasting Stress.

Unfavourable nutritional conditions during the neonatal critical period can cause both acute metabolic disorders and severe metabolic syndromes in later life. These phenomena have been tightly related to the epigenetic modification controlling the balance between satiety and hunger in the hypothalamus. In the present study, we investigated epigenetic modification associated with both the fasting stress effects and the short-term resilience to fasting stress in the hypothalamic paraventricular nucleus (PVN) of chicks. Fasting for 24 h at 3 days of age (D) (i.e. D3) significantly increased global methylation at lysine 27 of histone 3 (H3K27) and its specific histone methyltransferase (HMT) expression level in the PVN. Because global methylation could not fully reveal the changes at specific genes, the regulation of the gene for brain-derived neurotrophic factor (Bdnf), which was recently also found to have an anorexigenic effect, was evaluated as a potential target. Chromatin immunoprecipitation assay analysis revealed that tri- (me3) and di-methylated (me2) H3K27 exhibited an instant (on D4 only) and latent increase (on both D11 and D41), respectively, at the putative promoter of Bdnf after 24 h of fasting on D3. This indicated that fasting could regulate energy-expenditure-related genes via modifying methylation at H3K27, which we suspected might be a protective mechanism for keeping the inner environment homeostatic. To test this hypothesis, a short-term repetitive fasting stress was applied to chickens, which were fasted for 24 h either on D10 only or on both D3 and D10. It was found that pre-existing fasting on D3 could induce a short-term fasting resilience, which rescued the reduction of Bdnf expression from future fasting on D10. We call this phenomenon the 'molecular memory', which was mainly conducted by HMTs and H3K27me2/me3 in the PVN. In conclusion, chicks respond to fasting with dynamic methylation at H3K27 in the PVN during the neonatal critical period. This allows the PVN to form a 'molecular memory', which keeps the individual inner environment homeostatic and resilient to future fasting over the short term.

Lateral ventricle injection of the protein synthesis inhibitor anisomycin impairs long-term memory in a spatial memory task.

Although protein synthesis inhibition has been shown to affect long-term memory in a wide variety of animal species, cases have been reported in which protein synthesis inhibition failed to affect memory consolidation [S. Wittstock, R. Menzel, Color learning and memory in honey bees are not affected by protein synthesis inhibition, Behav. Neural Biol., 62 (1994) 224-229.]. Most findings argue that the critical time for protein synthesis is during or immediately after training. However, other reports show a second time window, hours after training, where protein synthesis inhibition can cause amnesia [F.M. Freeman, S.P.R. Rose, A.B. Scholey, Two time windows of anisomycin-induced amnesia for passive avoidance training in the day-old chick, Neurobiol. Learn. Mem., 63 (1995) 291-295.][G. Grecksch, H. Matthies, Two sensitive periods for the amnesic effect of anisomycin, Pharmacol. Biochem. Behav., 12 (1980) 663-665.]. In this study, we addressed two questions: (1) Is protein synthesis essential for spatial memory? and (2) At what injection time window(s) will protein synthesis inhibition cause spatial memory amnesia? We report that bilateral intraventricular microinjection of anisomycin (Ani) impairs consolidation of long-term memory, in the hippocampal-dependent Morris water maze spatial memory task. Memory was impaired in a dose-dependent manner without affecting short-term memory. Spatial memory was affected only if Ani was injected 20 min before performing the task and not in any other time window before or after the behavioral test. The inhibition did not affect pre-existing memories or the capability to memorize once the effect of the inhibition diminished.

Kir6.2 oligoantisense administered into the globus pallidus reduces apomorphine-induced turning in 6-OHDA hemiparkinsonian rats.

ATP-sensitive inwardly rectifying potassium channels (KATPs) couple cell metabolism with its membrane potential. The best characterized KATP is the pancreatic KATP which is an heteromultimer of Kir6.2 and SUR1 protein subunits. KATPs are found in a variety of excitable cells, including neurons of the central nervous system. Basal ganglia (BG), especially in the substantia nigra (SN) reticulata and the globus pallidus (GP), have a high density of KATPs. Pharmacological modulation of the KATPs within the BG alters GABAergic activity and produces behavioural changes. However, the relatively high concentrations of drugs used might not have been entirely selective for the KATPs and may have acted at presynaptic nerve terminals as well as on the post-synaptic neurons. As an alternative means of examining the role of KATPs in regulating motor behavior, we used oligoantisense technology to diminish selectively Kir6.2 formation in the GP neurons. We then examined the effect of reduction in Kir6.2 expression on apomorphine-induced turning behavior in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the SN. Two weeks after injection of 6-OHDA, contralateral circling in response to apomorphine (0.25 mg/kg sc) was recorded. Kir6.2 antisense oligodeoxyribonucleotide (ODN) was then administered daily for 6 days into the GP ipsilateral to the 6-OHDA injection. Responses to apomorphine were then tested again and the animals killed to determine the effect of the antisense ODN on Kir6. 2 mRNA. Administration of Kir6.2 antisense ODN significantly attenuated apomorphine-induced contralateral turning and specifically reduced Kir6.2 mRNA in the injected GP. These results are consistent with pharmacological experiments which suggest that KATP channels in the GP are involved in motor responses to apomorphine in 6-OHDA lesioned rats, localizing the effects to the GP neurons, probably through modulation of the GABAergic system.

