Protein synthesis in axons and terminals: significance for maintenance, plasticity and regulation of phenotype. With a critique of slow transport theory.

This article focuses on local protein synthesis as a basis for maintaining axoplasmic mass, and expression of plasticity in axons and terminals. Recent evidence of discrete ribosomal domains, subjacent to the axolemma, which are distributed at intermittent intervals along axons, are described. Studies of locally synthesized proteins, and proteins encoded by RNA transcripts in axons indicate that the latter comprise constituents of the so-called slow transport rate groups. A comprehensive review and analysis of published data on synaptosomes and identified presynaptic terminals warrants the conclusion that a cytoribosomal machinery is present, and that protein synthesis could play a role in long-term changes of modifiable synapses. The concept that all axonal proteins are supplied by slow transport after synthesis in the perikaryon is challenged because the underlying assumptions of the model are discordant with known metabolic principles. The flawed slow transport model is supplanted by a metabolic model that is supported by evidence of local synthesis and turnover of proteins in axons. A comparison of the relative strengths of the two models shows that, unlike the local synthesis model, the slow transport model fails as a credible theoretical construct to account for axons and terminals as we know them. Evidence for a dynamic anatomy of axons is presented. It is proposed that a distributed "sprouting program," which governs local plasticity of axons, is regulated by environmental cues, and ultimately depends on local synthesis. In this respect, nerve regeneration is treated as a special case of the sprouting program. The term merotrophism is proposed to denote a class of phenomena, in which regional phenotype changes are regulated locally without specific involvement of the neuronal nucleus.

Excess DNA in the nuclei of the subesophageal region of octopus brain.

Cytophotometric analyses of Feulgen-stained nuclei present in homogenates of vertical and subesophageal lobes of octopus brain have shown that the latter region contains larger nuclei with up to several times the amount of DNA present in vertical nuclei. No obvious relationship was found between DNA content and nuclear size. Except for a rather small minority, nuclei of the vertical lobe have a uniform size and the expected diploid amount of DNA. These parameters are not substantially dependent on body weight. In contrast, the DNA content of subesophageal nuclei increases progressively with body weight. The amount of DNA found in subesophageal nuclei does not seem to be a simple multiple of the diploid or haploid value.

Protein-synthesizing machinery in the axon compartment.

Contrary to the prevailing view that the axon lacks the capacity to synthesize proteins, a substantial body of evidence points to the existence of a metabolically active endogenous translational machinery. The machinery appears to be largely localized in the cortical zone of the axon, where, in vertebrate axons, it is distributed longitudinally as intermittent, discrete domains, called periaxoplasmic plaques. Studies, based on translation assays and probes of RNA transcripts in axon models such as the squid giant axon and selected vertebrate axons, provide evidence of locally synthesized proteins, most of which appear to be constituents of the slow axoplasmic transport rate groups. Metabolic and molecular biological findings are consistent with the view that the synthesis of proteins undergoing local turnover in the axonal compartment of macroneurons depends on the activity of an endogenous translational machinery. The documented presence of a metabolically active machinery in presynaptic terminals of squid photoreceptor neurons is also described. Finally, potential sources of axoplasmic RNAs comprising the machinery, which may include the ensheathing cell of the axon, as well as the cognate cell body, are also discussed.

Visualization of differential neurofilament phosphorylation in the pre- and postsynaptic axoplasm of the squid giant synapse: an electron spectroscopic study.

When inelastically scattered electrons with an energy loss specific for interaction with phosphorus atoms were used for visualization of sections of squid axons, bead-like domains of elongated proteins, presumably neurofilaments, exhibited distinct phosphorus signals. A marked asymmetry of these phosphorus signals was detectable between the pre- and the postsynaptic cytoskeleton of the giant synapse. Signals were very numerous and intense in the presynaptic terminal, while rare and weak in the postsynaptic axoplasm. The giant axon revealed a delayed appearance of phosphorus signals in its course from the cell bodies in the giant fibre lobe to its exit from the stellate ganglion. Numerous and intense phosphorylation signals were evident only in the peripheral giant axon. Asymmetry in the distribution of phosphorus signals between pre- and postsynaptic axoplasm paralleled differences in Ca(2+)-buffering mechanisms, as shown in a previous study. In the presynaptic terminal patterns of phosphorus signals correlated with precipitates which had formed after intra-axonal injection of calcium. Our observations suggest a role of phosphorylated neurofilaments in binding of calcium in the squid synapse.

