Injections of the retroviral vector CAG-GFP into the dentate gyrus of 6-to 7-week-old mice resulted in the labeling of newly generated granule cells with GFP6,15
. We observed axons and axon terminals in the hilus and the CA3 areas using confocal microscopy. We found that the mossy fiber boutons in CA3 were substantially larger at 28 and 75 days post viral injection (dpi) than those at 17 dpi (). At later time points, the mossy fiber boutons had long extensions, as previously described16
. The size of the GFP-labeled mossy fiber boutons at 75 dpi was similar to that of the mature neurons born during early postnatal development ((P
= 0.18) ). Notably, the size of the mossy fiber boutons in the hilus did not change significantly during the maturation of newborn neurons (P
= 0.65). Furthermore, the average size of the mossy fiber boutons in CA3 was significantly larger than that in the hilus (P
< 0.001, Student’s t
test), except for new neurons at 17 dpi.
Confocal microscopy of mossy fiber boutons from newborn neurons
We then used immuno-electron microscopy for GFP and serial-sectioning to analyze boutons that were formed by labeled axons. A total of 44 axonal boutons were sectioned, all of which formed synapses on either CA3 or hilar neurons (). We found synapses on thorny excrescences, dendritic spines or dendrites, which were unambiguously identified by the presence of at least four presynaptic vesicles within 100 nm of the active zone, a well-defined synaptic cleft, and postsynaptic density. We observed large mossy terminals synapsing with thorny excrescences () as well as boutons synapsing with spiny dendrites in the CA3 area () and with thorny excrescences in the hilus (). In addition, the thin GFP-positive axons (~0.1 to 0.2 μm in diameter) also formed en passant
synapses with thin dendritic shafts at each of the time points in both CA3 and the hilus (). In our serial sections, these thin dendrites were aspiny, suggesting that they arose from GABAergic interneurons17
. These aspiny dendrites were postsynaptic to both the GFP-positive axons and other thin axons that lacked GFP (). Furthermore, there were no intervening glial processes between the thin GFP-positive axons and the unlabeled axons. Further evidence of newborn granule cells forming synapses with GABAergic interneurons is provided below. Thin GFP-positive axons also synapsed onto the spines of thin dendrites, probably arising from spiny interneurons located in the stratum lucidum of CA3 (ref. 18
Electron micrographs illustrating the diversity of synapses made by newly generated neurons
We next examined the connectivity of GFP-positive mossy fibers that synapsed with dendrites of CA3 pyramidal cells (). At 17 dpi, 6 out of 8 GFP-positive mossy fiber boutons had formed synapses with the dendritic shafts of pyramidal cells (), whereas adjacent thorny excrescences and their spines19
were contacted by large, unlabeled mossy fiber boutons. The other two GFP-positive boutons at 17 dpi synapsed onto thorny excrescences that were shared with GFP-negative boutons. At 28 dpi, 10 out of 11 GFP-positive mossy fiber boutons shared both the thorny excrescence and spines with one or two GFP-negative mossy fiber boutons ( and ), whereas one bouton had synapsed directly with the dendritic shaft of a pyramidal neuron. At 75 dpi, 10 out of 12 GFP-positive mossy fiber boutons were entirely associated with one individual thorny excrescence with spines, and this synaptic complex was enveloped by astrocytic processes (). The other two boutons shared a spine with a GFP-negative terminal.
Temporal progression of the morphology of GFP-positive axon terminals in the CA3 area
The number of spines enveloped by each GFP-positive mossy fiber bouton increased significantly from 0.1 at 17 dpi (n
= 7) to 2 at 28 dpi (n
= 9) and 3.8 at 75 dpi (n
= 10) (17 versus 27 dpi, P
<0.05; 28 versus 75 dpi, P
< 0.05). In comparison, there were 2.9 spines per GFP-negative bouton (n
= 11; ). The total cross-sectional area of GFP-positive mossy fiber boutons also substantially increased over this time period, with the greatest increase occurring between 17 and 28 dpi (). Although some of the presynaptic vesicles were obscured by the immunostaining for GFP, which resulted in an underestimation of their total number, we found a significant increase in presynaptic vesicles from 60 at 17 dpi to 120 at 28 dpi, 179 at 75 dpi and 240 in GFP-negative boutons (17 versus 75 dpi, P
< 0.05; 75 dip versus GFP negative, P
< 0.005; ). Large dense-core and large clear-core vesicles were also present at all of the time points examined. The number of active zones also increased from 3 at 17 dpi to between 5 and 6 at 75 dpi and in GFP-negative boutons (17 versus 75 dpi, P
< 0.05; ). A model for the maturation of axonal boutons is shown in Supplementary Figure 1
We then investigated whether the output synapses formed by newly generated neurons are functional. The most direct approach would involve double patch recordings of presynaptic GFP-positive cells connected to target neurons in acute brain slices. However, such paired recordings are technically challenging, as each cell only contacts a small fraction of all possible targets20,21
and axons may be severed during slice preparation. To overcome this problem, we developed a retroviral vector carrying ChR2 (refs. 22,23
) and a fluorescent reporter gene (GFP or monomeric red fluorescent protein 1, mRFP1) with which to transduce the progeny of dividing progenitor cells of the dentate gyrus of adult mice. In these conditions, large populations of adult-born granule cells could be readily activated by blue light in acute hippocampal slices (Supplementary Fig. 2
online). Thus, ChR2 can now be used to evoke neurotransmitter release from the mossy fiber terminals of a large population of adult-born granule cells to find connected target neurons using whole-cell patch-clamp recordings.
