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— The DNA polymerase of the optic lobes of Octopus vulgaris Lam has been solubilized and some of its properties have been investigated. The enzyme catalyses the incorporation of [3H]thymidine triphosphate into a DNase-sensitive, acid-insoluble product in the presence of DNA, the four deoxynucleoside triphosphates and Mg2+. The pH optimum is between pH 9.2 and 9.6, the optimal concentration of Mg2+ is between 10 and 30 mM and the optimal temperature is in the range 27°-30°C. The content of enzyme per mg protein is highest in the vertical and optic lobes and lowest in the suboesophageal lobe.

The effect of a single electroconvulsive shock on protein synthesis in mouse brain cortex was studied by observing the incorporation into protein of intraperitoneally injected [3H]- or [14C]leucine. When the precursor was injected immediately after the electroshock there was a 50 per cent inhibition of the incorporation which was not seen with injections at times later than 10 min. To investigate a possible specificity, the cerebral cortices of experimental and sham control animals which had been injected with different isotopes were homogenized together and fractionated by differential centrifugation. Cell fractions were then separately extracted with phosphate buffer and with Triton X-100. The ratio of 3H to 14C in each fraction was compared with that of the total homogenate to reveal any specific effects due to the electroconvulsive shock. The treatment produced a slight inhibition of the incorporation of the isotope into the heavier particulate fractions (i.e. nuclei, mitochondria, synaptosomes) relative to that in the microsome and cell sap fractions. A possible explanation of these results is given with a discussion of the limitations of the technique.

Cerebral NAD(P)H:(acceptor) oxidoreductase I (diaphorase I) is inhibited by barbiturates and nervous depressants which compete with the substrate. Determination of K i values for each compound provides a quantitative measure of their inhibitory effect. An investigation has been made on the chemical structures required for inhibition. The importance of the -CO-NR- group and of the size substituent groups is discussed. Other groups of atoms can, however, substitute for -CO-NR-. Cyclic structures are not required for inhibition. A parallel investigation on the structural requirements for inhibition of NADH oxidase of heart mitochondria has revealed a general similarity in the chemical structures required. The possible nature of inhibitory groups involved in binding with the enzyme is discussed.

— Utilizing techniques of continuous and SDS-electrophoresis we have examined the saline-soluble and SDS-soluble (membrane-bound) proteins extracted from the main lobes of adult octopus brain and from the developing optic lobe of the same species. Several additional protein bands are present among the soluble and the membrane-bound proteins of the vertical lobe in comparison with the suboesophageal lobe. Since the former contains an essentially homogeneous population of small neurons, while the suboesophageal lobe is rich in large nerve cells, these protein bands have been attributed to the small neuronal type present in the vertical lobe. In the course of a 10,000-fold increment in body weight, from 0.4 g to 4 kg, there is a significant increase in the concentration of several soluble proteins extracted from the optic lobe. Three of these proteins increase to a marked degree. Among the membrane-bound proteins some show a moderate increase with age while other protein components of smaller molecular weight undergo a moderate decrease. The overall tissue concentration of the membrane-bound proteins increases between 0.4 g and 50 g body weight, slightly declining in animals of larger size.

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.

Publisher Summary This chapter discusses the involvement of brain DNA in learning and sleep. Newly-synthesized brain DNA is involved in the processing of neural information which follows the acquisition step. The chapter describes an experiment design based on the hypothesis that the information gathered by the brain during wakefulness is processed during the following period of sleep in two sequential steps taking place during synchronized sleep and eventually during paradoxical sleep. In the experiment, rats were exposed for four hours to a massive training session in a shuttle-box for a two-way active avoidance task. They had been previously injected intraventricularly with 3 [H]thymidine and were killed three hours after the end of training. During the latter period, they were left free to sleep in a EEG recording cage. In nonlearning animals, the concentration of radioactive DNA of the major brain regions, but not of liver, was found to be inversely proportional to the duration of paradoxical sleep recorded in the post-training period. No effect occurred in learning rats and in control rats. The results were interpreted to indicate the degradation of radioactive DNA synthesized during the training period in association to an information input unable to induce a meaningful pattern of adaptive behavioral response.

