Janelle C. LeBoutillier

Modulation of Presynaptic Plasticity and Learning by the H-ras/Extracellular Signal-Regulated Kinase/Synapsin I Signaling Pathway

Molecular and cellular studies of the mechanisms underlying mammalian learning and memory have focused almost exclusively on postsynaptic function. We now reveal an experience-dependent presynaptic mechanism that modulates learning and synaptic plasticity in mice. Consistent with a presynaptic function for endogenous H-ras/extracellular signal-regulated kinase (ERK) signaling, we observed that, under normal physiologic conditions in wild-type mice, hippocampus-dependent learning stimulated the ERK-dependent phosphorylation of synapsin I, and MEK (MAP kinase kinase)/ERK inhibition selectively decreased the frequency of miniature EPSCs. By generating transgenic mice expressing a constitutively active form of H-ras (H-rasG12V), which is abundantly localized in axon terminals, we were able to increase the ERK-dependent phosphorylation of synapsin I. This resulted in several presynaptic changes, including a higher density of docked neurotransmitter vesicles in glutamatergic terminals, an increased frequency of miniature EPSCs, and increased paired-pulse facilitation. In addition, we observed facilitated neurotransmitter release selectively during high-frequency activity with consequent increases in long-term potentiation. Moreover, these mice showed dramatic enhancements in hippocampus-dependent learning. Importantly, deletion of synapsin I, an exclusively presynaptic protein, blocked the enhancements of learning, presynaptic plasticity, and long-term potentiation. Together with previous invertebrate studies, these results demonstrate that presynaptic plasticity represents an important evolutionarily conserved mechanism for modulating learning and memory.

The pattern of dendritic development in the cerebral cortex of the rat.

The pattern of dendritic development of layer V pyramidal cells in the neocortex of the rat was studied using a variety of quantitative techniques in an attempt to determine what rules govern dendritic differentiation. Animals were sacrificed on postnatal days (P) 1, 3, 5, 7, 10, 15, 20, 25, 30 and 60, their brains impregnated with the rapid Golgi technique, and cells from the sensorimotor cortex examined for maximal apical and basilar dendritic field, number of dendritic branches at 20 micron intervals from the cell body, number of apical and basilar branch types (branching order), length of dendritic branch segments, and dendritic spine density. Primary dendrites are formed early in development, with no new ones formed after P7-10. Once a dendritic segment has bifurcated, all further development appears to occur at the tip, i.e. the trunk does not seem to undergo additional elongation, and new branches do not appear to form from the trunk. There is a plateau in dendritic differentiation close to the cell body after approximately P20; however, there is a continued increase in the length of terminal dendritic branches in the distal portions of the dendritic field into adulthood. During early development, dendrites bifurcate on reaching approximately 20-30 microns; however, during adulthood additional length is added to terminal dendrites without branching. Dendritic spines increase dramatically early in development, and then decline on proximal dendrites but continue to increase on terminal branches into adulthood. These results suggest that the terminal portion of the dendritic field remains plastic into adulthood, and that during development several general rules govern the pattern of dendritic differentiation.

Sequential changes in the synaptic structural profile following long‐term potentiation in the rat dentate gyrus: I. The intermediate maintenance phase

Long‐term potentiation (LTP), one of the most compelling models of learning and memory, has been associated with changes in synaptic morphology. In this study, LTP was induced and animals were sacrificed 1 h after the stimulation of the LTP group (induction / early maintenance phase). Synapses in the directly stimulated middle third of the dentate gyrus molecular layer (MML) were examined while synapses from the inner third of the dentate molecular layer (IML) of the LTP animals and both the MML and the IML of implanted animals served as controls. The total number of synapses per neuron, synaptic curvature, the presence of synaptic perforations, and the maximum length of the synaptic contact and active zone were examined. No overall change in the number of synapses per neuron was observed in the LTP tissue. LTP was associated with a significant increase in the proportion of perforated and irregular‐shaped synapses compared to controls. The increase in perforated synapses was particularly apparent in the proportion of concave perforated synapses. Nonperforated concave synapses were found to be significantly larger in potentiated tissue. The total synaptic length per neuron of synapses in a concave configuration was also significantly higher following potentiation. These results suggest that the specific structural profile associated with 1‐h post‐LTP induction, which differed from the profile observed at 24 h post‐induction, may represent a unique early phase of synaptic remodeling in a series of changes observed during LTP induction, maintenance, and decay. Synapse 36:286–296, 2000.