To determine whether new neurons establish functional synapses with their targets, we used brain slices (3–4 months after retroviral labeling) containing several dozen fluorescent cells to assess responses to light-driven mossy fiber stimulation in randomly selected putative postsynaptic cells. Cellular targets of mossy fibers are distributed in the hilus, GCL/hilar border and CA3 region, and include GABAergic interneurons, mossy cells and pyramidal neurons21
. We carried out whole-cell recordings in randomly selected neurons of these areas and determined their identity by considering electrophysiological and anatomical parameters, including location of the soma, morphology, firing properties and spontaneous synaptic activity (Supplementary Methods, Supplementary Fig. 3 and Supplementary Table 1
online). Briefly, interneurons are located in the hilus and the GCL/hilar border, spike with high frequency and have a pronounced afterhyperpolarization (AHP); mossy cells are located in the hilus, reach lower firing frequencies, lack AHP and show a high frequency of spontaneous synaptic activity; and pyramidal cells are found in the CA3 pyramidal layer and display intermediate spiking and AHP properties.
We tested light-induced postsynaptic responsiveness in 107 putative target cells that included 76 interneurons, 16 mossy cells, 11 pyramidal cells and 4 unclassified neurons (, an example of a successful recording obtained from a cell identified as a GABAergic interneuron of the GCL/hilar border is shown in ). Prolonged light stimulation of the slice elicited a high frequency of inward synaptic currents (). Repetitive light pulses of short duration delivered at low frequency (10 ms, 0.2 Hz) elicited postsynaptic currents (PSCs) that were time-locked to the stimulus (). For each cell, we averaged tens to hundreds of stimuli presentations to distinguish light-evoked PSCs from uncorrelated spontaneous activity (). PSCs were successfully evoked in 14 target cells () that corresponded to GABAergic interneurons (n = 11; two examples in ), one mossy cell (), one CA3 pyramidal cell () and one unclassified neuron.
Light-evoked neurotransmitter release from adult-born neurons expressing ChR2
Light-evoked synaptic responses should be subject to blockade by the appropriate receptor antagonists. Mossy fiber stimulation should typically evoke glutamatergic responses, although other possibilities have also been proposed, especially in developing neurons or after seizures24,25
. Polysynaptic inhibitory and excitatory signals can also be observed if mossy fiber activation recruits local interneurons or mossy or pyramidal cells, respectively26-28
. Both types of responses ultimately depend on glutamate release from mossy fibers and should therefore be blocked by the mixed AMPA/NMDA receptor antagonist kynurenic acid (Kyn, 4–10 mM). Light-evoked responses of adult-born neurons showed a pharmacological profile that was consistent with this notion, as Kyn consistently abolished evoked PSCs in a reversible manner (). The metabotropic GluR agonist DCG-IV, which reduces neurotransmitter release at mossy fiber terminals29
, also blocked PSCs (example shown in ; similar results were obtained in two experiments).
Pharmacological treatments demonstrate glutamate release by adult-born neurons
We also tested the effect of the GABAA receptor antagonist bicuculline methiodide (BMI, 20 μM) to identify putative feedforward inhibition. In 3 out of 5 cases, BMI elicited little or no changes in PSC amplitude, whereas subsequent addition of Kyn completely abolished the responses, confirming their glutamatergic nature (). In the remaining two cases, BMI blocked a substantial proportion of the PSC, but complete block was also observed in the presence of Kyn, consistent with feedforward GABAergic inhibition that is dependent on glutamate release from mossy fiber terminals ().