Markers Of Synaptogenesis Research Articles

The clinically available NMDA receptor antagonist, memantine, exhibits relative safety in the developing rat brain

The N-methyl- d-aspartate glutamate receptor (NMDAR) has been implicated in preterm brain injury (periventricular leukomalacia (PVL)) and represents a potential therapeutic target. However, the antagonist dizocilpine (MK-801) has been reported to increase constitutive neuronal apoptosis in the developing rat brain, limiting its clinical use in the developing brain. Memantine is another use-dependent NMDAR antagonist with shorter binding kinetics and has been demonstrated to be protective in a rat model of PVL, without effects on normal myelination or cortical growth. To further evaluate the safety of memantine in the developing brain, we demonstrate here that, in contrast to MK-801, memantine at neuroprotective doses does not increase neuronal constitutive apoptosis. In addition, there are no long-term alterations in the expression of NMDAR subunits, AMPAR subunits, and two markers of synaptogenesis, Synapsin-1 and PSD95. Evaluating clinically approved drugs in preclinical neonatal animal models of early brain development is an important prerequisite to considering them for clinical trial in preterm infants and early childhood.

Microglial Involvement in Neuroplastic Changes Following Focal Brain Ischemia in Rats

The pathogenesis of ischemic stroke is a complex sequence of events including inflammatory reaction, for which the microglia appears to be a major cellular contributor. However, whether post-ischemic activation of microglial cells has beneficial or detrimental effects remains to be elucidated, in particular on long term brain plasticity events. The objective of our study was to determine, through modulation of post-stroke inflammatory response, to what extent microglial cells are involved in some specific events of neuronal plasticity, neurite outgrowth and synaptogenesis. Since microglia is a source of neurotrophic factors, the identification of the brain-derived neurophic factor (BDNF) as possible molecular actor involved in these events was also attempted. As a means of down-regulating the microglial response induced by ischemia, 3-aminobenzamide (3-AB, 90 mg/kg, i.p.) was used to inhibit the poly(ADP-ribose) polymerase-1 (PARP-1). Indeed, PARP-1 contributes to the activation of the transcription factor NF-kB, which is essential to the upregulation of proinflammatory genes, in particular responsible for microglial activation/proliferation. Experiments were conducted in rats subjected to photothrombotic ischemia which leads to a strong and early microglial cells activation/proliferation followed by an infiltration of macrophages within the cortical lesion, events evaluated at serial time points up to 1 month post-ictus by immunostaining for OX-42 and ED-1. Our most striking finding was that the decrease in acute microglial activation induced by 3-AB was associated with a long term down-regulation of two neuronal plasticity proteins expression, synaptophysin (marker of synaptogenesis) and GAP-43 (marker of neuritogenesis) as well as to a significant decrease in tissue BDNF production. Thus, our data argue in favour of a supportive role for microglia in brain neuroplasticity stimulation possibly through BDNF production, suggesting that a targeted protection of microglial cells could represent an innovative approach to potentiate post-stroke neuroregeneration.

The neural substrates of amphetamine conditioned place preference: implications for the formation of conditioned stimulus–reward associations

Associations formed between conditioned stimuli and drug reward are major contributors in human drug addiction. To better understand the brain changes that accompany this process, we used immunohistochemistry for c-Fos (a neuronal activity marker), synaptophysin (a marker for synaptogenesis) and tyrosine kinase B receptor (a neurotrophic factor receptor that mediates synaptic plasticity) to investigate the neural substrates of amphetamine-induced conditioned place preference in rats. Conditioned place preference was induced by both 1.0 mg/kg and 0.3 mg/kg doses of amphetamine. Furthermore, amphetamine conditioning increased the density of c-Fos-immunoreactive cells and these cells were fully colocalized with the tyrosine kinase B receptor in the dentate gyrus, CA1 field and basolateral amygdala. Amphetamine conditioning increased the density of synaptophysin-immunoreactive varicosities in all brain regions studied, except the nucleus accumbens shell and dorsolateral striatum. The degree of conditioned place preference was highly correlated with c-Fos-immunoreactive cell density in the basolateral amygdala and with the density of synaptophysin-immunoreactive varicosities in all mesolimbic regions studied. The latter correlation was particularly impressive for the ventral pallidum and basolateral amygdala. The formation of conditioned stimulus-amphetamine reward associations is accompanied by tyrosine kinase B receptor expression in the basolateral amygdala and dentate gyrus, CA1 and CA3 fields of the hippocampus. These data therefore suggest that the formation of conditioned stimulus-reward associations requires, at least in part, activation of amygdalar-hippocampal circuits.

