Figure 5.
Astrocytes form tripartite synapses and D-serine is a co-agonist for NMDARs
(A) Illustration of imaging window for recording of Ca2+ responses in NMDAR1 expressing PI neurons.
(B) Protocol of drug application for (C) and (D). Order of application was randomized for each fly.
(C) Average traces for glycine, D-serine, and L-serine application in 0 or 4 mM Mg2+ (ngly = 8, 25; nD-ser = 25, 26; nL-ser = 28, respectively). Line is a moving average and shaded band is SEM.
(D) D-serine, but not L-serine, activates NR1+ PI neurons (from left to right, n = 25, 25, 8, 8, 25, 25, 23, 23, 28, and 28). ∗p < 0.05, ∗∗p < 0.001, Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test.
(E) Protocol for ketamine application with NMDA, TTX, and D-serine.
(F) Averaged traces for D-serine and NMDA (blue) and D-serine, NMDA, and ketamine (red) (n = 15).
(G) Ketamine inhibits D-serine induced activation of NR1+ PI neurons. ∗∗p < 0.01, Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test. Transparent dots indicate outliers that are >2 SD from the mean. When outliers are excluded from the analysis, the relationship still holds (see Figure S5F).
(H) Astrocytes tile the SMP. 3D representation of three astrocytes (shades of blue) reconstructed from EM data shown with the mushroom body neuropil (gray) and the SMP neuropil (yellow), shown in frontal and lateral views. Cell bodies are located outside the neuropil (yellow arrow heads), and processes in the neuropil have little overlap (∗).
(I) Astrocytes engulf synaptic boutons and contribute processes to tripartite synapses (TPSs). Grayscale EM data with labeled glutamatergic boutons (green) and glial processes (blue). Presynaptic densities (arrow) and synaptic cleft (red) are indicated. Left: example where glial processes contact a large proportion of a bouton’s membrane but not the synapse. Right: example of a glial process directly adjacent to the synaptic cleft and opposite the presynaptic density (white arrow head).
(J) Astrocytic processes are significantly closer to glutamatergic than cholinergic synapses in the SMP. Kernel density estimates (lines) of the probability distributions (points) of distances of Glu or Ach synapses or a random draw from both sets to 3 SMP based astrocytes. Only synapses in direct vicinity (2 μm radius around processes <600 nm thick) are considered. Statistical analyses shown in Figure S5.
(K) Glutamatergic synapses are overrepresented in tripartite synapses versus their representation in synapses in the general 2 μm vicinity of astrocytic processes. Ratios of synapses by predicted neurotransmitter usage are shown.
See also Figure S5 and Video S1 for anatomy of astrocyte processes engulfing a presynapse and contributing to TPS.