Supplemental Information. Evoked Axonal Oxytocin Release. in the Central Amygdala. Attenuates Fear Response

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1 Neuron, Volume 73 Supplemental Information Evoked Axonal Oxytocin Release in the Central Amygdala Attenuates Fear Response H. Sophie Knobloch, Alexandre Charlet, Lena C. Hoffmann, Marina Eliava, Sergey Khrulev, Ali H. Cetin, Pavel Osten, Martin K. Schwarz, Peter H. Seeburg, Ron Stoop, and Valery Grinevich

2 SUPPLEMENTAL FIGURES AND LEGENDS Figure S1, Related to Figure 1. Targeting of Virtually All OT Neurons of the SON, PVN, and AN in Lactating Rats (A-B) Expression of Venus in all subdivisions of the PVN (A) and in the main and retrochiasmatic parts of the SON (B). C, The additional injections between the PVN and SON resulted in Venus expression in various AN (accessory nuclei), as indicated by arrows. Coronal hypothalamic sections were stained with antibodies against GFP, visualized by FITC-conjugated secondary antibodies. Scale bar: 200 m. 2

3 Figure S2, Related to Figure 2. Distribution and Density of Venus-Labeled OT Fibers from the PVN and SON in Various Extrahypothalamic Forebrain Regions of Lactating Rats (A) Summarized distribution and density (number of fibers per mm 2 ) of Venus containing fibers, originating from the PVN or SON in different forebrain structures. Ipsi ipsilateral side; Con contralateral side. Colors (yellow, orange, red and violet) depict the ranges of fiber numbers in the analyzed structures, as presented in the lowest panel. Blue color indicates no detected fiber in the region. Within each color box the number of fibers per 3

4 mm 2 is presented. Note the absence of a number for the fiber density in the CeA after injection into the ipsilateral SON, although this structure is depicted in yellow color (i.e. contains <10 fibers). This is due to the fact that fibers in the CeA, originating from the SON, were marginally located at the ventro-lateral border of the nucleus (see also Fig. 3A, middle image), making it impossible to calculate the fiber density per mm 2 in this particular structure. For several structures with a pronounced gradient of fiber density in the anterior-posterior extent, the fiber number was averaged but the gradient was indicated ( - increase, - decrease). (B) Examples of Venus-positive fibers in select regions: AON anterior olfactory nucleus, FrA prefrontal cortex, Acb nucleus accumbens (shell), LS lateral septum, and CA1-v CA1 region of ventral hippocampus. Note the absence of Venus positive fibers originating from the SON within the AON and FrA. Brain sections were stained with GFP antibody and visualized by the DAB method. Scale bar = 50 m. 4

5 Figure S3, Related to Figure 3. Distribution, Density, Total, and Mean Length of Venus/OT-positive Fibers in the CeA of Virgin and Lactating Rats (A) Representative digital overlay of the fibers in CeA. Each color (red, blue or green) indicates fibers detected at 3 rostro-caudal levels in each of 3 rats per group. The phenotype of the axons in CeM (long, non-branching, lacking varicosities) is characteristic for the traversing axons (arrows). The fibers in the CeL form multiple short branches bearing prominent varicosities (arrowhead), indicating terminals in the CeL. The analysis was performed in the CeL, comprised by CeC, central central and CeL, central lateral subdivisions. st commissural stria terminalis. (B) A higher number of axon segments is detectable both in CeM and CeL in lactating rats, though the increase of 5

6 the fiber density is more pronounced in the CeL of lactating animals. (C) The total length of the fibers within CeM and CeL also increases in lactating animals. (D) The mean length of the fibers is similar in lactating rats and virgins. The significant difference in mean length of the axons between the CeM and CeL is present both in virgins and lactating rats. Due to our observation that the most prominent innervation of the CeA occurred after targeting of all OT nuclei, including AN (Figure 3A, right), we performed a quantitative analysis of the fiber density (per mm 2 ), total and mean length of fibers in lactating rats after infection of all OT nuclei (Figure S3). Within the CeA we discriminated medial (CeM) and lateral (CeL) subregions (Figure S3). Furthermore, we compared all the parameters on fiber density and length in lactating rats with those in virgin animals (Figure S3), as these were subject of our electrophysiological and behavioural studies. As seen in Figure S3, the overall density of fibers in virgin rats in the CeA is significantly lower than in lactating rats (S3B). The density of fibers in the CeM and CeL appeared to be similar in virgin rats. On the contrary, the number of fibers in lactating rats is higher in the CeM (~2.5 fold in comparison to the CeM in virgins), and even higher in the CeL (~4 fold in comparison to the CeL in virgins) (Figure S3B). The comparison of the fiber density between the CeL and CeM in lactating rats revealed that the density in the CeL is ~1.5 fold higher than in the CeM (Figure S3B). The estimated total length of the detected fibers was similar in the CeM and the CeL (Figure S3C), reflecting the presence of long fibers in the CeM and many short fibers in the CeL. When further quantified by estimating the mean length of individual fibers, this parameter was significantly higher in CeM than CeL (Figure S3D). Importantly, the morphology of fibers in the CeM and CeL exhibits a drastic difference between these CeA subregions: the majority of fibers in CeM had anatomical features of traversing fibers, streaming through the CeM (arrows in Figure S3A), while in CeL the fibers branched extensively and contained numerous varicosities (arrowheads in Figure S3A). This difference held for virgin and lactating animals. 6