The sequence complexity of nuclear and polysomal RNA from squid optic lobe and gill was measured by RNA-driven hybridization reactions with single-copy [3H]DNA. At saturation, brain nuclear and polysomal RNAs were complementary to 22.8 and 7.9% of the DNA probe, respectively. Assuming asymmetric transcription, the complexity of nuclear and polysomal RNA was equivalent to 2.5 X 10(8) and 8.8 X 10(7) nucleotides, respectively. Approximately 80-85% of the sequence complexity of brain total polysomal RNA was found in the polyadenylated RNA fraction. In contrast to these findings, nuclear and polysomal RNAs from gill hybridized to 9.1 and 2.9%, respectively, of the single-copy DNA, values that were 2.5-fold lower than those obtained in the CNS. Taken together, the results focus attention on the striking diversity of gene expression in the squid CNS and extend to the cephalopod mollusks the observation that nervous tissue expresses significantly more genetic information than other somatic tissues or organs.

Axoplasmic RNA from the giant axon of the squid (Loligo pealii) comprises polyadenylated [poly (A)+] RNA, as judged, in part, by hybridization to [3H]polyuridine and by in situ hybridization analyses using the same probe. The polyadenylate content of axoplasm (0.24 ng/microgram of total RNA) suggests that the poly(A)+ RNA population makes up approximately 0.4% of total axoplasmic RNA. Axoplasmic poly(A)+ RNA can serve as a template for the synthesis of cDNA using a reverse transcriptase and oligo(deoxythymidine) as primer. The size of the cDNA synthesized is heterogeneous, with most fragments greater than 450 nucleotides. The hybridization of axoplasmic cDNA to its template RNA reveals two major kinetic classes: a rapidly hybridizing component (abundant sequences) and a slower-reacting component (moderately abundant and rare sequences). The latter component accounts for approximately 56% of the total cDNA mass. The rapidly and slowly hybridizing kinetic components have a sequence complexity of approximately 2.7 kilobases and 3.1 X 10(2) kilobases, respectively. The diversity of the abundant and rare RNA classes is sufficient to code for one to two and 205, respectively, different poly(A)+ RNAs averaging 1,500 nucleotides in length. Overall, the sequence complexity of axoplasmic poly(A)+ RNA represents approximately 0.4% that of poly(A)+ mRNA of the optic lobe, a complex neural tissue used as a standard. Taken together, these findings indicate that the squid giant axon contains a heterogeneous population of poly(A)+ RNAs.

Knowledge of the complex relationship existing between sleep and brain biochemistry has been accumulating at a relatively slow pace. Progress in this field was thoroughly reviewed approximately 6 years ago,1,2 and there is little evidence that the rate of this progress has accelerated at all since then. Yet, such a goal should be considered an essential endeavor of the neurosciences. Although neurochemical studies cannot be taken as the only methodology leading to the goal, it is becoming increasingly evident that the final sentence of the chapter describing the role of sleep will not be written until we will know the type of operations a sleeping brain is performing at a molecular level. In turn, it is just as obvious that to design rewarding experiments at a molecular level, a general understanding of the biology of sleep is required, and questions should be asked within the framework of available hypotheses on the role of sleep. One should also remember that brain is not the only target organ for the functions of sleep. Other bodily activities are influenced by sleep, as is mental performance.

Isolated squid stellate nerves and giant fiber lobes were incubated for 8 hr in Millipore filtered sea water containing [3H]uridine. The electrophoretic patterns of radioactive RNA purified from the axoplasm of the giant axon and from the giant fiber lobe (cell bodies of the giant axon) demonstrated the presence of RNA species with mobilities corresponding to tRNA and rRNA. The presence of labeled rRNAs was confirmed by the behavior of the large rRNA component (31S) which, in the squid, readily dissociates into its two constituent moyeties (17S and 20S). Comparable results were obtained with the axonal sheath and the stellate nerve. In all the electrophoretic patterns, additional species of radioactive RNA migrated between the 4S and the 20S markers, i.e. with mobilities corresponding to presumptive mRNAs. Chromatographic analysis of the purified RNAs on oligo(dT)cellulose indicated the presence of labeled poly(A)+ RNA in all tissue samples. Radioactive poly(A)+ RNA represented approximately 1% of the total labeled RNA in the axoplasm, axonal sheath and stellate nerve, but more than 2% in the giant fiber lobe. The labeled poly(A)+ RNAs of the giant fibre lobe showed a prevalence of larger species in comparison to the axonal sheath and stellate nerve. In conclusion, the axoplasmic RNAs synthesized by the isolated squid giant axon appear to include all the major classes of axoplasmic RNAs, that is rRNA, tRNA and mRNA.

