Ultrastructural synaptic changes associated with neurofibromatosis type 1: a quantitative analysis of hippocampal region CA1 in a Nf1(+/-) mouse model.

Neurofibromatosis type 1 (NF1) is one of the most frequently diagnosed autosomal dominant inherited disorders resulting in neurological dysfunction, including an assortment of learning disabilities and cognitive deficits. To elucidate the neural mechanisms underlying the disorder, we employed a mouse model (Nf1(+/-) ) to conduct a quantitative analysis of ultrastructural changes associated with the NF1 disorder. Using both serial light and electron microscopy, we examined reconstructions of the CA1 region of the hippocampus, which is known to play a central role in many of the dysfunctions associated with NF1. In general, the morphology of synapses in both the Nf1(+/-) and wild-type groups of animals were similar. No differences were observed in synapse per neuron density, pre- and postsynaptic areas, or lengths. However, concave synapses were found to show a lower degree of curvature in the Nf1(+/-) mutant than in the wild type. These results indicate that the synaptic ultrastructure of Nf1(+/-) mice appears relatively normal with the exception of the degree of synaptic curvature in concave synapses, adding further support to the importance of synaptic curvature in synaptic plasticity, learning, and memory.

Eye-blink conditioning is associated with changes in synaptic ultrastructure in the rabbit interpositus nuclei.

Eye-blink conditioning involves the pairing of a conditioned stimulus (usually a tone) to an unconditioned stimulus (air puff), and it is well established that an intact cerebellum and interpositus nucleus, in particular, are required for this form of classical conditioning. Changes in synaptic number or structure have long been proposed as a mechanism that may underlie learning and memory, but localizing these changes has been difficult. Thus, the current experiment took advantage of the large amount of research conducted on the neural circuitry that supports eye-blink conditioning by examining synaptic changes in the rabbit interpositus nucleus. Synaptic quantifications included total number of synapses per neuron, numbers of excitatory versus inhibitory synapses, synaptic curvature, synaptic perforations, and the maximum length of the synapses. No overall changes in synaptic number, shape, or perforations were observed. There was, however, a significant increase in the length of excitatory synapses in the conditioned animals. This increase in synaptic length was particularly evident in the concave-shaped synapses. These results, together with previous findings, begin to describe a sequence of synaptic change in the interpositus nuclei following eye-blink conditioning that would appear to begin with structural change and end with an increase in synaptic number.

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.

Modeling behavioral recovery following lesion induction in the rat dentate gyrus.

Unilateral entorhinal lesions have enjoyed immense popularity as a model of recovery from damage. In part, the popularity has been supported the laminar organization of the hippocampal formation, which allows for the dissection of the contribution of individual afferent pathways to the recovery process. The commissural/associational pathway is of particular interest, since electrophysiological and gross anatomical data, although limited, have correlated sprouting in this pathway with behavioral recovery. Unfortunately, information relating recovery to synaptic structure is lacking. Addressing this issue, two analyses were conducted. Initially, a quantitative review of the literature reporting behavioral recovery following this type of lesion was conducted using meta-analytic techniques. Using this detailed information across decades of research, multiple linear regression analysis was conducted to address whether the morphological correlates of recovery could predict behavioral recovery. This resulted in an equation relating morphology and recovery that stood up well to several diagnostic tests. Moreover, this model suggests that synapse structure (in particular, synapse size and curvature, as well as terminal compartmentalization and the density of multi-synaptic terminals) holds a greater potential to predict behavioral recovery than increases in synapse number, which is typically seen as the optimal anatomical measure of recovery. This initial attempt to identify, quantify, and validate a model of lesion recovery is an important initial step in understanding how synaptic morphology may help mediate recovery of function.

Ultrastructural correlates of vesicular docking in the rat dentate gyrus.

To determine the extent to which CA1 synapses are typical of those found in other regions of the hippocampal formation, we have carried out a quantitative analysis of synapses in the middle molecular layer of the rat dentate gyrus, reconstructed from serial electron microscopy, and have compared these data with previous observations from CA1. In general, the morphology of synapses in areas CA1 and the dentate agree, other than an increased density of multisynaptic boutons. Thus, it seems that either area may form an equally effective model for the function of individual synapses in the hippocampal formation. In addition, the current study examines presynaptic curvature, which recent mathematical models have suggested may have profound effects on synaptic transmission. When synapses of distinct curvature profiles (i.e., presynaptically concave, convex, and flat) are examined, the average characteristics of these three synapse populations are distinct. In general, concave synapses have a greater number of morphologically docked vesicles, and thus, likely a greater probability of release. This, however, seems to be accounted for by the fact that these synapses are larger--the spatial density of docked vesicles remains identical across these curvature profiles. This study provides crucial data for further modeling of individual synapse function.

Changes in synaptic ultrastructure during reactive synaptogenesis in the rat dentate gyrus.