7 Figure S4, Related to Figure 4. Action Potential Frequencies and IPSC Amplitudes (A) Action potential frequencies of fluorescent neurons recorded in the PVN (blue, n=8) or SON (red, n=3) during 10, 20, 30 Hz or continuous application of BL. (B) (Left) cumulative distribution of IPSC amplitudes measured before and during the characteristic increase in frequency induced by blue-light exposure (BL). (Right) mean amplitudes for the same conditions (n=32 cells). Statistical significances: The double-sided Kolmogorov-Smirnov test was used for testing significance between cumulative distributions, and one-way ANOVA was used for bar charts. In none of the graphs could we detect a significant difference. 7

8 Figure S5, Related to Figure 5. Freezing Behavior and Estrous Cycle Freezing level measured at the end of second day of conditioning as a function of hormonal status. E, estrus (n=2); D, diestrus (n=2); P, proestrus (n=3); M, metestrus (n=4). We evaluated the dependence of freezing levels on hormonal cycles of female rats. To this purpose, the freezing level was measured at the end of the second day of conditioning as a function of the hormonal status. We found no significant influence of the stage of estrous cycle on freezing responses in our conditions (see Figure S5). 8

9 Figure S6, Related to Figure 6. Efficiency of PS-Rab and UPS-Rab In Vitro and In Vivo (A) Infection efficiency of UPS-Rab and PS-Rab in primary rat hippocampal cell cultures. Infection of primary hippocampal culture with UPS-Rab-mCherry (1 l/well containing ~ cells) led to the expression of mcherry in a fraction of the neurons. Infection of cell culture with raav expressing TVA coupled to tdtomato (1 l/well), followed by application of PS-Rab-EGFP (1 l/well), led to the infection of a fraction of neurons (yellow), comparable with that following infection with UPS-Rab-mCherry. Application of PS-Rab-mCherry (1 l/well) alone resulted in no infection. Infection of the culture with a mixture of raavs, expressing TVA-IRES-tdTomato and RG followed 9

10 by infection by PS-Rab-EGFP labeled virtually all neurons, reflecting monosynaptic transfer of PS-Rab-EGFP. Scale bar: 100 m. (B) PS-Rab-based back-labeling of extrahypothalamic neurons in areas known to innervate the CeA. Pregnant rats were injected into the CeA with raavs expressing TVA and RG, and later with PS-Rab-EGFP. They were analyzed on day 7 of lactation. Retrogradely labeled EGFP-positive neurons were found in well-defined input areas to CeA: cortical areas (AI agranular insular cortex; APir amygdalopiriform transition area: S1 primary somatosensory cortex); thalamic nuclei (MD mediodorsal thalamic nucleus; MG medial geniculate nucleus; PVA anterior part of paraventricular thalamic nucleus; SPF subparafascicular thalamic nucleus; VP ventral posterior thalamic nucleus), the bed nucleus of stria terminalis (BNST M&L medial and lateral BNST), and (D) the amygdala (ACo cortical amygdaloid nucleus; BM basomedial amygdaloid nucleus; CeA central amygdala primarily targeted nucleus; MeAD anterodorsal medial amygdaloid nucleus). Brain sections were stained with GFP antibody and visualized by FITC secondary antibodies. Scale bar = 400 m. (C) Back-labeling of OT neurons after injection of PS-Rab into the Acb or NTS. Pregnant rats were injected into Acb or NTS with raavs expressing TVA and RG followed later by PS-Rab-EGFP and killed on day 7 of lactation. Example of EGFP-positive backlabeled neuron (arrow) in the accessory magnocellular nuclei after injection of PS-Rab- EGFP into the Acb; the example cell appeared in yellow due to colocalization of EGFP (green) and OT (red) signals (left panel). Note the presence of EGFP-positive axonal endings in the posterior pituitary (right panel), which contain OT-immunoreactivity (inset in right panel). Example of EGFP-positive back-labeled neuron (arrow) in the PVN after injection of PS-Rab-EGFP into the NTS; the cell appeared yellow due to colocalization of GFP (green) and OT (red) signals (left panel). Note the absence of EGFP-positive axonal endings in the posterior pituitary (right panel). Brain sections were stained with EGFP and OT antibodies, and visualized by FITC (green) and CY3 (red) secondary antibodies, respectively. Scale bars in m: 100 and 10 (inset). (D) No endocytotic uptake of UPS-Rab-mCherry by axonal terminals of magnocellular hypothalamic neurons in the posterior pituitary of lactating rats. Pregnant rats were injected into the posterior pituitary with a mixture of UPS-Rab-mCherry and raav, expressing GFP under the control of the GFAP promoter, and were analyzed on day 7 of lactation. Infection by the raav resulted in the appearance of GFP signal in pituicytes. 10