Role of phospholipase D1 in neurite outgrowth of neural stem cells

Employing neural stem cells from the brain cortex of E12 rat embryos, we investigated the possible role of phospholipase D (PLD) in the synaptogenesis and neurite formation of neural cells during differentiation. Expression level of PLD1 increased during neuronal differentiation of the neural stem cells, resulting in increased PLD activity. Expression level of synapsin I, a marker of synaptogenesis, also increased as the differentiation of neural stem cells progressed. To figure out the effect of PLD on synapsin I expression, we treated the neural stem cells with phorbol myristate acetate (PMA) to stimulate PLD activity. Increased PLD activity induced by PMA treatment resulted in elevated synapsin I expression and neurite outgrowth during neuronal differentiation. To further confirm the role of PLD in neurite outgrowth, we transfected the dominant-negative form of rat PLD1 cDNA (DN-rPLD1) into neural stem cells to downregulate PLD activity. Overexpression of DN-rPLD1 showed the complete inhibition of neurite outgrowth of neural stem cells under differentiation condition. While transfection of DN-rPLD1 did not affect the synapsin I expression, overexpression of rPLD1 resulted in increased synapsin I expression of the neural cells. These results suggest that PLD1 plays a critical role in neurite outgrowth during differentiation of the neural stem cells. In conclusion, this is the first evidence to show that PLD1 acts as an important regulator of neurite outgrowth in neural stem cell by promoting neuronal differentiation via increase of synapsin I expression.

Improvement in neurological outcome after administration of atorvastatin following experimental intracerebral hemorrhage in rats.

Atorvastatin, a beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitor, improves neurological functional outcome, reduces cerebral cell loss, and promotes regional cellular plasticity when administered after intracerebral hemorrhage (ICH) in rats. Autologous blood was stereotactically injected into the right striatum in rats, and atorvastatin was administered orally beginning 24 hours after ICH and continued daily for 1 week. At a dose of 2 mg/kg, atorvastatin significantly reduced the severity of neurological deficit from 2 to 4 weeks after ICH. The area of cell loss in the ipsilateral striatum was also significantly reduced in these animals. Consistent with previous study data, higher doses of atorvastatin (8 mg/kg) did not improve functional outcome or reduce the extent of injury. Histochemical stains for markers of synaptogenesis, immature neurons, and neuronal migration revealed increased labeling in the region of hemorrhage in the atorvastatin-treated rats. Analysis of the data in this study indicates that atorvastatin improves neurological recovery after experimental ICH and may do so in part by increasing neuronal plasticity.

Spatio-temporal changes in neurofilament proteins immunoreactivity following kainate-induced cerebellar lesion in rats.

1. Spatio-temporal changes in phosphorylated (pNFP) and nonphosphorylated (npNFP) neurofilament proteins were assessed immunocytochemicaly in adult rat cerebellum, 2-30 days following unilateral injection of kainic acid (KA) or physiological saline (s.c.). 2. Analysis of the staining intensity and pattern demonstrated that injection of both KA and physiological saline elicited significant and long-lasting increase of pNFP and npNFP immunoreactivity, at the ipsilateral, and to lesser extent at the contralateral side of lesion. 3. Kainate intoxication induced abundant expression of pNFP and npNFP in cerebellar white matter, as well as in all layers of perilesioned cortex. Higher pNFP expression was evidenced in the Purkinje cell layer, particularly at cell bodies, initial segments, and proximal dendrites, which normally do not contain pNFP. In addition, synaptophysin immunocytochemistry was used as a marker of synaptogenesis and plasticity. 4. Spatio-temporal pattern of NFP and synaptophysin expression suggests that perilesioned cortex undergoes dynamic changes following brain demage and possess a reparative capacity to abridge the consequences of brain trauma.