Ribosomes and polyribosomes are present in the squid giant axon: an immunocytochemical study.

Ribosomes and polyribosomes were detected by immuno-electron microscopy in the giant axon and small axons of the squid using a polyclonal antibody against rat brain ribosomes. The ribosomal fraction used as antigen was purified by ultracentrifugation on a sucrose density gradient and shown to contain ribosomal RNAs and native ribosomes. The polyclonal antibody raised in rabbits reacted with at least ten proteins on immunoblots of purified rat brain ribosomes as well as with a set of multiple ribosomal proteins prepared from the squid giant fiber lobe. Immunoreactions were performed on cryostat sections of the stellate nerve cut at a distance of more than 3 cm from the stellate ganglion, using pre-embedding techniques. Ribosomes and polyribosomes were identified within the giant axon and small axons using electron microscopic methods, following binding of peroxidase-conjugated anti-rabbit IgG secondary antibody. Polysomes were more frequently localized in peripheral axoplasm, including the cortical layer of the giant axon, and were generally associated with unidentified cytoskeletal filaments or with dense matrix material. The immunochemical demonstration of ribosomes and polyribosomes in the giant axon and small axons of the squid confirms similar observations in the squid and the goldfish obtained with the method of electron spectroscopic imaging, and strongly supports the view that a local system of protein synthesis is present in axons. The immunochemical method here described offers an alternative tool for the selective identification of ribosomes, and is likely to prove of value in the analyses of other axonal systems.

Induction of cerebellar hsp72 in rats learning a two-way active avoidance task.

The involvement of brain heat shock proteins in learning was examined by Western analyses in rats trained for an active avoidance task, and in passive and active controls. Expression of the constitutive hsp73 was intense in brain, liver, and kidney of all rats. Conversely, expression of the inducible hsp72 occurred in the cerebellum of most trained rats, but not in passive or active controls. Significant correlations were present between avoidances and cerebellar scores determined 8 h after training. Induction of hsp72 may therefore be attributed to learning in the cerebellum, while in other brain regions, liver and kidney stress-related stimuli may play a prevalent role.

Messenger RNAs in synaptosomal fractions from rat brain.

Synaptosomal fractions from rat brain have been analyzed with semi-quantitative RT-PCR methods to determine their content of mRNAs coding for presynaptic, postsynaptic, glial, and neuronal proteins. Each mRNA was determined with reference to the standard HPRT mRNA. In our analyses, mRNAs were considered to be associated with synaptosomes only if their relative amounts were higher than in microsomes prepared in a polysome stabilizing medium, rich in Mg(++) and K(+) ions, or in the homogenate. According to this stringent criterion, the following synaptosomal mRNAs could not be attributed to microsomal contamination and were assumed to derive from the subcellular structures known to harbor their translation products, i.e. GAT-1 mRNAs from presynaptic terminals and glial processes, MAP2 mRNA from dendrites, GFAP mRNA from glial processes, and TAU mRNA from neuronal fragments. This interpretation is in agreement with the involvement of extrasomatic mRNAs in local translation processes.

Rapid important paper Messenger RNA in squid axoplasm.

Using a translation assay we have shown that the axoplasm of the squid giant axon contains significant amounts of mRNA coding for a heterogeneous group of prot sets of proteins specified by glial and neuronal perikaryal mRNA. Messenger RNA is associated with the "microsomal" fraction of the axoplasm. The possible involvement of axoplasmic mRNA in protein synthesis remains to be ascertained. It is known that axoplasmic proteins are synthesized by the isolated giant axon, presumably by the surrounding glia cells.

Environmental stimuli modulate the circadian rhythm of [3H-methyl]thymidine incorporation into brain DNA of male rats.

A circadian rhythm of DNA synthesis is present in most organs of adult mammals, with peak levels (acrophase) generally in the rest period. We have recently reported that in the rat brain the acrophase of the rhythm of thymidine incorporation into DNA occurs on the contrary during the active period. To determine whether the brain waking activity was exerting a modulatory action we measured the circadian rhythm of incorporation in the brain and kidney of young adult male rats housed in conditions of sensory and social enrichment or impoverishment for four days. Biochemical and autoradiographic data show that the brain rhythm persists in the enriched condition, but is abolished in the impoverished condition. On the other hand, the rhythm of incorporation in the kidney is maintained in both conditions. These results suggest that the permanence of the brain oscillation is selectively dependent on the complexity of the sensory and social stimulation.

