Increases in the Number of Multiple Synapse Boutons Associated with Learning and Memory

Studies reviewed above indicate that learning of a new behavior is typically accompanied by synaptogenesis in a learning-relevant region of the mammalian brain, which is manifested by an increase in the number of synapses. Another ultrastructural synaptic alteration involving synaptogenesis is multiple-synapse bouton (MSB) formation, which is characteristic of various forms of plasticity (reviewed in 26). Each MSB establishes separate synaptic contacts with two or more discrete postsynaptic elements (Figure 23.2) instead of only one synapse with a single postsynaptic element as is the case for a single-synapse bouton. We next consider studies estimating MSB numbers after learning.

3.2.1. Effect of Acrobatic Task Acquisition

One such study27 used the behavioral paradigm of acrobatic motor learning developed by Black et al.15 and summarized above (see Section 3.1.1.). Adult rats were trained to traverse an elevated obstacle course for 30 days. Control animals were given a voluntary motor exercise on a wheel or were kept inactive in their home cages. The paramedian lobule of the cerebellar cortex was examined. The numerical density of parallel fiber varicosities establishing synaptic contacts with two Purkinje cell spines was assessed per unit volume of the molecular layer, and the numerical density of Purkinje cells was estimated per unit volume of cell body layer, using the method of physical disector. MSB number per Purkinje cell was calculated from the density values. MSBs were counted if they were observed contacting two postsynaptic spines in a single section plane. This approach to counting of MSBs greatly underestimates their occurrence21, and the magnitude of the underestimation depends on MSB size, shape, and orientation. Nevertheless, the results of this experiment indicate that rats undergoing acrobatic motor learning have more MSBs per neuron than active or inactive controls. The formation of additional MSBs probably contributes to the increase in overall synapse number detected earlier in the molecular layer of adult rat cerebellar cortex following acrobatic learning15,16,18,19. Additionally, the establishment of a second synaptic contact between a preexisting parallel fiber varicosity and another Purkinje cell spine was interpreted as an indication of a selective, learning-related strengthening of this particular pathway that may represent a mechanism of neural encoding27.

3.2.2. Effect of Trace Eyeblink Conditioning

The work from our laboratory has demonstrated that the total number of all synapses and of axospinous synaptic junctions in the rabbit CA1 stratum radiatum remains stable 24 h after trace eyeblink conditioning25. It was possible, however, that learning- induced synaptogenesis under such conditions might be confined to a specific subset of synaptic connections formed by MSBs. The validity of this supposition was tested in a study26 using the behavioral protocol of trace eyeblink conditioning outlined above (see Section 3.1.5.). Modern stereological techniques and serial section analyses were employed for obtaining unbiased estimates of the total number of MSBs in the stratum radiatum of hippocampal subfield CA1. All MSBs entirely included in section series were identified as axonal swellings and with two or more separate spines. The

Figure 23.2. Electron Micrographs of Consecutive Serial Sections (A-C) Through the Rat CA1 Stratum Radiatum Demonstrating a Multiple-Synapse Bouton. The bouton (labeled MSB in (A)) makes synapses with two spines (labeled SP1 and SP2 in (A)).

numerical density of MSBs per unit volume was assessed with physical disectors, and the total MSB number was calculated as the product of MSB numerical density and CA1 stratum radiatum volume. It was found that conditioned rabbits had significantly more MSBs (18%) as compared with either pseudoconditioned or untrained controls, whereas the difference on this measure between the two control groups was small (2.8%) and not statistically significant. These data demonstrate that trace eyeblink conditioning is associated with the formation of MSBs. For MSBs to be formed, some axonal terminals must make new synaptic contacts with additional dendritic spines. Therefore, hippocampus-dependent associative learning promotes a specific synaptogenesis resulting in the formation of MSBs.

The latter structural synaptic change, which is not accompanied by a gain in total synapse number25, can be explained based on the phenomena of spine protrusive motility and turnover that were initially observed in brain slices maintained in vitro (reviewed in 28) and then confirmed by in vivo experiments (reviewed in 2, 3). We will consider, as an example, the data reported by Svoboda and colleagues29, 30 who imaged apical dendritic segments of mouse neocortical pyramidal neurons for periods of days to months. Under conditions of stable sensory input (i.e., visual and whisker-related stimuli), some spines were observed for only a few days or less before retracting into parent dendrites. Other spines formed de novo and grew toward axonal varicosities. Despite such a turnover of spines, their density per unit length of dendritic branch remained stable, whereas the retraction or elongation of dendritic and axonal processes did not occur. Subsequent serial section electron microscopy of the dendritic segments imaged in vivo revealed that spine retraction was associated with synapse elimination and spine outgrowth with synapse formation. Although the remarkably rapid turnover and associated motility of transient spines are most prominent during early postnatal development, they are also retained in adulthood, though to a lesser degree. In the somatosensory cortex, for example, the transient spine fraction reaches ~65% in 16-25-old-day mice but still amounts to ~25% in middle-aged (6-month-old) mice. Interestingly, plasticity of cortical synaptic fields following partial sensory deprivation was accompanied by a marked increase in spine turnover, but not by a change in spine density.

These data, taken together, suggest at least two models of structural plasticity that may underlie the learning-related addition of MSBs26. According to one model, newly formed spines establish synaptic contacts with single-synapse boutons activated by conditioning stimulation; simultaneously, some spines contacting nonactivated single-synapse boutons are retracted into parent dendrites. An alternative model postulates that some existing postsynaptic spines contacting nonactivated single-synapse boutons leave their presynaptic partners, relocate to boutons activated by conditioning stimulation, and synapse with them. In either case, some boutons would lack synaptic contacts, and such boutons without synapses are indeed encountered in the CA1 stratum radiatum31.

The structural reorganization of synaptic connectivity following trace eyeblink conditioning may have different functional implications, depending on the origin of the postsynaptic spines contacting the newly formed MSBs. It has been documented with the aid of three-dimensional reconstruction from serial sections that MSBs in the CA1 stratum radiatum can synapse with spines arising from the same or different dendrites32, 33. In our study, however, we could not identify parent dendrites of many spines that were postsynaptic to MSBs. If these spines emerged from the same dendrite, the strength of the conditioned synaptic input to target CA1 neurons would be amplified. If, on the other hand, multiple spines synapsing with additional MSBs emanated from dendrites of different neighboring neurons, this might contribute to the synchronous activation of the latter and thus to the assembly of functional multineuronal units tuned to the synaptic input activated by conditioning stimulation. In either case, the synaptic plasticity that develops as a result of trace eyeblink conditioning would enhance synaptic inputs to and outputs from CA1 pyramidal neurons.

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