Three-dimensional relationships between hippocampal synapses and astrocytes

Abstract

Recent studies show that glutamate transporter-mediated currents occur in astrocytes when glutamate is released from hippocampal synapses. These transporters remove excess glutamate from the extracellular space, thereby facilitating synaptic input specificity and preventing neurotoxicity. Little is known about the position of astrocytic processes at hippocampal synapses. Serial electron microscopy and three-dimensional analyses were used to investigate structural relationships between astrocytes and synapses in stratum radiatum of hippocampal area CA1 in the mature rat in vivo and in slices. Only 57 +/- 11% of the synapses had astrocytic processes apposed to them. Of these, the astrocytic processes surrounded less than half (0.43 +/- 22) of the synaptic interface. Other studies suggest that astrocytes extend processes toward higher concentrations of glutamate; thus the presence of astrocytic processes at particular hippocampal synapses might signal which ones are releasing glutamate. The distance between nearest neighboring synapses was usually (approximately 95%) <1 microgram. Astrocytic processes occurred along the extracellular path between 33% of the neighboring synapses, neuronal processes occurred along the path between another 66% of the neighboring synapses, and only 1% of the synapses were close enough such that neither astrocytic nor neuronal processes occurred between them. These morphological arrangements suggest that the glutamate released at approximately two-thirds of hippocampal synapses might diffuse to other synapses, unless neuronal glutamate transporters are more effective than previously reported. The findings also suggest that physiological recordings made from hippocampal astrocytes do not uniformly sample the glutamate released from all hippocampal synapses.

Fig. 1.

Astrocytic profiles and 3D reconstructions illustrating their relationships to synapses in the middle of stratum radiatum of hippocampal area CA1. a, Astrocytic profiles are illustrated (blue) on a single thin section in the vicinity of 11 synapses (arrows). The identity of each of the astrocytic profiles occurring on this single EM section was confirmed by viewing serial sections. On this one section, three synapses have astrocytic profiles at their perimeters (arrowheads). To identify whether astrocytic processes occurred at the perimeter of the other synapses, they were viewed through serial sections, and four more of the synapses were found to have astrocytic profiles at their perimeters, for a total of seven.b–d, 3D reconstructions illustrate astrocytic profiles (blue), boutons (green), spines (gray), and PSDs (red). Astrocytic profiles surround (b) 50% of the perimeter of this macular synapse and (c) 3% of the perimeter of a perforated synapse, both occurring on SSBs. In d, three synapses occur with a single presynaptic bouton, called a multiple synapse bouton, and a single astrocytic process surrounds 75, 64, or 100% of the perimeter of each synapse fromleft to right, respectively. Scale bars: 1 μm (shown in b for b–d).

Fig. 2.

The axon–spine interface.a, Schematic illustration of the PSD, the nonsynaptic interface, and the perimeter of the axon–spine interface vieweden face. b, Five 3D reconstructions of the axon–spine interface arranged clockwise in order of increasing PSD size. These reconstructions demonstrate the variability in the sizes and shape of the PSDs and the nonsynaptic interfaces. Scale bar: 1 μm. c, Larger synapses have larger nonsynaptic interfaces as well.

Fig. 3.

Percentage of synapses with astrocytic profiles at the perimeter of the axon–spine interface. (Height of bar = the mean across 4 series volumes; individual values are superimposed; totaln = 229 synapses.)

Fig. 4.

Fraction of the perimeter of individual hippocampal synapses that is surrounded by astrocytic profiles (mean ± SD; n = 131 synapses). The amount of astrocyte surrounding the perimeter at macular synapses (n = 103) was greater than at perforated synapses (n = 28; *p < 0.01). None of the other differences reached statistical significance.

Fig. 5.

Five pairs of nearest neighboring (NN) synapses. EM (a) and schematic (b) illustrations of the extracellular path among a cluster of neighboring synapses that have neuronal (N, dotted) processes or neuronal membranes of the axon–spine interface between them. NN path lengths (gray) denoted by double arrowssignify mutual NN paths, whereas those with single arrows signify one-way NN paths. Path lengths inb from left to right are 0.85, 0.47, and 0.15 μm. c, d, EM and schematic representation of NN synapses that have both neuronal and astrocytic (A, striped) profiles along that path between them, which is 1.1 μm. In e andf, the two synapses are immediately adjacent to one another with almost no distance (<0.01 μm) between them. In this case, the two NN synapses are on an MSB; in the only other case where synapses were this close to one another, the NN synapses were on two different boutons. Scale bar (shown in b): a, b, 1 μm; (shown in d) c–f, 1 μm.

Fig. 6.

Percentage of synapses on SSBs and MSBs.a, Of the 229 synapses evaluated, 187 were on SSBs and 42 were on MSBs. b, Only 32 of the synapses on MSBs had the NNs within the series volume: 14 had their NN on the same MSB, whereas 18 had their NNs on a different bouton.

Fig. 7.

Distances and composition of processes along the paths between NN synapses. a, These measurements were obtained for 141 synapses. The mean edge-to-edge path length was 0.42 ± 0.2 μm, and the mean center-to-center path length was 0.65 ± 0.3 μm. b, Of these, the path between 48 NN synapses had both astrocytic and neuronal membrane along it, whereas 91 had neuronal but no astrocytic membrane; only two pairs of NN synapses had neither astrocytic nor neuronal membrane between them.

Fig. 8.

Hypothetical model depicting how the differential distribution of astrocytic processes at hippocampal synapses might reflect synaptic activity. a, A previously releasing synapse (Synapse 1) has an astrocytic process bordering its perimeter where glutamate might otherwise escape from the axon–spine interface. A synapse not releasing glutamate (Synapse 2, dark gray) has no astrocytes bordering its cleft. b, The astrocytic process has grown toward Synapse 2 (no longer shaded) as changes in synaptic function have caused it to release sufficient glutamate so that some escapes from its perimeter.