11 The red signal from UPS-Rab-mCherry was detected neither in pituicytes nor in the axonal terminals of magnocellular neurons. Consistently, red signal from UPS-RabmCherry was not found in OT neurons (stained for OT, green) in the hypothalamus. Pituitary sections were stained with GFP and dsred antibodies, visualized by FITCconjugated antibodies; hypothalamic sections were stained with OT antibody visualized by FITC. Scale bars in m: 400 (pituitary), 200 (hypothalamus). We determined the infectious efficiency of PS-Rab-EGFP and its unpseudotyped variant SAD G-mCherry (termed UPS-Rab-mCherry) in cell culture (Figure S6A). Besides hypothalamic labeling, we observed neurons expressing only green fluorescence in various extrahypothalamic structures previously reported as sources of innervation of the CeA (Swanson and Petrovich, 1998), further attesting to the correct monosynaptic retrograde transfer of the virus (Figure S6B). Next we tested this approach in the nucleus accumbens (Acb) and nucleus of solitary tract (NTS), both identified hypothalamic OT neuron targets. While injections of both raavs followed by PS-Rab-EGFP in the Acb back-labeled OT neurons especially in the AN (Figure S6C, upper panel), the injection in the NTS resulted in the appearance of EGFP-positive neurons in the PVN, but not AN or SON (Figure S6C, lower panel). In accordance to previous anterograde labelings (see also Figure 2 and S2), these results confirmed the efficient retrograde transsynaptic labeling with this method. Thus, these findings confirm the formation of functional monosynaptic hypothalamic projections into the CeA as well as to the Acb and NTS. To exclude the possibility of rabies virus uptake by damaged axons or by endocytosis, we also tested the virus directly into the posterior pituitary, where permanent and extensive exocytotic and endocytotic events from OT (and VP) axons take place without the presence of synaptic contacts (Hsu and Jackson, 1996; Klyachko and Jackson, 2002). For this control experiment we used the unpseudotyped rabies virus (UPS-Rab), the infectious efficiency of which was assessed in cultured hippocampal neurons (Figure S6D). In contrast to PS-Rab, UPS-Rab can infect cells directly, in absence of TVA receptor expression, as was seen in cultured neurons (Figure S6A) and thereby can report about the presence of unspecific uptake. UPS-Rab-mCherry was injected into the posterior pituitary of pregnant rats in the same volume and infection time, as done previously with PS-Rab. Correct injection was confirmed by mixing the UPS-Rab-mCherry with a raav expressing GFP from the GFAP promoter (Figure S6A); 11

12 GFAP is a typical marker of pituicytes (Wittkowski, 1998). As a result, we found GFP expression in pituicytes of the posterior lobe, but did not observe any mcherry positive axonal terminals (Figure S6D, upper panel). Consistently, there were no back-labeled, mcherry-positive neurons in the PVN, SON or AN (Figure S6D, lower panel). Thus, the potential axonal damage during injection of viruses or endocytotic events does not permit rabies virus uptake, at least in neuroendocrine magnocellular neurons. 12

13 Table S1. Animal Groups, Types of Applied Viruses, and Viral Vectors, Targeted Structures, Duration of Viral Infection, and the Figures Showing the Experimental Results 13

