Supporting Information

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Supporting Text
Supporting Table 1
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8

Fig. 6. Intrinsic electrophysiological properties of isolated interneurons and their comparison with electrical characteristics in vivo. (A) Whole-cell patch clamp with a 5-ms depolarizing current injection was used to characterize the passive membrane properties, excitability and synaptic connectivity of interneurons in vitro (n > 15 cells per parameter per group). (B) Spontaneous inhibitory postsynaptic current (sIPSC) amplitude distributions showed enhanced synaptic efficacy as a factor of survival time. (C) Comparison of known morphological and electrophysiological properties of cholecystokinin (CCK)⁺ interneurons in vitro and in vivo. CB₁R, CB₁ cannabinoid receptor; VIP, vasoactive intestinal polypeptide; VGLUT, vesicular glutamate transporter; AP, action potential.

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Fig. 7. Endocannabinoid actions on synaptogenesis of isolated interneurons. Endocannabinoid-induced synaptogenesis was assessed by Western blot detection of vesicular GABA transporter (VGAT), synaptophysin, presynaptic markers of inhibitory synapses, and the developmentally regulated neurofilament b-III-tubulin (1). Interneuron cultures were lysed in RIPA buffer containing protease and phosphatase inhibitors, denatured in Laemmli buffer and protein samples were analyzed by SDS/PAGE with rabbit anti-VGAT (1:4000), rabbit anti-synaptophysin (1:1,000; both from Synaptic Systems, Goettingen, Germany), and mouse anti-b-III-tubulin (1:1,000, Promega) primary antibodies as described in ref. 1. All blots were scanned in a Storm 840 fluorimager and quantifications were done with IMAGEJ 1.32j (National Institutes of Health). (A) Anandamide (AEA) alone or in combination with brain-derived neurotrophic factor (BDNF) did not affect the expression of either synaptic markers. In contrast, BDNF significantly increased synaptophysin expression at 6 days in vitro. b-III-tubulin concentrations were significantly increased by combined treatment. Representative blots revealing differences in expression of synaptic markers. Samples from identical numbers of neurons were loaded. (B) Summary of densitometric analysis from triplicate experiments. o, P < 0.05; oo, P < 0.01 vs. control. Data were expressed as mean ± SEM.

1. Berghuis, P., Dobszay, M. B., Sousa, K. M., Schulte, G., Mager, P. P., Hartig, W., Gorcs, T. J., Zilberter, Y., Ernfors, P. & Harkany, T. (2004) Eur. J. Neurosci. 20, 1290–1306.

Fig. 8. De novo brain-derived neurotrophic factor (BDNF) synthesis and release are not involved in endocannabinoid-induced TrkB receptor activation. (A) Real-time quantitative PCR analysis for BDNF mRNA transcripts was performed on total RNA extracted from primary cortical cultures with or without 180-min anandamide (AEA) stimulation. mRNA extracted from adult somatosensory cortex was used as positive control. Data were normalized to 18S rRNA contents in each sample. Note that AEA does not induce BDNF expression even after extended periods of stimulation. (B–B'′) CB₁ cannabinoid receptors (CB₁Rs) and TrkB receptors exhibit predominant cell surface localization after cotransfection in PC12 cells. Intracellular localization of both receptors suggests their active trafficking to the plasma membrane. 2-Arachidonoylglycerol (2-AG) and AEA (data not shown) triggers the clustering of CB₁R and TrkB receptors (appear in yellow color, arrowheads). [Scale bars: 10 mm (B′), 8 mm (B), 1.75 mm (2-AG).] (C) AEA induces TrkB receptor activation in PC12_TrkB/CB1R cells that is blocked by AM251. Crude fractions were analyzed to ensure identical CB₁R expression after cell transfection. (D) To ascertain that an up-regulation of BDNF expression contributed to AEA-induced TrkB activation in PC12_TrkB/CB1R cells, we performed real-time quantitative PCR analysis for BDNF mRNA. AEA stimulation did not affect BDNF mRNA transcript levels after 15 or 30 min. Next, we extended these studies by showing with Western blotting that BDNF protein concentrations did not change in culture media of PC12_TrkB/CB1R cells after incubation with AEA. Recombinant BDNF was used as positive control. Collectively, these data confirm that AEA-induced TrkB activation does not involve de novo BDNF transcription or BDNF release from preexisting intracellular pools.