Low-dose spiral computed tomography for measuring abdominal fat volume and distribution in a clinical setting.

OBJECTIVES: Computed tomography (CT) has been used to measure body composition, however, a technique with reduced radiation exposure has not yet been introduced. This study tested a low-dose spiral CT technique on a phantom to determine its validity and reproducibility. The method was then applied for volume and distribution measurements in patients. DESIGN: Construction and measurement of a phantom followed by measurement of patients referred to CT for clinical indications. SETTING: Radiology Department, University Hospital. SUBJECTS: Twenty-four post-gastrectomy patients. INTERVENTION: A 22 cm phantom with a known amount of water and fat was scanned using high- and low-dose technique, standard and double table speed during a volumetric scan. The low-dose technique was implemented in the patient group. Total volume, total fat and four defined compartmental fat volumes in the truncal area were measured. RESULTS: The mean fat volume measured using the low-dose CT technique in the phantom was 0.2% above the actual fat content. The coefficient of variation for this method was 5%. By using low-dose, double speed instead of standard-dose technique, radiation exposure to the skin was decreased by more than 90% (equivalent to 4 mGy) of what is used in diagnostic imaging. The patient scans showed that no significant differences in BMI and total measured volume existed between female and male patients, but percent fat and percent subcutaneous fat were significantly larger in women (P = 0.006 and 0.002, respectively), as were percent intraabdominal and mediastinal fat in men (P = 0.002 and 0.003 respectively). CONCLUSIONS: Low-dose spiral CT accurately measures fat volume in vitro, and can be used in vivo for compartmental fat measurements.

Colonography using multislice CT.

Computed tomography (CT) represents the preferred imaging modality for imaging the large bowel when virtual endoscopic reconstructions are desired. Using the spiral acquisition technique, it has become possible to scan the entire abdomen within a single breathhold, however, slice thicknesses of 5 mm or more are necessary should the breathhold not last longer than 30-40 s. With the advent of multislice CT, contiguous 1-mm slices can be obtained through the entire abdomen while even shortening the breathhold to 25-30 s. The improved speed and spatial resolution of multislice CT results in remarkably sharp virtual reconstructions allowing detection of polyps with sizes less than 3 mm. The disadvantages must still be considered including a dataset consisting of up to 800 images representing a new challenge for postprocessing hard- and software.

CT colonography: data acquisition and patient preparation techniques.

The success of a virtual endoscopy is essentially dependent on the image quality of the corresponding 3-dimensional reconstructions. Before loading image data on a post-processing computer, certain prerequisites concerning the source data must be met. To carry out a CT colonography, the source data must be of good quality. High spatial resolution in all geometrical directions, continuous data acquisition without gaps, and artefact-free images are pivotal factors influencing source data. A generally applicable rule is that the size of the smallest detectable polyp is limited by the nominal slice thickness, emphasizing the ultimate importance of the initially chosen primary slice collimation. Furthermore, calculation of an endoluminal view is impossible without sufficient distension of the bowels. Thorough patient preparation that accommodates the technical circumstances for post-processing is also required for attaining a high sensitivity in polyp detection.

New insight in eggshell formation.

The matrix proteins that participate in crystalization fulfill important functions during the formation of the calcified tissues and contribute to the biomechanical properties of the mature product. We suggest that osteopontin (OPN) is part of an array of macromolecules synthesized and secreted by the cells adjacent to the mineralization front that self-assemble outside the cell and direct crystal formation. The OPN meets the theoretical requirements for involvement in the mineralization process. The phosphorylated residues of acidic phosphoprotein have been shown to exist in the protein as reactive monoesters that are available for interaction with other ions, among them crystal constituents such as calcium ions. In addition, sulfation of OPN was also found to be associated with mineralization of other tissues. In contrast to the calbindin gene, whose expression is dependent on the calcium flux, the regulation of OPN synthesis is at least in part dependent on the mechanical strain imposed by the resident egg. These results demonstrate the complexity of the regulation of the matrix genes governing eggshell formation.