Post-trial sleep sequences including transition sleep are involved in avoidance learning of adult rats.

High resolution computerized EEG analyses, and behavioral observations were used to identify slow wave sleep (SS), paradoxical sleep (PS) and transition sleep (TS) in adult male Wistar rats exposed to a session of two-way active avoidance training. Of the four sleep sequences that could be identified, two included TS (SS-->TS-->W and SS-->TS-->PS), while the other two did not (SS-->W and SS-->PS). Comparison of post-trial sleep variables between fast learning rats (FL, reaching criterion in the training session), slow learning rats (SL, reaching criterion in the retention session the following day), and non learning rats (NL, failing to reach criterion) indicated that the total amounts of SS, TS and PS of the SS-->TS-->PS sequence was markedly higher in FL rats than in SL rats. In addition, in comparison with the corresponding baseline period, the average duration and total amount of SS and TS episodes of the SS-->TS-->PS sequence increased in FL rats, while the number of SS-->TS-->W sequences decreased. On the other hand, the average duration of SS episodes increased in the SS-->TS-->W and SS-->W sequences of SL rats, and in the SS-->W and SS-->TS-->PS sequences of NL rats. Correlative analyses between number of avoidances and post-trial sleep variables demonstrated that avoidances were directly correlated with the duration of SS episodes of the SS-->TS-->PS sequence and with the duration of TS episodes of the SS-->TS-->W sequence, but inversely correlated with the number and amount of SS episodes of the SS-->W sequence and with the duration and amount of SS episodes of the SS-->PS sequence. On the whole, the data supported the view that TS-containing sleep sequences are involved in long-term storage of novel adaptive behavior, while sleep sequences lacking TS are involved in the maintenance of innate behavioral responses.

The sequential hypothesis of the function of sleep.

In addition to modulatory roles concerning bodily functions, sleep is assumed to play a main processing role with regard to newly acquired neural information. Elaboration of memory traces acquired during the waking period is assumed to require two sequential steps taking place during slow wave sleep (SWS) and eventually during paradoxical sleep (PS). This view is suggested by several considerations, not the least of which concerns the natural sequence of appearance of SWS and PS in the adult animal. While the involvement of PS in memory processing is well documented, the involvement of SWS is supported by the results of baseline and post-trial EEG analyses carried out in rats trained for a two-way active avoidance task or a spatial habituation task. Together with control analyses, these data indicate that the marked increase in the average duration of post-trial SWS episodes does not reflect the outcome of non-specific contingent factors, such as sleep loss or stress, but is related to memory processing events. Several considerations have furthermore led to the proposal that, during SWS, after a preliminary selection step, the first processing operation consists in the weakening of non-adaptative memory traces. The remaining memory traces would then be stored again under a better configuration during the ensuing PS episode. This view is in agreement with several relevant features of sleep, including the EEG waveforms prevailing during SWS and PS, as well as the ontogenetic sequence of appearance of SWS and PS. Some theoretical considerations on the role of sleep are also in agreement with the sequential hypothesis. More recent data indicate that the learning capacity of rats is correlated with several baseline EEG features of sleep and wakefulness. They include the average duration of PS episodes and of SWS episodes followed by wakefulness (longer in fast learning rats), and the waking EEG power spectrum of fast learning rats whose output is more balanced in the frequency range below 10 Hz than in slow learning and in non-learning rats. Additional EEG data suggest that fast learning rats may accomplish 'on line' processing of newly acquired information according to a sequence of events not dissimilar from the one proposed by the sequential hypothesis.

Distributed changes in rat brain DNA synthesis with long-term habituation and potentiation of the perforant path-granule cell synapse.