Blockade of N-methyl- d-aspartate receptors by phencyclidine causes the loss of corticostriatal neurons

Perinatal administration of the N-methyl- d-aspartate (NMDA) receptor antagonist phencyclidine (PCP) has been reported to produce regionally selective apoptotic cell death in the frontal cortex. The development of certain behavioral abnormalities following PCP treatment suggested that extracortical regions such as the striatum also could be affected. In this study, perinatal PCP treatment caused a marked reduction in striatal, but not hippocampal, staining for polysialic acid–neural cell adhesion molecule (PSA-NCAM), an NMDA-regulated molecule important in synaptogenesis. In order to isolate striatal influences to the cortex, this investigation was continued in vitro using corticostriatal slices. For these experiments we cultured coronal corticostriatal slices from postnatal day 7 rats. After 4 days in vitro, PCP was added for 48 h and then washed out for 24 h before harvesting the tissue. Similar to what was observed in vivo, we found that PCP treatment results in a marked reduction in striatal staining for PSA-NCAM. No change was observed in the mature form of NCAM. In striatal synaptoneurosomes, immunoblot analysis confirmed that the levels of PSA-NCAM and synaptophysin, a molecule often used as a marker of synaptogenesis, were substantially down-regulated by PCP. These effects were prevented by M40403, a superoxide dismutase mimetic that also prevented the PCP-induced terminal dUTP nick-end labeling of DNA fragments that was observed selectively in the cortex. These data suggest that PCP causes cell death by apoptosis selectively in the cortex, but not in the striatum, following either in vivo treatment of perinatal rat pups or in vitro treatment of corticostriatal slices. Further, cortical apoptosis induced by PCP negatively impacts striatal synaptogenesis, a process important in normal neural development. This deficit is probably caused by a reduction in corticostriatal neurotransmission. It is possible that the dysregulation of striatal synaptogenesis contributes to the behavioral abnormalities observed following perinatal PCP administration in vivo.

Estradiol increases pre- and post-synaptic proteins in the CA1 region of the hippocampus in female rhesus macaques (Macaca mulatta).

The role of estrogen (E) in promoting learning and memory in females has been well studied in both rodent and primate models. In female rats, E increases dendritic spine number, synaptogenesis, and synaptic proteins in the CA1 region of the hippocampus, an area of the brain that mediates learning and memory. In the present study, we used radioimmunocytochemistry to examine whether E and progesterone were capable of modulating the levels of pre- and postsynaptic proteins in the CA1 region of the female nonhuman primate hippocampus. It was found that E increased syntaxin, synaptophysin (presynaptic), and spinophilin (postsynaptic) levels in the stratum oriens and radiatum of the CA1 region, whereas combined E and progesterone treatment decreased these synaptic proteins to the levels found in untreated, spayed controls. Furthermore, progesterone treatment alone significantly increased synaptophysin levels in the stratum oriens and radiatum of the CA1 region. The levels of these synaptic proteins were unaltered by hormone treatment in the dentate gyrus, suggesting that this steroid-induced plasticity is hippocampal region specific. As these synaptic proteins are important components of the synaptic apparatus and reliable markers of synaptogenesis, it appears that E-induced increases in cognitive function of higher order mammals may be mediated in part by the effect of E on hippocampal synaptogenesis and synaptic plasticity.