Using a translation assay it has been shown that the axoplasm extruded from the squid giant axon contains mRNA. The translation pattern of axoplasmic mRNA, as resolved on SDS-polyacrylamide gels, is different from the translation patterns of stellate nerve and giant fibre lobe (nerve cell bodies) RNA, although many protein bands are the same in axoplasm and the nerve cell bodies, as might be expected. Comparison of the axoplasmic proteins reveals a significant degree of correspondence. Axoplasmic mRNA is associated with the “microsomal” fraction of the axoplasm. Using reverse transcriptase, axoplasmic RNA can be transcribed into cDNA. The results of hybridization kinetics of axoplasmic cDNA with its template RNA show that the diversity of axo- plasmic RNA is considerable higher than indicated by the translation assays. On the other hand, the complexity of axoplasmic RNA is about 30-fold lower than that of total giant fibre lobe RNA and about 150-fold lower than that of optic lobe polysomal poly(A)+ mRNA. The latter results strongly supports the view that the high complexity of brain mRNA reflects the marked cellular and subcellular heterogeneity of the tissue.

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.

In the adult rat, brain DNA is in a state of turnover, which also involves a neuronal perikaryal fraction. Brain DNA synthesis is modulated by learning in upward or downward shifts, depending on the nature of the learning task. The concentration of radioactive brain DNA synthesized during training is inversely dependent on the amount of post-training paradoxical sleep, but only in non-learning rats. These results are taken as evidence that brain DNA is synthesized during learning in relation to the process of information storage, and may eventually be degraded in relation to the removal of that information. The latter process requires paradoxical sleep, and occurs if the information proves to be without adaptive value. It is proposed that transposition mechanisms underly the observed brain DNA turnover and the corresponding events of information processing. The data also support the sequential hypothesis of sleep function, which holds that the information gathered by brain during wakefulness is processed during sleep in two major steps occurring sequentially during synchronized and pardoxical sleep.

— The process of protein synthesis in the brain of Octopus vulgaris Lam has been examined after systemic administration of [3H]leucine and upon incubation of the tissue in sea water containing the radioactive precursor. After injection of [3H]leucine in the branchial heart, the radioactivity of the TCA-soluble fractions of the three main brain divisions reached a maximum in about 30 min and decreased thereafter, while incorporation into the protein fractions was complete in approx. 2 h. Per unit wet weight the radioactivity of brain proteins was higher than that of most other organs. In vitro the rate of incorporation of [3H]leucine in the protein fraction of the optic lobe remained low for more than 1 h, but increased several fold thereafter. Preincubation of the tissue in sea water abolished the lag period. Similar effects were observed in the vertical lobe as well as in the optic lobe of young and adult octopuses but not in the white body, a non-nervous organ. The process of protein synthesis in the optic lobe is markedly inhibited by puromycin, cycloheximide and chloramphenicol. Electrophoretic analysis on polyacrylamide gels indicated that the soluble proteins labelled in vitro and in vivo are similar.