Effects of lead exposure during development on neocortical dendritic and synaptic structure

Abstract The effect of lead (Pb) on neocortical dendritic and synaptic development was examined in rats. Newborn pups were indirectly exposed to Pb by placing 4% Pb carbonate in their mothers diet from postnatal days 1 to 25. The mean brain weight of the Pb-treated animals was reduced 13.2%; neocortical thickness was reduced 13.9%. For analysis of dendritic development, layer V pyramidal cells from area 3 of the sensorimotor cerebral cortex were examined using the Scholl method to determine the number of dendritic branches at 20-μm intervals from the cell body. Although there were no differences in the number of processes leaving the cell body, reductions in dendritic branches were observed at distances greater than 40 μm, reaching significance at 80 and 100 μm. In addition the length of the primary apical dendrite was reduced 5.6% in Pb-treated animals. Synaptic parameters were examined in the molecular layer of the occipital cortex in ethanol phosphotungstic acid-stained tissue. There was a 22.7% reduction in the number of synapses per 15,000× field. No significant differences were observed in the following synaptic parameters measured at 300,000×: presynaptic length and thickness, postsynaptic length and thickness, and cleft width.

Synaptic development in the human fetus: A morphometric analysis of normal and Down's syndrome neocortex

Postmortem tissue was obtained from six normal and four Downs syndrome brains ranging in age from 12 to 40 weeks postconception. Tissue was processed for electron microscopy using routine osmium and EPTA staining procedures, and to examine synaptic development, photomicrographs were systematically taken throughout the molecular layer of the sensorimotor neocortex. The number of EPTA-stained synapses were consistently greater than the number of osmium-stained synaptic contacts. A progressive increase in synaptic density throughout the range of ages examined was observed for both normal and Downs syndrome tissue. There was also an increase with developmental age in apparent measures of synaptic maturity, e.g., an increased ratio of mature to primitive contacts and asymmetrical to symmetrical contacts. In normal tissue, pre- and postsynaptic membrane lengths were observed to increase with the ages studied, whereas synaptic membrane widths appeared to attain mature values by 29 weeks postconception. Cleft width remained fairly constant to 28 weeks postconception. Although direct statistical comparisons could not be made, in Downs tissue synaptic parameter development was generally less consistent and the parameters appeared to be reduced during the later stages of development studied.

Development and plasticity of the hippocampal-cholinergic system in normal and early lead exposed rats

A review of previous evidence suggested the possibility of a functional association between the effects of early lead (Pb) exposure, hippocampal damage and cholinergic deficiency. To further assess this possibility, Long-Evans hooded rat pups were exposed to Pb for the first 25 postnatal days via the maternal milk. Dams were fed either 4.0% PbCO3 or a Na2CO3 control diet throughout this period. At 30 and 115 days of age, the brains of Pb and control animals were processed for acetylcholinesterase histochemistry. Morphometric evaluation of the molecular layer of the hippocampal dentate gyrus indicated that while absolute increases in the dimensions of the afferent systems to the hippocampal dentate gyrus are observed between 30 and 115 days of age, no significant rearrangement in the pattern of lamination occurs during this time. No effects of Pb were seen on the development of the cholinergic innervation of this brain region at either of these ages. Unilateral perforant path transections performed on Pb and control animals at 100 days of age indicated reduced cholinergic plasticity in the molecular layer of the hippocampal dentate gyrus of Pb exposed animals, as indicated by AChE histochemistry. These findings indicate that a decrease in neuroanatomical plasticity may be a critical brain mechanism underlying the learning deficits observed following exposure to Pb.

Synaptic structural changes during development and aging.

Although a great deal is known about the development of synaptic number, comparatively little is known about the effects of development, and particularly aging, on the structure of the synapse. The present study examined synaptic structure in the molecular layer of the motor-sensory neocortex during early development (postnatal days (P) 1, 3, 5, 7, 10, 15, 20, 30), adulthood (P60, 90), and old age (28 months). Tissue was stained with osmium tetroxide (osmium) or ethanol phosphotungstic acid and the following synaptic characteristics were quantified: (1) presynaptic element length, area, thickness, maximal projection height and smoothness, and number and size of vesicles adjacent to the presynaptic element; (2) postsynaptic element length, area, and thickness; and (3) cleft width. There is an early developmental increase in synaptic element length, followed by an increase in thickness into adulthood. During development the height and width of the presynaptic dense projections increase, after which they remain stable. While the number of adjacent synaptic vesicles increases throughout the lifespan, there is a parallel decrease in their size. During the period of rapid synaptogenesis in this brain region there are no decreases in any of the synaptic structural parameters examined, indicating that newly generated synapses are either formed the same size as the existing mature synapses, or are extremely plastic and grow very rapidly. Unlike age-associated changes in synaptic number, no changes were found in synaptic structure during aging.

