Evaluating synapses using unbiased stereology and electron microscopy (physical disector). a, A set of 4 electron micrographs overlaid with counting frames and taken from corresponding areas of 4 ultramicrotome serial sections in the BLA. Blue asterisks mark the “tops” of synapses. Tops were counted if they were within the counting frame or on the inclusion (green lines) but not the exclusion lines (red lines). Each micrograph was used as the reference and the look-up section with its matching pair. Tops of synapses are labeled according to their membrane specialization (as, asymmetric) and their postsynaptic target (sp, spine). The scale bar in the lower right panel equals 1 μm and is valid for all four panels. b, High power view of the area within the black box drawn on the left side of each of four sections in a. In panel 2, an asymmetric axospinous synapse is considered a top of a synapse because a synaptic cleft, postsynaptic density, and neurotransmitter vesicles are apparent in panel 2 but not 1. Similarly, in panel 3, an asymmetric axospinous synapse is considered a top of a synapse because a synaptic cleft, postsynaptic density, and neurotransmitter vesicles are apparent in panel 3 but not 2. The scale bar in panel 4 equals 500 nm and is valid for panels 1-4.

AMPH CPP produced structural indices of increased excitatory input. a, Electron micrographs demonstrating asymmetric, presumably excitatory, synapses in the BLA. In the left panel, the arrow points to a spine apparatus. In the middle panel, an asymmetric synapse on a spine head and an asymmetric synapse on the shaft of the dendrite are marked. In the right panel, an electron micrograph depicts a multisynaptic bouton in the BLA. Abbreviations: as, asymmetric specialization; sp, spine; d, dendrite; MSB, multisynaptic bouton. The scale bar equals 500 nm and is valid for all three panels. b, Pie chart illustrating the percentage of single synaptic boutons classified as axospinous, axodendritic, or synapses onto unknown postsynaptic targets (axo-unknown). c, The total number of asymmetric synapses and the number of asymmetric axospinous synapses were greater for rats in the AMPH CPP group at 1 h and 24 h after the CPP test compared to animals in the delayed pairing and saline groups. There were no differences in the total number of asymmetric synapses and the number of asymmetric axospinous synapses for rats in the delayed pairing group compared to the saline group at either 1 h or 24 h after the CPP test. Data are expressed as the mean ± S.E.M. d, The number of asymmetric axodendritic synapses and MSBs was greater for animals in the AMPH CPP group at 1 h and 24 h after the CPP test compared to animals in the delayed pairing and saline groups. There was no difference in the number of asymmetric axodendritic synapses and MSBs for rats in the delayed pairing group compared to the saline group at either 1 hr or 24 h after the CPP test. Data are expressed as the mean ± S.E.M. * p < 0.05 versus the delayed pairing and saline groups.

AMPH CPP increased other in vivo measures of excitatory synaptic drive. a, The amplitude of measured spontaneous EPSPs was increased by AMPH CPP. There was no difference in the amplitude of measured spontaneous EPSPs between neurons of the delayed pairing and saline groups. Data are expressed as the mean ± S.E.M. b, To obtain a measure that is less biased by analysis procedures for identifying synaptic events, the fluctuation of the membrane potential was used as an additional index of the frequency and amplitude of spontaneous synaptic events. The standard deviation (SD) of the mean membrane potential was increased in the AMPH CPP group (represented graphically as the distribution of the membrane potential). There was no difference between neurons in the delayed pairing and saline control groups in the SD of the membrane potential. Data are expressed as the mean ± S.E.M. c, To further provide evidence for a synaptic change after AMPH CPP, the paired-pulse ratio of a stimulated synaptic input was measured. The paired-pulse ratio, in the presence of DNDS, was increased for the AMPH CPP rats, which is consistent with increased synaptic drive. There was no difference in the paired-pulse ratio between the delayed pairing and saline groups. Data are expressed as the mean ± S.E.M. * p < 0.05 versus the delayed pairing and saline groups.

The formation of associations between context and drug modify the activity of the BLA. Neuronal activity in the BLA determines its contribution to behavior. a, Synaptic inputs (1) drive the activity of BLA neurons and its contribution to behavior (2). b, However, after repeated context-drug associations, there is an increase in the number of excitatory inputs to BLA neurons (1), resulting in increased BLA neuronal activity and drug-seeking behavior (2).

Behavioral conditioning with AMPH produced AMPH CPP. Animals treated with AMPH and immediately paired with a specific chamber spent more time in the AMPH-paired than the unpaired chamber during the CPP test. Animals treated with the delayed pairing regimen or with saline alone did not exhibit a preference for one chamber over the other during the CPP test. Data are expressed as the mean ± S.E.M. * p < 0.01 versus the delayed pairing and saline groups.

AMPH CPP produced functional indices of increased excitatory input. a, In parallel with an increase in the number of asymmetric synapses, there was an increase in the frequency of in vivo spontaneous synaptic events in animals conditioned with AMPH but not for animals in the saline-treated or delayed pairing control groups. The frequency of spontaneous events was observed as a decrease in the inter-event interval of events detected from individual BLA pyramidal neurons. b, The inter-event interval was significantly smaller for neurons in the AMPH CPP group compared to the delayed pairing and groups. There was no difference in the inter-event interval for neurons in the delayed pairing group compared to the saline group. Data are expressed as the mean ± S.E.M. c, Most spontaneous synaptic events occurred during barrages of synaptic activity. The number of events per barrage, or burst, of activity was greater in neurons of the AMPH CPP compared to the delayed pairing and saline groups. Barrages of EPSPs are depicted along with a raster plot for each trace to show the occurrence of each event. The mean number of EPSPs per burst did not differ between neurons of the delayed pairing and saline groups. Data are expressed as the mean ± S.E.M. * p < 0.05 versus the delayed pairing and saline groups.

Intracellular blockade of IPSPs with DNDS. a, Inclusion of 500 μM DNDS in the recording electrode resulted in a decrease in the occurrence of spontaneous hyperpolarizing events (examples of IPSPs at *; baseline frequency 0.83 ± 0.21 Hz; DNDS frequency 0.03 ± 0.01 Hz), measured between -60 to -50 mV. Also apparent are the action potentials evoked by spontaneous events (action potentials are truncated), indicative of their excitatory nature. b, DNDS blocked evoked IPSPs, visible as the downward deflection in these overlaid traces of the response to synaptic stimulation (at arrow) at a range of membrane potentials (−85 to −55 mV).