Results of previous RNA-DNA hybridization experiments have demonstrated that the mammalian brain expresses 2-to 5-fold more of the haploid genome than other somatic tissues or organs. The striking complexity of gene expression in brain raises fundamental questions regarding the ultimate function of this large amount of genetic information and the degree to which it participates in the development and maintenance of tissue-specific structure and function. Here, we review our recent results obtained from a comparative study of the diversity of gene expression in brain. In this work, RNA-DNA saturation hybridization was used to estimate the sequence complexity of nuclear and polysomal RNA from rat, goldfish and squid brain. Additionally, the data were compared to the complexity of RNA from a typical non-neural tissue of each of these animal species. Our findings suggest that, as is the case in mammals, the diversity of gene expression in the CNS of teleosts and cephalopod mollusks is greater than in non-neural tissue. Importantly, however, the differences in the complexity of goldfish and squid brain RNA relative to that of non-neural tissue is significantly less than that observed in several mammalian species.

The possible involvement of brain DNA in learning received consideration some time ago, when nucleic acids appeared to be suitable candidates for the role of information storage molecules (Hyden, 1959; Gaito, 1961). DNA however was quickly put aside in view of its role as depository of genetic information and because of its presumed metabolic stability (Briggs and Kitto, 1962). On these counts RNA remained as a better candidate, at least until it was shown that brain had the same major RNA components of other organs (Vesco and Giuditta, 1967), thus leaving little room for brain specific RNA memory molecules. More convincing hypotheses were then advanced and RNA acquired a new role in learning as intermediate in the process of protein synthesis (Briggs and Kitto, 1962). It is ironical (and relevant) to note that both conclusions were based on incorrect assumptions. Indeed, a large population of brain-specific RNA transcripts came to be discovered in the early seventies following the application of hybridization techniques (Hahn and Laird, 1971; Brown and Church, 1971; Grouse et al., 1972). Their role in brain activity remains still to be clarified (Chaudari and Hahn, 1983; Kaplan, 1983). As to DNA, our views on its functional capacities have now become substantially more flexible in order to accomodate newly discovered features such as amplification, transposition, magnification and genomic rearrangement (Lewin, 1983).

The biosynthesis of RNA during sleep has been studied in two purified nuclear fractions separated from rabbit cerebral cortex after subarachnoidal injection of radioactive orotate. The biochemical parameters have been referred to the percent EEG synchronization recorded during the period of incorporation (1 hr). The content of radioactive RNA per nucleus increases significantly with percent synchronization in the fraction of large nuclei (of neuronal and astroglial origin). While sedimentation and electrophoretic analyses of this RNA are consistent with the hypothesis of an enhanced turnover of rRNA during wakefulness, the accumulation of labelled RNA which is observed during sleep may be due to a modified turnover of nuclear heterogeneous RNA. On the other hand, in the fraction of small nuclei (mostly of oligodendroglial origin) the content of radioactive RNA per nucleus and the pattern of sedimentation of labelled RNA show no dependence on the electrical state of the cortex. These data indicate that in the cerebral cortex the sleepwakefulness transition is accompanied by a differential cellular response in RNA turnover.

The possible involvement of cerebral DNA synthesis in the learning process was examined in rats injected intracerebrally with 3H thymidine. During the period of incorporation (4.5 hr) one rat was trained to an active avoidance task while a second animal was kept in the same experimental room. In comparison with control rats paired to learning animals, the concentration of PCA-soluble radioactivity and of radioactive DNA of the cerebral cortex increased in all animal groups, i.e., control rats paired to non-learning animals, learning rats and non-learning rats. No change occurred in liver. In the cerebral cortex the slope of the regression line obtained by plotting the concentration of radioactive DNA versus the concentration of PCA-soluble radioactivity was lower in learning rats than in the group of pooled control animals. A comparable effect was noted in the hippocampus. In non-learning animals a similar decrease was present in the cerebral cortex and in cerebellum. In addition, it was found that in learning animals the percent incorporation was inversely related to the total number of avoidances only in the cerebral cortex. In non-learning rats a similar inverse relationship was present in the cerebral cortex and in cerebellum. In the former region the regression line of learning rats was shifted upwards in comparison with the regression line of non-learning animals. These results are interpreted to indicate that the incorporation of 3H-thymidine into cerebral DNA is directly related to the level of stress and is increased by learning.