Neurobehavioral development following aluminum administration in infant rabbits

Aluminum (Al) is known to be a neurotoxic agent in some species, inducing neurofibrillary tangles, dendritic atrophy, and behavioral deterioration, and has been implicated as a possible agent in human Alzheimers disease and dialysis dementia. This study was conducted to assess the neurotoxic effects of Al in infant rabbits, and to compare the effects to those previously observed to follow exposure in the adult animal. Aluminum tartrate (2 microM) or physiologic saline was injected into the right lateral ventricle of 2-day-old (day P3) New Zealand white rabbits. The animals were trained in a step-down active avoidance task on P12 and retested 1 day later. They were killed on P20, and their hippocampal CA1 pyramidal cells examined for neurofibrillary tangles or prepared with the rapid Golgi stain for an examination of dendritic development. Additional animals were similarly infused with 1 or 3 microM Al for qualitative and some quantitative observations. No overt neurologic signs were observed in the 1- or 2-microM groups, however, most of the 3-microM group died between P10 and P20. Although there were no significant differences between the 2-microM and control animals on either learning or retention of the active avoidance task, deficits in retention of the task were observed in the 3-microM group. Neurofibrillary tangles in CA1 pyramidal cells were observed with dosages of 1 microM and higher. In the 2-microM group, the pattern of dendritic arborization in CA1 pyramidal cells was consistent with that expected for cells retarded in their development. These results have implications in terms of developmental differences in the neurobehavioral effects of Al.

Altered NMDA Sensitivity and Learning Following Chronic Developmental NMDA Antagonism

We have previously shown that chronic developmental administration of N-methyl-D-aspartate (NMDA) antagonists reduces synaptic development; however, on withdrawal from NMDA antagonism, there is a rebound period during which synaptogenesis exceeds control levels. The current research was undertaken to explore this period of withdrawal, using the noncompetitive antagonist phencyclidine (PCP), examining 2 behavioral measures in which the NMDA receptor is implicated: 1. NMDA-induced seizures, and 2. learning and memory in the Morris water maze. Using a protocol identical to that previously used to examine synaptic development, male Long-Evans rats were given 1 daily SC injection of either 10 mg/kg PCP or its physiological saline vehicle for a period of 15 days, beginning on postnatal Day 5 (P5) and ending on P20. Animals were then assessed for either sensitivity to NMDA-induced seizures on P21, P26, P36, or P56, or they were assessed for their acquisition performance and initial heading in the Morris water maze on P23, P26, P30, P38, and P75. Chronic treatment with PCP resulted in greater behavioral ratings of seizure activity after NMDA administration, observed 1 (P21), 5 (P26), and 15 (P36) days after the last injection of PCP, indicating increased sensitivity of the NMDA receptor/channel complex during this period after withdrawal from developmental NMDA antagonism. PCP-treated animals also required significantly more trials to reach criterion in the Morris water maze on P23, P26, and P30, and displayed significantly less accurate initial swim headings on all test days. The results are discussed in terms of the role of the NMDA receptor-channel complex in development and learning/memory processes.

Hippocampal mossy fiber pathway development in normal and postnatally lead-exposed rats.

Abstract Lead (Pb)-induced alterations in the postnatal development of the hippocampal formation have recently been suggested as underlying many of the behavioral changes observed after early Pb exposure. This study was thus designed to assess hippocampal mossy fiber pathway (MFP) development in both normal and postnatally Pb-exposed animals. From postnatal days 1 (P1) to 25, Long-Evans hooded rat pups were exposed to Pb through the maternal milk of dams fed 4.0% PbCO 3 , by weight, in ground laboratory chow. Control pups suckled dams receiving a Na 2 CO 3 control diet. On P25 and P60, weight-matched pairs of Pb-treated and control animals were selected for morphometric evaluation of the MFP following Timms silver sulfide staining. An increase in the overall length, as well as a reduction in width of the MFP in the CA3 region of the hippocampus of control animals were observed between P25 and P60. Examination of the MFP of P25 Pb-exposed animals indicated severe reductions in the development of this pathway for all parameters measured. At P60, significant reductions in MFP development were still observed in Pb-exposed animals; however, a large degree of recovery in the later-forming parts of the MFP was evident.