The involvement of brain deoxyribonucleic acid (DNA) synthesis in adaptive neural events was studied in the adult rat during long-term habituation (LTH) or potentiation (LTP) of the perforant path-granule cell synapse. Male Long-Evans rats were given 50 muCi [3H]thymidine intraventricularly under urethane anesthesia. Soon thereafter, field excitatory postsynaptic potential (EPSP) slope and population spike were monitored from the right dentate gyrus before and at various times (5, 10, 15, 60 min) following the delivery to the ipsilateral perforant bundle of a low frequency (LFS: 1.0 Hz, 160 s) or a high-frequency train (HFS: 400 Hz, 200 ms), repeated once after 5 min. Unstimulated implanted rats served as controls. DNA synthesis was evaluated by the incorporation of the radioactive precursor into DNA of several brain areas at the end of a 1 h incorporation period. In CA1, LTH and LTP increased DNA synthesis by 30% on the stimulated side. In the entorhinal cortex, LTH but not LTP increased DNA synthesis (by 30%) on the stimulated side. Conversely, in the frontal cortex, LTP but not LTH increased DNA synthesis (by 100%) on both sides. Long-lasting changes in synaptic efficacy covaried non-linearly with DNA synthesis in mono- and polysynaptically stimulated hippocampal regions, and in functionally associated neocortical areas. The co-variations of population spike amplitude were positive for LTH and negative for LTP in the dentate gyrus and frontal cortex of both sides, and in CA3/CA1 of the stimulated side, indicating higher DNA synthesis at lower values of LTH and LTP, and viceversa. Further, regional cross-correlation analyses revealed a high degree of synchronization among brain sites, following low- or high-frequency train pulses, indicating that (i) extra-target sites participate on the stimulated and on the contralateral side, and (ii) small distributed changes take place across the sampled neural networks. A modulatory role of information flow on brain DNA synthesis is inferred to take place in a diffuse, distributed manner.

Trains of sleep sequences are indices of learning capacity in rats.

In previous work dealing with the identification of four sleep sequences (SS-->W, SS-->PS, SS-->TS-->W and SS-->TS-->PS) in the baseline session of adult male Wistar rats [Mandile P, Vescia S, Montagnese P, Romano F, Giuditta A. Characterization of transition sleep episodes in baseline EEG recordings of adults rats, Physiol Behav 1996;60:1435-1439], we have shown that those containing an intervening episode of transition sleep (TS) strongly correlate with the number of avoidances scored the following day [Vescia S, Mandile P, Montagnese P, Romano F, Cataldo G, Cotugno M, Giuditta A. Baseline transition sleep and associated sleep episodes are related to the learning ability of rats, Physiol Behav 1996;60:1513-152]. More recently, clusters of sleep sequences (trains) separated by waking intervals longer than 60 s have been identified in the baseline session of the same rats [Piscopo S, Mandile P, Montagnese P, Cotugno M, Giuditta A, Vescia S. Identification of trains of sleep sequences in adult rats, Behav Brain Res, this volume], and distinguished in homogeneous or mixed trains according to the presence of a single sleep sequence or more than one sequence. Mixed trains have been further separated into trains containing the SS-->TS-->W sequence (+TSW trains) and trains lacking it (-TSW trains). Analysis of the distribution of variables of baseline trains (and of their sleep sequences and components) among fast learning (FL), slow learning (SL), or non-learning (NL) rats, indicates that variables of +TSW trains prevail in FL rats, while variables of -TSW trains prevail in NL rats. In addition, variables of +TSW trains correlate with the number of avoidances of the training session, while variables of -TSW trains do not significantly correlate, or show inverse correlations. Interestingly, sleep sequences such as SS-->W or SS-->TS-->W show direct or inverse correlations with avoidances depending on whether they are included in +TSW trains or in -TSW trains. The data are interpreted to suggest that the outcome of brain operations performed during a sleep sequence may selectively condition the appearance of later sequences within a time interval shorter than a given threshold. An analogous mechanism may be responsible for the aggregation of sleep components in sleep sequences.

Identification of trains of sleep sequences in adult rats.

In previous studies based on high resolution EEG analyses of the 7 h baseline session of 18 adult male Wistar rats [6,14], we have identified four sleep sequences initiating with slow wave sleep (SS) and terminating with waking (W) or paradoxical sleep (PS). Two of these sequences contained an intervening episode of transition sleep (TS). Several variables of these sequences (SS-->W, SS-->TS-->W, SS-->TS-->PS, and SS-->PS) were selectively correlated with the capacity of rats to learn a two-way active avoidance task the following day, and were differently distributed in fast learning, slow learning and non learning rats [21]. The temporal organization of different sleep components in sequences suggested that a comparable temporal organization might concern the different sleep sequences, albeit on a longer time scale. We have now used waking periods longer than 60 s to separate clusters of baseline sleep sequences (trains) in the same rats. Trains containing the same sleep sequence (homogeneous trains) have been distinguished from trains containing different sleep sequences (mixed trains). In addition, mixed trains including the SS-->TS-->W sequence (+TSW trains) have been separated from mixed trains lacking that sequence (-TSW trains). Mixed trains of the +TSW type were longest and most numerous, while homogeneous trains were shortest and least abundant. Mixed trains of the -TSW type displayed intermediate values. Several variables of sleep sequences and sleep components differed within mixed trains and among mixed and homogeneous trains. The data indicate that baseline sleep sequences aggregate in relatively long strings in a non random fashion. The mechanism of this association is discussed.