The information acquired by brain during wakefulness (W) may be processed in two sequential steps occurring during synchronized sleep (SS) and paradoxical sleep (PS), respectively. On the assumption that brain molecules synthesized during the acquisition step undergo a comparable sleep processing, we have designed an experiment aimed at the verification of the sequential hypothesis. Groups of adult female Wistar rats received [3H-methyl] thymidine by intraventricular injection 30 min before being exposed to a 4 hr session of a two-way active avoidance training. Animals failing to achieve the learning criterion were further allowed a period of 3 hr during which they were left free to sleep, or were deprived of PS or of total sleep. Control rats were similarly treated, but were left in their home cages in the same training room during the period of acquisition. The results of correlative study among behavioral, sleep and biochemical variables demonstrate that the specific radioactivity of DNA in the cerebral cortex, cerebellum and brainstem is correlated with several variables of postacquisition sleep, mostly SS parameters. The correlations depend on the previous waking experience of the rats. The data substantiate the two main consequences of the hypothesis, i.e., (1) the involvement of SS in brain information processing; and (2) the dependence of the operations performed by the sleeping brain on the nature of the previous waking experience. The results also provide some insight into the kind of processing which occurs in the sleeping brain.

The sequential hypothesis on sleep function assumes that the information gathered by brain during the waking period is processed during sleep in two main steps occurring during synchronized sleep (SS) and, eventually, during paradoxical sleep (PS). To verify the main consequences of the hypothesis, i.e., (1) that SS is involved in brain information processing; and (2) that the structure of sleep is dependent on the nature of the previous waking experience, an experiment was designed involving rats exposed to a training session (two-way active avoidance) but failing to learn (NL), and rats left in their home cages in the same training room (C). The structure of sleep, determined by EEG techniques in the postacquisition period (3 hr), was different in NL rats in comparison to C rats, chiefly because SS episodes were markedly longer in the former group. A more detailed analysis indicated that, in NL rats, SS episodes not followed by PS increased their duration first, while those followed by PS became longer in the second half of the sleep period. Comparable results were obtained in the comparison of NL and C subgroups deprived of PS at the end of the acquisition period by chlomipramine treatment. The data support the sequential hypothesis and provide evidence for a primary role of SS in brain information processing.

Administration of electroconvulsive shocks in rats induces a marked inhibition of the process of thymidine incorporation into cortical DNA. The inhibitory effect reaches its maximum after 10–15 min. Percentage incorporation returns to normal values within one hour of the treatment. These findings may suggest the participation of a metabolically active fraction of DNA in neural functions.

Publisher Summary This chapter discusses DNA turnover in rat cerebral cortex. A process of DNA turnover has been shown to occur in rat brain and attributed to repair reactions. This interpretation has not found support in other observations. The chapter present a study that reconsidered the problem by injecting groups of rats with 3H-tyymidine given intracranially at the same time of the day. After attaining a maximum labeling at 5 h, cerebral DNA loses more than 60% of its radioactivity at 13 h. From 13 to 73 h, it remains essentially stable except for a second slight but significant decrease which takes place between 41 and 49 h. A comparable initial loss of radioactive DNA occurs in a purified fraction of neuronal perikarya prepared from the cerebral cortex by the method of Satake and Abe. The concentration of radioactive DNA in liver does not change between 5 and 33 h. A significant later decrease is followed by an opposite trend continuing until 73 h. These results indicate that neuronal cells contribute to the marked DNA turnover observed in cerebral cortex.

Male Wistar rats weighing between 200 and 350 g were deprived of paradoxical sleep (PS-D) by placing them on small platforms surrounded by water. In comparison with rats kept in similar cages but without water, PS-D animals manifested a marked decrease in body and liver weight, which depended on animal size and o time of exposure to the condition of PS-D. A comparable weight loss occurred in the spleen, while more limited effects were present in brain and cerebellum. Exposure to the condition of PS-D induced a pronounced increase in the concentration of liver DNA and RNA. The latter effect occurred considerably earlier. On the other hand, only limited and uncertain variations were noted in the DNA concentration of brain and cerebellum. Body and liver changes were readily reversed after a period of recovery lasting two days.