Heat stress attenuates new cell generation in the hypothalamus: a role for miR-138.

The anterior hypothalamus (Ant Hyp) of the brain serves as the main regulator of numerous homeostatic functions, among them body temperature. Fine-tuning of the thermal-response set point during the critical postnatal sensory-developmental period involves neuronal network remodeling which might also be accompanied by alterations in hypothalamic cell populations. Here we demonstrate that heat stress during the critical period of thermal-control establishment interferes with generation of new cells in the chick hypothalamus. Whereas conditioning of the 3-day-old chicks under high ambient temperatures for 24h diminished the number of newborn cells in anterior hypothalamic structures 1 week after the treatment, mild heat stress did not influence the amount of new cells. Phenotypic analysis of these newborn cells indicated a predominant decrease in non-neuronal cell precursors, i.e. cells that do not express doublecortin (DCX). Furthermore, heat challenge of 10-day-old previously high-temperature-conditioned chicks abolished hypothalamic neurogenesis and significantly decreased the number of cells of non-neural origin. As a potential regulatory mechanism for the underlying generation of new cells in the hypothalamus, we investigated the role of the microRNA (miRNA) miR-138, previously reported by us to promote hypothalamic cell migration in vitro and whose levels are reduced during heat stress. Intracranial injection into the third ventricle of miR-138 led to an increase in the number of newborn cells in the Ant Hyp, an effect which might be partially mediated by inhibition of its direct target reelin. These data demonstrate the role of ambient temperature on the generation of new cells in the hypothalamus during the critical period of thermal-control establishment and highlight the long-term effect of severe heat stress on hypothalamic cell population. Moreover, miRNAs, miR-138 in particular, can regulate new cell generation in the hypothalamus.

MiR-138 promotes the migration of cultured chicken embryonic hypothalamic cells by targeting reelin.

Neuronal network remodeling during critical periods of sensory development might be accompanied by alterations in hypothalamic cell populations. MicroRNAs play a central role in regulating neuronal function, including neural stem cell proliferation, and neuronal migration, maturation and integration into viable circuits by modulating different mRNA targets. Here we investigated the role of miR-138 in cell proliferation and migration in a neuron-enriched hypothalamic cell culture prepared from chicks on embryonic day 16. Ectopic expression of miR-138 enhanced hypothalamic cell migration, but did not affect cell proliferation. As a potential mechanism for miR-138's effect on cell migration, we investigated reelin (Reln) as a direct target of miR-138. Luciferase reporter assay and Ago2-immunoprecipitation experiments confirmed direct binding of miR-138 to the Reln 3'-untranslated region. Ectopic miR-138 abolished Reln levels in hypothalamic cells and enhanced their migration, similar to Reln-antisense DNA. Furthermore, inhibition of Reln expression by miR-138 led to decreased phosphorylation level of the key component of Reln-regulated signaling cascades, Disabled 1. These findings describe miR-138 as a novel regulator of hypothalamic cell migration, acting at least in part via inhibition of Reln expression and leading to the inactivation of Reln signals.

Taste memory: the role of protein synthesis in gustatory cortex.

Application of the protein synthesis inhibitor anisomycin to the rat gustatory cortex before and during training impairs conditioned taste aversion (CTA) to saccharin. No behavioral impairment is observed if the inhibitor is applied to an adjacent cortical area or to one cortical hemisphere only. The consumption of saccharin and of total fluid, as well as behavioral recognition of saccharin, is not affected. Preexposure of rats to saccharin several days before training markedly inhibits CTA to that taste. Injection of anisomycin to the gustatory cortex immediately prior to the preexposure period attenuates the latent inhibition. These results suggest that protein synthesis in the gustatory cortex is required for normal acquisition of the memory of taste.

Modulation of protein tyrosine phosphorylation in rat insular cortex after conditioned taste aversion training.

Protein tyrosine phosphorylation is a major signal transduction pathway involved in cellular metabolism, growth, and differentiation. Recent data indicate that tyrosine phosphorylation also plays a role in neuronal plasticity. We are using conditioned taste aversion, a fast and robust associative learning paradigm subserved among other brain areas by the insular cortex, to investigate molecular correlates of learning and memory in the rat cortex. In conditioned taste aversion, rats learn to associate a novel taste (e.g., saccharin) with delayed poisoning (e.g., by LiCl injection). Here we report that after conditioned taste aversion training, there is a rapid and marked increase in tyrosine phosphorylation of a set of proteins in the insular cortex but not in other brain areas. A major protein so modulated, of 180 kDa, is abundant in a membrane fraction and remains modulated for more than an hour after training. Exposure of the rats to the novel taste alone results in only a small modulation of the aforementioned proteins whereas administration of the malaise-inducing agent per se has no effect. To the best of our knowledge, this is the first demonstration of modulation of protein tyrosine phosphorylation in the brain after a behavioral experience.