Estradiol increases hippocampal syntaxin protein levels in ovariectomized primates

Objective: Estrogen (E) has beneficial effects on learning and memory and may aid in the prevention of neurodegenerative diseases such as Alzheimer’s disease (AD). E has been found to increase spine density and synaptogenesis in the rat and nonhuman primate hippocampus, an area of the brain implicated in learning and memory and damaged in AD patients. Recent work in our laboratory has shown that E increases pre-(syntaxin and synaptophysin) and post-synaptic(spinophilin) protein levels in the female rat hippocampus. These proteins have been shown to be reliable markers of synaptogenesis. This study determined whether the effect of E on syntaxin observed in rats also occurs in nonhuman primates. Due to the similarity in neural organization and function of humans and nonhuman primates, elucidation of the synaptic proteins contributing to the hormone-induced structural plasticity in the primate hippocampus has immediate translational benefits for human mental health.

Differential expression of syntaxin-1 and synaptophysin in the developing and adult human retina.

Synaptophysin and syntaxin-1 are membrane proteins that associate with synaptic vesicles and presynaptic active zones at nerve endings, respectively. The former is known to be a good marker of synaptogenesis; this aspect, however, is not clear with syntaxin-1. In this study, the expression of both proteins was examined in the developing human retina and compared with their distribution in postnatal to adult retinas, by immunohistochemistry. In the inner plexiform layer, both were expressed simultaneously at 11-12 weeks of gestation, when synaptogenesis reportedly begins in the central retina. In the outer plexiform layer, however, the immunoreactivities were prominent by 16 weeks of gestation. Their expression in both plexiform layers followed a centre-to-periphery gradient. The immunoreactivities for both proteins were found in the immature photoreceptor, amacrine and ganglion cells; however, synaptophysin was differentially localized in bipolar cells and their axons, and syntaxin was present in some horizontal cells. In postnatal-to-adult retinas, synaptophysin immunoreactivity was prominent in photo-receptor terminals lying in the outer plexiform layer; on the contrary, syntaxin-1 was present in a thin immunoreactive band in this layer. In the inner plexiform layer, however, both were homogeneously distributed. Our study suggests that (i) syntaxin-1 appears in parallel with synapse formation; (ii) synaptogenesis in the human retina might follow a centre-to-periphery gradient; (iii) syntaxin-1 is likely to be absent from ribbon synapses of the outer plexiform layer, but may occur at presynaptic terminals of photoreceptor and horizontal cells, as is apparent from its localization in these cells, which is hitherto unreported for any vertebrate retina.

Synaptophysin immunocytochemistry with thermal intensification: a marker of terminal axonal maturation in the human fetal nervous system

Synaptophysin is a protein of synaptic vesicles and may be demonstrated in tissue sections of human brain and spinal cord by immunocytochemistry using a monoclonal antibody. Synaptophysin immunoreactivity was studied in paraffin-embedded sections of the central nervous system (CNS) in 14 normal human fetuses and neonates ranging in age from 8 to 41 weeks gestation, and in three brains with heterotopic neurons or malformations. A progressive expression of synaptophysin is seen in axonal terminals within grey matter in various parts of the CNS, beginning in the ventral horns of the spinal cord and brainstem tegmentum at 12–14 weeks. In the cerebellum, the molecular layer shows a band of reactivity from 18 weeks; by term two parallel bands of synaptophysin are seen in the molecular layer and reactivity also is demonstrated in the Purkinje and internal granular layers. In the cerebral neocortex, the molecular zone has weak synaptophysin reactivity as early as 10 weeks, though reactivity is not detected in the deep layers of the cortical plate until 19 weeks and in layers 2–4 until 25 weeks gestation. Synaptophysin reactivity is strong at the surface of neurons but not detected in their somatic cytoplasm; coarsely beaded reactivity within the neuropil probably corresponds to synaptic vesicles in terminal axons. Similar granular synaptophysin reactivity is seen around heterotopic neurons in the subcortical white matter, in dysgenesis of the cerebellar cortex and in the residual anencephalic forebrain. Thermal intensification by heating the incubating solution in a microwave oven often enhances immunoreactivity because of more complete antigen retrieval and is recommended for tissue stored in formalin or in paraffin for long periods. Synaptophysin provides a useful tissue marker of synaptogenesis during normal development and in cerebral dysgeneses, and may provide useful correlations with functional imaging of the brain in living patients. Used in conjunction with other neuronal markers, the expression of synaptophysin in terminal axons of distant neurons, in temporal relation to the maturation of the neurons they innervate, may provide clues to the pathogenesis of epilepsy in early infancy.