Female Wistar rats weighing 200 g were implanted with cortical electrodes and two intraventricular cannulae. Five days later they were given 3H-thymidine and exposed to shuttle-box training for four hours. They were then left free to sleep in the following three hours during which their EEG activity was recorded. In comparison with control animals (C), learning (L) and non-learning (NL) rats exhibited an increase in SS. In comparison to the EEG recording made the previous day, all animal groups displayed an increase in SS, but only NL rats suffered a decrease in PS(%). The specific radioactivity of DNA measured in several brain regions was tendentially lower in NL rats, but significance was achieved only in the cerebellum in the comparison between NL rats and C rats. No change occurred in liver. More marked and significant decrements in the DNA specific radioactivity of all brain regions were observed in the subgroup of NL rats displaying relatively high values of PS time in comparison to the analogous subgroups of C and L animals. Comparable decrements were present with regard to the subgroup of NL rats endowed with relatively low PS time. Less widespread and more limited changes were observed in the concentration of acid-soluble radioactivity. In addition, several significant correlations were detected by Spearman's analysis among behavioral, biochemical and sleep parameters. The results are consistent with the interpretation that the selective decrease in brain radioactive DNA observed in NL rats reflects a loss of DNA synthesized during the training period. The loss is related to the amount of post-training PS and is associated to a lengthening of the mean duration of PS episodes. It may be concluded that the loss of newly-synthesized brain DNA reflects the elimination of molecules associated with neural information devoid of adaptive value.

We have examined the incorporation of [3H-methyl]thymidine into DNA extracted from several brain regions of rats learning a reverse handedness task, of control rats allowed to use their preferred paw, and of control rats left in their home cages. In learning animals, decrements in percent incorporation were observed in the visual cortex, remaining brain, hippocampus and entorhinal cortex. In the latter two regions less marked decreases were present in the active control group. No variation occurred in the sensory-motor cortex. In learning rats the specific radioactivity of neuronal DNA was markedly decreased in the hippocampus and remaining brain. In the former region, a less marked decrease was present in active control rats. In subcellular fractionation studies it was observed that decreases in DNA specific radioactivity prevailed in the mitochondrial fraction isolated from the hippocampus and visual cortex of learning rats. Brain radioactive DNA was widely distributed among fractions differing in their degree of repetitiveness. Its pattern of distribution did not coincide with that of bulk DNA and differed significantly among behavioural groups. The results suggest a non random origin of newly-synthesized brain DNA and its involvement in learning.

The pattern of growth of the optic lobe of Octopus vulgaris has been investigated from 0.1 to 6.0 gms body weight by measuring the weight of the lobe and the number of nuclei present in the tissue homogenate. In the normal habitat, the rate of increase in nulcear number relative to increase in body weight is 1.32 up to 1.5–1.8 gms, but undergoes a three-fold decrease above this size. On the other hand, the rate of increase in tissue weight remains essetially constant throughout the same period of development, with the exception of the interval between 1.0 g and 1.5 gms during which it rises markedly. Nuclear density increases steadily up to approximately 1.2 gms and approaches a constant value above this size. In octopuses reared in transparent containers or under an opaque cover the rates of increment in number of nuclei and in tissue weight are generally lower than normal between 0.2 g and 1.5 gms. In both experimental conditions the wave of acceleration in the rate of increase of tissue weight occurring between 1.0 g and 1.5 gms in normal animals appears earlier particularly in octopuses reared under an opaque cover. At body weights larger than 1.5–2.0 gms the rates of increment in number of nuclei and tissue weight become higher than normal in octopuses reared in transparent boxes, but change in the opposite direction when rearing takes place in opaque containers. In the former condition, the nuclear density of newly added tissue starts decreasing earlier than normal but reaches a higher level at body weights above 2 gms, while in octopuses reared under an opaque cover the decrease occurs at approximately normal timing but attains lower values at higher body weights.

After the intracranial injection of [methyl-3H]thymidine the specific activity of rat cortical DNA increases rapidly, reaching a maximum at about 5 h. More than half of the radioactive DNA disappears from the tissue in the following few hours. During the same period of time the concentration of radioactive DNA in liver remains essentially constant. Minor variations occur in both organs after 41 h. An apparent rapid turnover of DNA is also present in a fraction of purified neuronal perikarya prepared from the cerebral cortex.