Table 1. Antibodies used to characterize cultured interneurons

CB₁R, CB₁ cannabinoid receptor; VGAT, vesicular GABA transporter; VGLUT, vesicular glutamate transporter; GFAP, glial fibrillary acidic protein; GAD, glutamic acid decarboxylase. For details and procedures, see Supporting Materials and Methods.

Supporting Materials and Methods

It is generally accepted that the membranes of living cells form impermeable barriers and thereby exclude the possibility of using antibodies recognizing, e.g., intracellular domains of transmembrane proteins (1). Therefore, we used an N-terminal CB₁ cannabinoid receptor (CB₁R) antibody to isolate interneurons. Because two trypsin cleavage sites exist within the extracellular N-terminal 77-aa sequence of the rat CB₁R, at positions 14 and 54, we tested whether enzymatic digestion of embryonic tissues affects isolation yields. No difference in isolated cell numbers was found when enzymatic digestion of embryonic tissues was followed by cell sorting with N-terminus-specific anti-CB₁R antibodies (n = 7) as compared with mechanical dissociation. Isolation with antibodies preabsorbed with their corresponding fusion protein suppressed isolation yields 40,000 ± 3,000 cells per embryo (n = 2). Whereas selective cell isolation procedures target neurons with uniform, mostly multipolar morphologies, control experiments with preadsorbed antibodies result in cellular debris and astrocytes showing nonselective uptake of antibody conjugates.

Isolated cells were plated at low density (≈20,000 cells per well) and their identities were analyzed at 6 days in vitro. We used a standard panel of immunocytochemical methods to analyze the heterogeneity of our cultures (2, 3). Cultures were exposed to selected combinations of the following primary antibodies to reveal the presence of inhibitory interneurons, excitatory pyramidal cells, astrocytes, and oligodendrocytes (Table 1). Primary antibodies were applied at 4°C overnight and visualized using combinations of Cy2-, Cy3-, and Cy5-tagged secondary antibodies raised in donkey (1:400 in 2% BSA; Jackson ImmunoResearch). All specimens were counterstained with Hoechst 33,342 (Sigma) to aid cell counts. Control experiments for CB₁R and vesicular glutamate transporter (VGLUT)3 antibodies involved their preadsorption with corresponding fusion proteins (1 and 20 mg/ml, respectively), and yielded no detectable immunolabeling. Cell counts of b-III-tubulin, CB₁R, myelin basic protein (MBP), or glial fibrillary acidic protein (GFAP)-immunoreactive (ir) cells were performed at ´63 primary magnification on an Axioplan 2 microscope (Zeiss) equipped with filter sets for analyzing Cy2- or Cy3-stained specimens and expressed as the percentage of nuclei stained with Hoechst 33,342. All neurons examined expressed vesicular GABA transporter (VGAT), cholecystokinin, VGLUT3, and CB₁R. Quantification of the number of glutamic acid decarboxylase (GAD) immunoreactive cells demonstrated that virtually all neurons (>96%) harbored the GABA synthesis enzyme. The high purity of interneuron cultures was also confirmed electrophysiologically, as excitatory spontaneous postsynaptic currents were not observed. To determine the number of glial cells, cultures were grown in DMEM/F12 supplemented with B27 (2%) known to suppress glia proliferation. Our cultures predominantly contained b-III-tubulin-ir neurons, whereas MBP-ir oligodendrocytes (0.6 ± 0.4%) and GFAP-ir astrocytes (6.2 ± 1.0%) known to express CB₁Rs (4, 5) were only occasionally present.

The authors thank Dr. R. H. Edwards the guinea pig anti-VGLUT3 antibody.