RE-STRUCTURING OF SYNAPSES 24 HOURS AFTER INDUCTION OF LONG-TERM POTENTIATION IN THE DENTATE GYRUS OF THE RAT HIPPOCAMPUS IN VIVO M. G. STEWART,*² E. HARRISON,* D. A. RUSAKOV,*‡ G. RICHTER-LEVIN§ and M. MAROUN§ *Department of Biology, The Open University, Walton Hall, Milton Keynes MK7 6AA, UK §Department of Psychology, University of Haifa, Mount Carmel, Haifa 31905, Israel ‡Dept of Neurophysiology, National Institute for Medical Research, Mill Hill, London, UK Abstract —In male rats, long-term potentiation was induced unilaterally in the dentate gyrus, either by high frequency (200 Hz) or theta rhythm stimulation. Structural synaptic changes were examined 24 h after induction using quantitative electron microscopy. A disector technique was employed in order to estimate the density of synapses (using 70–80-nm sections) and of granule cell nuclei (using 2-mm sections) in the middle, and inner molecular layer in both hemispheres. Synaptic height and total lateral areas of synaptic active zones per unit tissue volume were assessed via assumption-free stereological techniques coupled with image analysis. The results obtained indicated that both synaptic density and number (corrected per neuron) of axo-spinous, but not axo-dendritic, synapses were 40% higher in the middle, but not inner molecular layer of the potentiated hemisphere compared to the contralateral (control hemisphere). No significant inter-hemispheric difference was found in the volume densities of lateral areas of active zones. These data suggest that 24 h after long-term potentiation induction, active zones of existing axo-spinous synapses either split forming separate contacts, or decrease in size while new synapses are formed.  2000 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: axo-spinous, dentate, morphology, potentiation, synapse restructuring. Learning and memory are believed to involve use-dependent changes in synaptic weight. The cellular machinery, which underlies such changes, has been extensively studied using the physiological paradigm of long-term potentiation (LTP) of synaptic transmission in the hippocampus. 5 However, the cellular basis of LTP expression still represents the subject of considerable debates. 1,6,30,32–36 Whilst it is reasonable to assume that long-lasting changes of synaptic efficacy must be supported by structural alterations at least at the level of membrane domains, 48 no universal agreement exists concern- ing the significance of these alterations. Nevertheless, the plausibility of real-time, function-dependent changes in the appearance of living synaptic elements (dendritic spines) has been demonstrated in vitro. 29,31,40,41 At the electron microscope level, however, technical limitations may restrict the sample size 46 and identification of potentiated synapses is still an unresolved issue. Therefore, a large proportion of ultrastructural studies concerning hippo- campal LTP, have explored a relatively homogeneous popu- lation of perforant path synapses on granule cell dendrites (confined mostly to the medial molecular layer of the dentate gyrus). Van Harreveld and Fifkova 47 demonstrated an increased width of dendritic spine profiles in potentiated tissue 6 min to 23 h after the induction of LTP. Wenzel et al. 51 showed a set of synaptic changes induced by high- frequency stimulation of the perforant path, whilst Desmond and Levy 12,13 demonstrated an increase in the number of axo-spinous synapses with concave profiles of synaptic active zones (AZs) 60 min after LTP was induced. In parallel, it was reported that high-frequency stimulation of the perforant path results in an increased number of synaptic vesicles located in the proximity of the AZ membrane, 4,13 and in formation of spinule-like membrane invaginations in pre-synaptic ter- minals. 42 In area CA1 of the hippocampus, a profound (up to 48%) increase in the number of axo-dendritic spines was found following the induction of LTP with high-frequency stimulation in vitro. 10 There is no reason to believe that many individual synaptic features (e.g. spinules or AZ curvature) cannot be analysed qualitatively in regular, single section electron micrographs. However, in such micrographs, the occurrence of each struc- tural element is proportional to its size, and thus can be misleading. This bias can be partly overcome by employing an analytical stereological correction factor 13,14 or, more comprehensively, by adopting a ‘design-based’ stereological technique, the disector. 43 In studies of synaptic morphology in the dentate gyrus, one prominent correlate of LTP in vivo was found, using the disector technique, approximately 1 h after the tetanus: an increased number of completely partitioned AZs at axo-spinous synapses. 19–22 Buchs and Mu ¨ller, 8 showed a greater proportion of perforated synaptic densities 30– 40 min after induction of LTP in organotypic hippocampal slices. In general, it has been argued that changes in the shape of synaptic elements, rather than changes in synaptic numbers, are indicative of the early stage of LTP (up to 1–2 h) at perforant path synapses. 8,14 However, a serial section study has indicated an increased density of dendritic spines in the medial molecular layer 30 min after tetanisation. 2,47 More recently, Andersen and F. Soleng 3 demonstrated an increase in spine density in granule cell dendrites in the middle Synaptic correlates of hippocampal LTP 221 221 Neuroscience Vol. 100, No. 2, pp. 221–227, 2000  2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00 PII: S0306-4522(00)00295-5 Pergamon www.elsevier.com/locate/neuroscience ²To whom correspondence should be addressed. Tel.: + 44-1908-653448, fax: +44-1908-654167. E-mail address: m.g.stewart@open.ac.uk (M. G. Stewart). Abbreviations: LTP, long-term potentiation; AZ, active zone (of synapse); MML, middle molecular layer; IML, inner molecular layer; N V , synapse density; Hsyn, synapse height; SAZ, lateral area of individual active zones.