The problem of the role played by DNA in the functional activity of the nervous system can be traced back to a number of experimental observations dealing chiefly with the content of DNA in brain cells and with the occurrence of a process of DNA turnover in adult brain. Somewhat later developments have concerned the physicochemical properties of brain DNA and the enzymes involved in DNA synthesis and degradation. In view of the heterogeneous cellular composition of brain, all of these studies have become more meaningful with the progressive refinement of methods allowing analysis of single cells or the preparation of homogeneous populations of cell types.

By centrifugation of a 2.0 M sucrose homogenate of rat brain cortex in a discontinuous sucrose gradient two classes of nuclei differing in size and morphological appearance have been separated. Large nuclei are presumably derived from neurones and astroglia, while small nuclei originate largely from oligodendroglia. Both nuclear fractions are essentially free of cytoplasmic contaminants and have a low degree of cross-contamination. The method is rapid and reproducible and gives an overall yield of 30% nuclei. The main RNA species of high molecular weight extracted from the large nuclei sediment at 28 S and 18 S. Similar species are present in the small nuclei. Incorporation studies with [14C]orotate administered in vivo indicate that the content of radioactive RNA per nucleus increases at similar rates in both nuclear classes up to 20–30 min, while there is a progressive subsequent prevalence in the large nuclei which reaches a maximum within 4 h. At this time the average nuclear content of radioactive RNA is almost 3 times higher in the large than in the small nuclei. Analysis of the labelled nuclear RNA by sedimentation in sucrose gradients shows that heterogeneous and ribosomal RNAs are present in both nuclear classes. After 30 min of incorporation preribosomal species can be detected in the large nuclei, while heterogenous RNA prevails in the small nuclei. On the other hand, mature radioactive 18 S and 28 S rRNA appear after 60 min in the small nuclei, but only after 180 min in the large nuclei. At earlier times the main radioactive preribosomal species of the latter nuclei sediment in the region of 30 S and 20 S.

— The pattern of RNA biosynthesis in rabbit cerebral cortex was examined during periods of physiological sleep and wakefulness monitored with EEG techniques. The types of newly synthesized RNA were analysed by sedimentation on sucrose gradients. In experiments lasting 60 and 105 minutes the labelling ratio of RNA sedimenting in two regions of the gradient (28 S-50 S50 S) increased with decreasing percentages of EEG synchronization. Analysis of the radioactivity distributions of five homogeneous experiments in terms of moments confirmed the dependence of the RNA synthetic pattern on the electric state of the cortex. The possible nature of the RNA species involved in this effect is discussed.

THE uptake of macromolecules by neurones and glia may be relevant to an understanding of the relationships between these cells and to the concept of the glia-neurone unit1. Protein uptake has been studied biochemically using suspensions of animal cells2, but little morphological evidence has been gathered from neuronal systems3–5. This problem can be investigated conveniently using the giant axon of the squid; biochemical analyses can be carried out on samples of axoplasm after incubation of intact axons with a labelled macromolecule. To do this we have used125I-albumin as a model protein because of its commercial availability and the lack of reincorporation of free 125I-tyrosine into protein2.

A quick method is described for determining the number of cells in the brain ofOctopus vulgaris. The method is based on nuclear counts of fresh homogenates. The total number of brain cell nuclei increases with age from approx. 4 · 107 at 10–20 g body weight to approx. 17 · 107 at about 1 kg. Within this body-size range the overall increase in the vertical and optic lobes is 5- and 6-fold and in subordinate brain centres 3- and 4-fold. Almost no further increase was found above 1 kg. The average DNA content per nucleus is higher than expected from diploid cells and increases with age in all the main divisions of the brain except the vertical lobe which has a diploid content throughout life (10.4 pg). The overall number of nuclei per mg wet tissue declines between early and late ontogeny. The decline is chiefly in the suboesophageal lobes; in the vertical lobes there is no change in nuclear concentration.