Synaptic reorganization following kainic acid-induced seizures during development

Prolonged seizures in the adult brain causes neuronal loss in the hippocampus and aberrant growth (sprouting) of granule cell axons (mossy fibers) in the supragranular zone of the fascia dentata and stratum infrapyramidale of CA3. There is considerable evidence that these changes in neuronal growth following seizures are age related, with younger animals having fewer reactive changes following prolonged seizures than older animals. However, there is little information available regarding the age at which seizures in the developing brain result in alterations in axonal growth and synapse formation. In this study, we evaluated the effects of kainic acid (KA)-induced seizures during development on synaptic reorganization using the expression of growth-associated protein-43 (GAP-43), a marker for synaptogenesis and Timm stain which detects the presence of zinc in granule cell axons. Age specific doses of KA were used to induce seizures of similar intensity at various ages (postnatal days (P) 12, 21, 25, 35, 45, 60) in Sprague–Dawley rats. Up to the age of P25, there were no differences in either Timm or GAP-43 staining between animals with KA seizures and controls. In P25 and older KA-treated rats, Timm staining was found in the supragranular layer of the dentate gyrus. This staining increased with age at the time of KA injection. Seizures in adult (P60), but not younger rats also resulted in increased staining in the suprapyramidal layer of the CA3 subfields. Changes in GAP-43 were delayed compared to the Timm staining with no differences between KA-treated animals and controls until P35 when a band of GAP-43 immunostaining appeared in the supragranular inner molecular layer, progressively increasing in intensity and thickness with time. This study demonstrates that seizure-induced reactive synaptogenesis is age-related. Since both Timm and GAP-43 reflect different aspects of reactive synaptogenesis, used in combination these methods provide useful information about the structural changes following seizures during development.

Expression of microtubule-associated protein-2a and other novel microtubule-associated protein-2 transcripts in human fetal spinal cord.

In human fetal spinal cord (HFSC), six additional microtubule-associated protein-2 (MAP-2) transcripts are generated by alternative splicing of two recently described exons, exon 8 and exon 13. The following three translated proteins are detected by western blot analysis: MAP-2b expressing exon 8 (MAP-2b + 8; MAP-2a), MAP-2b expressing exon 13 (MAP-2b + 13), and MAP-2c expressing exon 8 and exon 13 (MAP-2c + 8 + 13). The finding that MAP-2b + 8 is expressed in HFSC demonstrates for the first time the presence of MAP-2a in human fetal CNS. Immunocytochemical studies show that exon 8-specific antibody and exon 13-specific antibody stain independent and overlapping populations of neurons in the lumbar region of the HFSC. Antibody 13-immunopositive neurons have predominantly cytosolic staining, whereas in the antibody 8-immunoreactive neurons staining was observed in the cytosol, dendrites, and some synapses. The prenatal expression of MAP-2a, which has been used as a marker of synaptogenesis, not only demonstrates the presence of a mature MAP-2 isoform in HFSC, but suggests that MAP-2a is important during human fetal as well as postnatal synaptogenesis.

Light and electron microscopic analysis of synaptic development in Macaca monkey retina as detected by immunocytochemical labeling for the synaptic vesicle protein, SV2.