Advances in stereology, combined with continuing relevance to aging, as well as recovery from disease and injury make the reexamination of reactive synaptogenesis (RS) overdue. Moreover, recent mathematical models have suggested novel aspects of morphology, such as compartmentalization, may have profound effects on synaptic transmission. Given these novel findings, their correlation with other models of synaptic plasticity, and their potential significance for behavioral function, the precise nature of these changes need to be explored through quantitative morphometry. Towards this goal, the synaptic morphology of the dentate gyrus was assessed via serial electron microscopy at 3, 6, 10, 15, and 30 days following unilateral entorhinal cortex lesions. Foremost, the results showed that degree of curvature is a plastic feature of synapses. During RS, concave synapses showed an immediate/long-lasting increase in curvature, suggesting their importance in the compensation response. Flat synapses showed unique changes in growth, having implications for development and activation following synaptogenesis. Moreover, changes in size and curvature showed a different dynamic depending on proximity from damage. In the directly denervated MML, synapses showed an increase in curvature proportionate to increases in size. In the neighboring IML, however, these changes were independent-increases in curvature far surpassed synaptic growth.

Comparative analyses of synaptic densities during reactive synaptogenesis in the rat dentate gyrus.

Advancements in the field of synaptic plasticity have created the need for a reexamination of classic paradigms using new and more precise techniques. One prime candidate for such a reexamination is the process of reactive synaptogenesis (RS). Since the time course of RS was initially outlined in the 1970s and 1980s, advances in stereology have allowed for better characterization of synaptic ultrastructure. Thus, a reexamination was undertaken in the hippocampal dentate gyrus by assessing the densities and proportions of several synaptic subtypes in Long-Evans hooded rats at 3, 6, 10, 15 and 30 days following induction of unilateral lesions of the entorhinal cortex. Although initial synaptic loss in the denervated region was similar to previous reports, recovery during the first 30 days is not as dramatic as previously observed. Following lesioning, concave and perforated synapses retained pre-lesion density despite massive degeneration, underscoring their theoretical importance in plasticity and maintenance of neural function. Convex synapses showed opposite changes, having implications for excitation/inhibition imbalance following lesion induction. These complementary alterations in synaptic structures support ultrastructural changes as a means for compensation following synaptic loss. Nearby areas also seem to participate in this response, with a striking similarity to other models of plasticity, such as long-term potentiation.

Complementary techniques for unbiased stereology of brain ultrastructure.

Estimating synaptic density is central to any examination of synaptic change. Barring techniques involving exhaustive sectioning, the traditional method for this estimation is the double dissector technique, which derives a ratio of synapses per neuron. This ratio, however, may not always equate to an absolute number of synapses. In contrast, the unbiased brick, while free of these problems, is limited by changes in reference space and cannot provide information on synaptic connectivity. Thus, the ideal analysis is to derive both synaptic measures. When these measures agree, one can discount changes in extraneous factors influencing synaptic counts.

Unique changes in synaptic morphology following tetanization under pharmacological blockade.

Long-term potentiation (LTP) in the hippocampus has been associated with changes in synaptic morphology. Whether these changes are LTP-dependent or simply a result of electrophysiological stimulation has not yet been fully determined. This study involved an examination of synaptic morphology in the rat dentate gyrus 24 h after electrophysiological stimulation sufficient to induce LTP. In one group, ketamine, a competitive NMDA antagonist, was injected prior to stimulation to block the formation of LTP. Synaptic morphological quantification included estimating the total number of synapses per neuron, determining synaptic curvature and the presence of synaptic perforations, and measuring the maximal PSD profile length of the synapses. The results indicated that most of the changes observed following the induction of LTP (increases in the proportion of concave-shaped synapses, increases in perforated concave synapses, and a decrease in the length of nonperforated concave synapses) are not observed under ketamine blockade, suggesting that they are LTP-specific and not simply the result of tetanic stimulation. Ketamine was associated, however, with several novel structural changes including a decrease in the length of the perforations in the concave perforated synapses, a reduction in the number of convex perforated synapses, and a nonlayer-specific increase in synaptic length compared to controls. Based on previous research, this combination of morphological characteristics is potentially less efficacious, which suggests that synapses that are tetanized but not potentiated, due to pharmacological blockade, appear to undergo opposing, compensatory, or homeostatic changes. These results support the suggestion that synaptic morphology changes are both stimulation- and area-specific, are highly complex, and depend on the specific local physiology.

The role of synaptic morphology in neural plasticity: structural interactions underlying synaptic power.

The study of synaptic plasticity has revealed a common cascade of ultrastructural events across several paradigms. Most notable of these paradigms are development, long-term potentiation (LTP), and adult reactive synaptogenesis (RS). These plastic neural events are discussed in terms of major categories of synaptic morphological change--synaptic density, curvature, and perforations, as well as the size of synaptic elements. The potential functional implications of these morphological changes are reviewed, along with considerations based on recently developed mathematical models of synaptic function. These considerations are then incorporated into the common structural alterations observed during multiple forms of synaptic activation, producing a sequential model supporting increased efficacy associated with neural plasticity. The data suggest that during a plastic challenge, synapses move through a continuum of morphological change, dependent upon the interaction of structural parameters and their effect on various aspects critical to synaptic efficacy. This complex interplay of morphological alterations and synaptic types over time and location may form a critical aspect of neural plasticity.