Circadian oscillations of DNA synthesis in rat brain.

The possibility that the synthesis of brain DNA undergoes a circadian fluctuation was examined in male adult Wistar rats, kept under natural lighting conditions or born and raised under artificial lighting conditions. Groups of rats were taken every 4 h during the 24 h, injected subcutaneously with [methyl-3H]thymidine and killed 4 h later. By cosinor analysis, the DNA specific activity of cerebral hemispheres and brainstem was found to show a significant 24 h rhythm with the peak at the beginning of the dark period (waking period). By contrast, in kidney, the peak of the circadian rhythm of DNA specific activity occurred during the light period (sleep period), in agreement with literature data. On the other hand, in 4-week-old rats, born and raised in artificial lighting conditions, brain DNA specific activity followed a 12 h rhythm, in agreement with the lack of a significant diurnal oscillation of the sleep--waking structure. It is concluded that brain DNA synthesis undergoes a circadian fluctuation in association with the circadian rhythm of waking.

Paradoxical sleep deprivation of the mother enhances DNA synthesis in fetal rat brain: autoradiographic and biochemical evidence.

Pregnant rats were deprived of paradoxical sleep for 3 days starting on the 18th gestational day. The condition of PS-D was imposed by confinement on a small platform surrounded by water or by daily injections of clomipramine. Four hours before the killing rats received a s.c. injection of [3H]-thymidine. The amount of radioactive DNA determined by autoradiography in several regions of fetal brain was found to be markedly increased under both experimental conditions in comparison with the control fetal brain. Considerably more limited effects were observed in kidney. Comparable changes of lower magnitude were obtained by comparing the specific radioactivity of DNA samples purified by chlorophorm extraction and digestion with RNase and proteinase K. The results fully confirm our previous data obtained under similar experimental conditions but based on the analysis of an acid-washed DNA fraction.

Effect of paradoxical sleep deprivation on dna synthesis in fetal rat brain.

We have investigated the effect of PS-D induced in gestating rats by treatment with clomipramine or with the platform technique on the process of DNA synthesis taking place in fetal organs. This parameter was taken as a biochemical index of ongoing cellular proliferation. In brain and, to a minor extent, in liver and kidney the rate of fetal DNA synthesis was markedly increased in both experimental groups. The effect was more prominent in the clomipramine group. PS-D treatment of gestating rats, notably by the platform technique, left long-lasting effects in the offspring with regard to organ weight and DNA concentration as well as to learning capacity. It is concluded that the occurrence of PS in gestating rats may exert a significant influence on fetal development.

Unscheduled brain DNA synthesis, long-term potentiation, and depression at the perforant path-granule cell synapse in the rat.

We investigated the effect of long-term potentiation (LTP) of the perforant path-granule cell synapse, on the synthesis of DNA in the target area and in polysynaptically stimulated hippocampal (CA3/CA1) and cortical areas (entorhinal, temporal, and occipital cortices) in the rat. The contralateral nonstimulated side was used as a control. The degree of LTP was indexed by the field EPSP and population spike amplitude recorded in the dentate area of the stimulated side before and after high frequency stimulation (250 Hz, 250 ms) every 30 min. DNA synthesis was evaluated in tissue homogenates after a 3-h period of incorporation of 3H-thymidine. DNA synthesis was significantly lower in the stimulated side in the hippocampal cortex CA3/CA1 (-25%), and in the entorhinal cortex (-50%), but not in the dentate area. In addition, the occurrence of preparations without expression of LTP allowed the analysis of unscheduled brain DNA synthesis (UBDS) in a supposedly long-term depression (LTD) subgroup. UBDS was higher in the group without LTP (no-LTP group) than in that with a significant LTP expression (LTP-group) on both sides of the brain. Furthermore, correlative analyses revealed that UBDS covaried with LTP of the EPSP (but not of population spike) in the dentate area and in extratarget hippocampal subregions on both sides and in dorsal cortex on the stimulated side. Further, regional crosscorrelation analyses revealed a high degree of coupling among brain sites following LTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term habituation to spatial novelty modifies posttrial synchronized sleep in rats.