Memory and long-term potentiation (LTP) dissociated: normal spatial memory despite CA1 LTP elimination with Kv1.4 antisense.

Long-term potentiation (LTP) in the hippocampal slice preparation has been proposed as an in vitro model for long-term memory. However, correlation of LTP with memory in living animals has been difficult to demonstrate. Furthermore, in the last few years evidence has accumulated that dissociate the two. Because potassium channels might determine the weight of synapses in networks, we studied the role of Kv1.4, a presynaptic A-type voltage-dependent K+ channel, in both memory and LTP. Reverse transcription-PCR and Western blot analysis with specific antibodies showed that antisense oligodeoxyribonucleotide to Kv1.4 microinjected intraventricularly into rat brains obstructed hippocampal Kv1.4 mRNA, "knocking down" the protein in the hippocampus. This antisense knockdown had no effect on rat spatial maze learning, memory, or exploratory behavior, but eliminated both early- and late-phase LTP and reduced paired-pulse facilitation (a presynaptic effect) in CA1 pyramidal neurons without affecting dentate gyrus LTP. This presynaptic Kv1.4 knockdown together with previous postsynaptic Kv1.1 knockdown demonstrates that CA1 LTP is neither necessary nor sufficient for rat spatial memory.

Virtual endoscopy of the small bowel: phantom study and preliminary clinical results.

The purpose of this study was to determine the optimal scanning technique for lesion detection in a small bowel phantom and to evaluate the virtual endoscopy (VE) technique in patients. A small bowel phantom with a fold thickness of 7 mm and length of 115 cm was prepared with nine round lesions (3 x 1 mm, 2 x 2 mm, 2 x 3 mm, 2 x 4 mm). Spiral CT parameters were 7/7/4, 3/5/2, 3/5/1, 1.5/3/1 (slice thickness/table feed/reconstruction interval). VE was done using volume rendering technique with 1 cm distance between images and 120 degrees viewing angle. Two masked readers were asked to determine the number and location of the lesions. Seven patients underwent an abdominal CT during one breathhold after placement of a duodenal tube and filling of the small bowel with methyl cellulose contrast solution. VE images were compared with the axial slices with respect to detectability of pathology. With the 7/7/4 protocol only the 4-mm lesions were visualised with fuzzy contours. The 3/5/2 protocol showed both 4-mm lesions, one 3-mm lesion and one false positive lesion. The 3/5/1 protocol showed both 4-mm and both 3-mm (one uncertain) lesions with improved sharpness, and no false positive lesions. One 2-mm and one 1-mm lesion were additionally seen with the 1.5/3/1 protocol. Path definition was difficult in sharp turns or kinks in the lumen. In all patients, no difference was found between VE and axial slices for bowel pathology; however, axial slices showed 'outside' information that was not included in VE. We conclude that the 3/5/2 protocol may be regarded as an optimal compromise between lesion detection, coverage during one breathhold, and number of reconstructed images in patients; round lesions of 4 mm in diameter can be detected with high certainty.

[Virtual bronchoscopy]. / Virtuelle Bronchoskopie.

Flexible bronchoscopy represents a clinically well-established invasive diagnostic tool. Virtual bronchoscopies, calculated from thin-slice CT sections, allow astonishing immitations of reality although principal differences exist between both technologies: the fact that colour representation is artificial and concommitant interventions are impossible limits the clinical use of virtual bronchoscopy. However, its value increases when calculations can be attained within minutes due to technological advancements, and when virtually any chest CT is suitable for further postprocessing. Indications, findings and the clinical role of virtual bronchoscopy are discussed.

Na(+)-K(+)-ATPase gene expression in the avian eggshell gland: distinct regulation in different cell types.