The development of synapses has been followed in Macaca monkey fetal and infant retina using immunocytochemical labeling for the transmembrane synaptic vesicle glycoprotein, SV2. Electron microscopy (EM) was used to verify the presence of morphological synapses at selected ages. EM immunocytochemical labeling in adult retina showed that all synaptic types contained SV2 in inner (IPL) and outer (OPL) plexiform layers. In fetal retina, SV2 expression and the appearance of morphological synapses were closely related in time, demonstrating that SV2 is a reliable marker for synaptogenesis. SV2 expression appears along a foveal to peripheral gradient. Both SV2 and synapses appear in the foveal IPL at Fd50-55, and reach the retinal edge by Fd90-103. Cone ribbon synapses and SV2 labeling are not present in the foveal OPL until Fd60. Photoreceptors in the far periphery contain SV2 by Fd119-125. This pattern demonstrates an "inner to outer" direction of synaptogenesis. Cones show SV2 labeling before rods at the same retinal eccentricity. In the cone-dominated fovea, SV2 labeling and bipolar cell ribbon-containing terminals are present at Fd55 when amacrine cell conventional terminals are very scarce, indicating that bipolar synapses precede amacrine synapses in monkey foveal IPL. SV2 labeling and bipolar terminals appear first in the outer IPL which contains "OFF" ganglion and bipolar processes in the adult, suggesting that "OFF" midget bipolar cells may form the first synapses. Both SV2 immunocytochemical labeling and EM morphology find that monkey retina follows a generalized inner before outer, and cone before rod synaptic developmental pattern, similar to that in other mammals. The cone-dominated fovea initiates synaptogenesis, and shows a different synaptic sequence from rod-dominated peripheral retina.

Coordinate and Noncoordinate Regulation of Synaptic Vesicle Protein Genes during Embryonic Development

Because the formation of a mature nerve terminal requires the accumulation of large quantities of synaptic vesicles, the expression of synaptic vesicle proteins would be expected to correlate with synaptogenesis. However, previous studies have provided conflicting evidence on this point. We have examined the developmental pattern of expression of mRNA and protein for three RNAs derived from two genes coding for synaptic vesicle membrane proteins. For these experiments, we cloned a chick p65 (synaptotagmin) cDNA using a reduced stringency screen with a rat p65 cDNA probe. We examined p65 expression in chick forebrain in conjunction with that of synaptophysin II. RNase protection assays for p65 and the two isoforms of synaptophysin II (Bixby, 1992) show essentially coordinate increases of these three mRNAs in embryonic forebrain during the peak period of synaptogenesis (E17 to E20). However, each of the three mRNAs has a distinct temporal pattern of expression during the early stages of embryogenesis. In the ciliary ganglion, upregulation of synaptophysin II mRNA correlates very well with synaptogenesis. Our results suggest that the regulation of expression of vesicle membrane protein mRNA can serve as a marker for synaptogenesis, despite temporal differences in early expression patterns. In contrast to mRNA expression, assays for vesicle protein expression show a relatively steady rise in both p65 and synaptophysin II throughout the embryonic period, without a sharp increase corresponding to that seen in message levels. These results suggest that the expression both of the p65 and of the synaptophysin II proteins is post-transcriptionally regulated.

Morphological correlates of long‐term potentiation imply the modification of existing synapses, not synaptogenesis, in the hippocampal dentate gyrus

This report evaluates two morphological markers of synaptogenesis following the induction of long-term potentiation (LTP) in the dentate gyrus of the anesthetized rat. These two morphological features, polyribosomes and multiple synaptic contacts, are known to increase in number with synaptogenesis in the mature hippocampus. The analysis focused on the middle third of the dentate molecular layer. As shown previously, this is the region of primary synaptic activation in our electrophysiological protocol and the region of localized morphological changes with LTP. Here the incidence of a polyribosome at the base of a dendritic spine declined 57% with LTP. In addition, the number of multiple synaptic contacts decreased 18% there with LTP. Both decreases were more pronounced immediately following conditioning stimulation than at later intervals. Because both morphological features decrease with LTP but increase with synaptogenesis, the data do not support the hypothesis that new synapses form with LTP. Instead, the data add further support to the view that the strengthening of existing excitatory synapses underlies LTP.