To assess the role of posttrial synchronized sleep in the processing of a nonassociative task, adult male Sprague-Dawley rats with chronically implanted cortical electrodes for EEG recording were exposed to a Làt-maze, and horizontal (HA; corner crossing) and vertical (VA; rearings) activities were monitored during two 10-min test trials made at a 3-h (experiment 1) or 24-h (experiment 2) interval. EEG conventional recording was taken during 3 h under baseline conditions (day 1), and following exposure to the maze (day 2), and analyzed as to the amount (a), number (n), and mean duration (d) of synchronized sleep (SS) episodes followed by wakefulness (SS-->W) or by paradoxical sleep (SS-->PS). In both experiments there was a significant intertrial decrement (long-term habituation: LTH) for horizontal activity (LTH-HA), vertical activity (LTH-VA), and emotionality (LTH-E). In experiment 1, in comparison to baseline values, the posttrial SS-->PS(a) increased, mainly for the appearance of SS-->PS episodes in the 1st h. SS-->W(a) also increased in the first h. Correlative analyses among behavioral and sleep parameters showed that SS-->PS(n) and (d) covaried positively with LTH-HA relative to the entire test, and with LTH-VA relative to the second part of the test in the third h. Negative correlations were present between SS-->PS(n) and (d), and LTH-E. In experiment 2, exposed rats showed a lower SS-->PS(n) in the first hour and an increased SS-->PS(d) in the second hour. No change was observed as to SS-->W episodes.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain RNA synthesis, long-term potentiation and depression at the perforant path-granule cell synapse in the guinea pig.

The effects of long-term changes in synaptic efficacy at the perforant path-granule cell synapse on the de-novo synthesis of ribonucleic acid (RNA) were investigated in hippocampal and cortical areas in anaesthetized Guinea pig preparations. Two experiments were run with stimulating and recording microelectrodes aimed at the perforant bundle and dentate gyrus hilus on both sides. In Experiment 1, a low-frequency (LFS; 0.02 Hz, 3 h) or high-frequency stimulation (HFS; 400 Hz, 250 ms) was delivered to the left perforant bundle with the contralateral side as control. In Experiment 2, animals received LFS or HFS trains with implanted nonstimulated animals used as controls. The latency and amplitude of the field postsynaptic potentials (FPSP) and population spike (POPS) were monitored under baseline conditions and following stimulation over a 3 h period. In addition, two HFS groups were tested with few (HFS-F: every 15 min) or several test stimuli (HFS-S: every 3 min). In both experiments RNA synthesis was determined by measuring the amount of 3H-5,6-uridine incorporated into the RNA 3 h after bilateral intraventricular injection. In Exp. 1 the LFS group showed a higher synthesis of RNA than both HFS groups. The rate of RNA synthesis did not differ between the stimulated and nonstimulated side. In Exp. 2 the HFS groups showed a decreased RNA synthesis. In the HFS-F group, it pertained to the dorsal dentate area, CA1, subiculum, cingulate and dorsal cortices bilaterally, and to the ventral dentate area and CA3 on the nonstimulated side. In contrast, the HFS-S group showed decreased RNA synthesis at the dorsal dentate area and dorsal cortex on the stimulated side, and at CA1, subiculum, and cingulate cortex bilaterally. The decrease was stronger in the HFS-F than in the HFS-S group. Moreover, the subgroup with a low (0-60%) and that with a high (61-240%) level of long-term potentiation of FPSP revealed lower and higher RNA synthesis, respectively, both in homosynaptic target areas, and in heterosynaptic sites. Further, correlative analyses between FPSP, POPS and RNA synthesis revealed a complex pattern, depending upon the type of stimulation and on the brain side. Finally, cross-correlation analyses revealed a high degree of coupling among brain sites in the stimulated groups, indicating distributed covariant changes in RNA synthesis across different brain sites. Thus, changes in synaptic efficacy covary with changes in RNA synthesis, and presumably exert a modulatory role on gene expression.