The avian eggshell gland (ESG) is a tissue specialized in transporting the Ca(2+) required for eggshell formation and represents a unique biological system in which the calcification process takes place in a circadian fashion. With the use of RNA fingerprinting, a set of genes differentially induced at the time of calcification was detected, one of which was identified as the alpha(1)-subunit of Na(+)-K(+)-ATPase. The gene was expressed in a circadian manner in both cell types populating the ESG, but in different temporal patterns, suggesting distinct mechanisms of regulation. Ca(2+) flux and mechanical strain were found to regulate gene expression in the inner glandular epithelium and the pseudostratified epithelium facing the lumen, respectively. Mechanical strain also affected gene expression in cell layers facing the lumen in other parts of the oviduct. Only the alpha(1)-isoform, not the alpha(2)- or alpha(3)-isoform, of Na(+)-K(+)-ATPase was expressed in the ESG. In summary, we demonstrate that the alpha(1)-subunit Na(+)-K(+)-ATPase gene is expressed in different epithelial cell types in the ESG and is regulated by various mechanisms, which may reflect the disparity in the physiological roles of the cells in the process of eggshell formation.

Overexpression of urokinase-type plasminogen activator in transgenic mice is correlated with impaired learning.

Transgenic mice designated alpha MUPA overproduce in the brain murine urokinase-type plasminogen activator (uPA), an extracellular protease implicated in tissue remodeling. We have now localized, by in situ hybridization, extensive signal of uPA mRNA in the alpha MUPA cortex, hippocampus, and amygdala, sites that were not labeled in counterpart wild-type mice. Furthermore, biochemical measurements reveal a remarkably high level of enzymatic activity of uPA in the cortex and hippocampus of alpha MUPA compared with wild-type mice. We have used the alpha MUPA mice to examine whether the abnormal level of uPA in the cortex and the limbic system affects learning ability. We report that alpha MUPA mice perform poorly in tasks of spatial, olfactory, and taste-aversion learning, while displaying normal sensory and motor capabilities. Our results suggest that uPA is involved in neural processes subserving a variety of learning types.

Safe and easy power injection of contrast material through a central line.

Power-assisted injection of contrast material into an antecubital vein is commonly used in CT and has been proven superior to manual injection. Power-assisted injection through a central line bares the risk of rupturing the line because manual control over the pressure applied by the power injector is lacking. We present a simple safety device which allows manual control of the pressure by means of an interposed three-way stopcock combined with a small syringe for pressure equalization.

Spatial learning induced changes in expression of the ryanodine type II receptor in the rat hippocampus.

Calcium signaling critical to neural functions is mediated through Ca(2+) channels localized on both the plasma membrane and intracellular organelles such as endoplasmic reticulum. Whereas Ca(2+) influx occurs via the voltage- or/and ligand-sensitive Ca(2+) channels, Ca(2+) release from intracellular stores that amplifies further the Ca(2+) signal is thought to be involved in more profound and lasting changes in neurons. The ryanodine receptor, one of the two major intracellular Ca(2+) channels, has been an important target for studying Ca(2+) signaling in brain functions, including learning and memory, due to its characteristic Ca(2+)-induced Ca(2+) release. In this study, we report regional and cellular distributions of the type-2 ryanodine receptor (RyR2) mRNA in the rat brain, and effects of spatial learning on RyR2 gene expression at mRNA and protein levels in the rat hippocampus. Using in situ hybridization, reverse transcription polymerase chain reaction, and ribonuclease protection assays, significant increases in RyR2 mRNA were found in the hippocampus of rats trained in an intensive water maze task. With immunoprecipitation and immunoblotting, protein levels of RyR2 were also demonstrated to be increased in the microsomal fractions prepared from hippocampi of trained rats. These results suggest that RyR2, and hence the RyR2-mediated Ca(2+) signals, may be involved in memory processing after spatial learning. The increases in RyR2 mRNA and protein at 12 and 24 h after training could contribute to more permanent changes such as structural modifications during long-term memory storage. Zhao, W., Meiri, N., Xu, H., Cavallaro, S., Quattrone, A., Zhang, L., Alkon, D. A. Spatial learning induced changes in expression of the ryanodine type II receptor in the rat hippocampus.

Late memory-related genes in the hippocampus revealed by RNA fingerprinting.

Although long-term memory is thought to require a cellular program of gene expression and increased protein synthesis, the identity of proteins critical for associative memory is largely unknown. We used RNA fingerprinting to identify candidate memory-related genes (MRGs), which were up-regulated in the hippocampus of water maze-trained rats, a brain area that is critically involved in spatial learning. Two of the original 10 candidate genes implicated by RNA fingerprinting, the rat homolog of the ryanodine receptor type-2 and glutamate dehydrogenase (EC 1.4.1.3), were further investigated by Northern blot analysis, reverse transcription-PCR, and in situ hybridization and confirmed as MRGs with distinct temporal and regional expression. Successive RNA screening as illustrated here may help to reveal a spectrum of MRGs as they appear in distinct domains of memory storage.