Prenatal terbutaline exposure in the rat: Selective effects on development of noradrenergic projections to cerebellum

Terbutaline, used in the treatment of premature labor and asthma, crosses the placenta and can stimulate β 2-adrenergic receptors in the fetus. This study examines the effects of prenatal exposure to terbutaline (10 mg/kg SC on gestational days 17, 18 and 19) on the development of noradrenergic projections in brain regions of the fetal and neonatal rat, using synaptosomal uptake of [ 3H]norepinephrine as a marker for synaptogenesis. Although terbutaline exposure did not compromise body or brain region growth, uptake was adversely affected selectively in the cerebellum, a region which also displays close coupling of fetal β 2-receptors to control of cell development near term. These results thus provide biochemical evidence that terbutaline may be a neurobehavioral teratogen.

Delays in growth and biochemical development of rat brain caused by maternal methadone administration: are the alterations in synaptogenesis and cellular maturation independent of reduced maternal food intake?

Food consumption of control pregnant and nursing rats was matched to that of methadone-treated dams and the patterns of growth and of biochemical development of the brain in the offspring were compared, using tyrosine hydroxylase activity as a marker for synaptogenesis of catecholamine neurons and the developmental pattern of ornithine decarboxylase activity as an index of delay of cellular maturation. Although body growth was slowed significantly, the degree of deficit was attenuated compared to previously reported experiments with non-pair-fed controls. Reduced brain weight and slowing of synaptogenesis also were evident in the methadone-exposed pups, and in this case, the effect was not attenuated with the use of pair-fed controls. Reduced brain weight and slowing of synaptogenesis also were evident in the methadone-exposed pups, and in this case, the effect was not attenuated with the use of pair-fed controls. Delays in the normal maturational decline of brain ornithine decarboxylase activity were prominent in the methadone group compared to offspring of pair-fed controls, just as reported previously in comparisons using non-pair-fed dams. These results indicate the part of the general growth deficit seen in perinatally-addicted pups is related to maternal undernutrition, but that delays in central synaptogenesis or in the pattern of biochemical maturation of the brain are relatively independent of the nutritional deficit.

Molecular forms of acetylcholinesterase in chick muscle and ciliary ganglion: Embryonic tissues and cultured cells

Molecular forms of acetylcholinesterase were studied in chick muscle and ciliary ganglion cultures using sedimentation analysis in sucrose gradients. The high molecular weight form of the enzyme cannot be used as a marker for synaptogenesis in cocultures of muscle and ganglia since (i) it was always present together with the 11 S and 6.5 S forms in muscle or ganglia cultured alone and (ii) it did not increase in the cocultures where pronounced muscle contractions occurred due to synapse formation. In vivo studies showed that the heavy form of the enzyme was present in the ciliary ganglion but not in the muscle as early as the fifth embryonic day.

A chemical study on the development of the human forebrain and cerebellum during the brain ‘growth spurt’ period. I. Gangliosides and plasmalogens

Following upon previous studies on the lipid composition of the developing human brain, a further study is presented with the main object of tracing the chemical changes underlying the period of brain ‘growth spurt’. Gangliosides and plasmalogens were selected as approximate markers of synaptogenesis and myelination, respectively, and these lipids were compared in cerebrum and cerebellum to establish the time, if any, at which their rate of accretion increases in a significant way. In the forebrain the rate of increase in concentration of these lipids accelerated at about the 32nd week of gestational age. Although there were too few postnatal cases to draw very firm conclusions, it seemed that the ganglioside concentration levelled off at about two months postnatal age and that the plasmalogen concentration reached a plateau between the 4th and the 6th postnatal months. In the cerebellum the concentration of gangliosides was clearly lower than that in the forebrain until about one year of age, the maximum rate of increase occuring between the last weeks of gestation and the second postnatal month. The plasmalogen concentration was somewhat higher in the cerebellum than in the forebrain but the concentration profile was similar to that followed by the gangliosides. In clear contrast with the concentration profiles in the cerebrum, in the cerebellum both lipids apparently continued to increase up to the second postnatal year. A mainly perinatal period of vulnerability is suggested for the forebrain, and a more prolonged one (probably until the second year of life) for the cerebellum.