14 Table S2. Stereotactic Coordinates and Volumes of Injected Viruses Used for Targeting Various Brain Structures n.a. = not applicable; n.i. = not injected. 14

15 SUPPLEMENTAL EXPERIMENTAL PROCEDURES Cell Culture Hippocampal neurons were from embryonic E18 hippocampi of Sprague-Dawley rats. After cultivating in 24-well plates for one week, the neurons were infected with 1ml per well of either raav expressing TVA or 1 ml of raav expressing TVA plus 1ml raav expressing RG. Six days later the same cells were infected with 1 l of RS-Rab-EGFP and cultured for five more days. In parallel, cultured cells were infected only with 1 l of UPS-Rab-mCherry and analogously cultured for five days. DAB-Based Visualization of Venus and Counting Venus-Immunoreactive Fibers in Thirty-Four Forebrain Regions For the analysis of long-ranged projection of OT neurons, the coronal vibratome sections (50mm) of whole forebrains from 6 rats on day 14 of lactation (previously injected on day 10 of pregnancy with OT promoter-venus raav either into the SON, n = 3 or into the PVN, n = 3) were stained with rabbit anti-gfp antibodies (Molecular Probes; 1:5.000) visualized by diaminobenzidine (DAB). To analyze the distribution of Venuspositive fibers, a set of 34 brain areas was chosen according to the distribution of OTR (Gimpl and Fahrenholz, 2001). In two of three rats injected into the SON and two rats out of three injected into the PVN the innervation pattern in each brain structure (3 sections/structure; selected according Paxinos and Watson, 1998) was assigned to colourcoded categories: no fibers; up to 10 fibers; up to 50 fibers; > 50 fibers, and plexus/basket formation. In two additional rats (one rat injected in the SON and one rat into the PVN) we determined the number of fibers per mm 2. The determination of fiber densities in the 34 brain structures was performed with the aid of a Neurolucida 3D reconstruction system and NeuroExplorer Software package (MicroBrightField, Colchester, VT, USA, and NeuroExplorer, Littleton, MA, USA). Per structure, depending on its anteriorposterior extent, 3 to 6 neighboring sections were analyzed and averaged. In structures such as the nucleus accumbens (shell), dorsal pallidum, ventral part of dentate gyrus and paraventricular nucleus of thalamus, with a pronounced gradient of fibers in the anteriorposterior extent, the fiber number was also averaged but the gradient was taken into account (see Figure S2). At magnifications of 1x4x and 1.6x4x and 1.6x20x, a counting grid covering an area of 1 mm 2 and divided into 16 large squares or 64 smaller squares 15

16 was interposed, the fibers were counted in 2 to 4 representative squares, and the numbers were extrapolated for 1 mm 2. Qualitative and Quantitative Analyses of OT Projections in the CeA We analyzed 3 virgin and 3 lactating rats that had received injections of OT promoter - Venus raav into all nuclei (the PVN, SON and AN; see Table S1). Venusimmunoreactivity was visualized by DAB, followed by the enhancement of DAB by Nickel, as described by Adams (1981). The fiber counting to achieve the number of fibers per mm 2 in two regions of the CeA, CeM and CeL (the latter comprising the central central and central lateral subdivisions) was performed in serial 50 µm sections at 3 rostro-caudal Bregma (-1.80 mm, mm, mm), Neurolucida 3D reconstruction system and NeuroExplorer Software package (MicroBrightField, Colchester, VT, USA, and NeuroExplorer, Littleton, MA, USA). The estimation of the length of the fibers was performed using ImageJ software. To get a better overview of the fiber distribution in the CeA, the original gray scale images of the CeA were pseudo-colored in Photoshop (red, blue, green color codes each of the three virgin rats numbered 1, 2 and 3, and the same for lactating rats) and digitally overlaid at 3 rostro-caudal levels of CeA. The quantitative data were evaluated using the GraphPad Prism program (GraphPad Software, Inc, USA). Differences between groups were examined using One-Way ANOVA. P values <0.05 were considered statistically significant. Hormonal Cycle Assessment: Virgin female Wistar rats without preference for the stage of estrous cycle were used for both electrophysiological and behavioural experiments. In a number of behavioral experiments the effects of hormonal cycle were assessed (Figure S5). In this case the cycle was determined by collecting vaginal smears during 3 consecutive days 3 hours before the beginning of the dark phase on anaesthetized females (isoflurane 3%), using a micropipette filled with 40 μl of 0.9% NaCl solution and pipetting 3 times. A light microscope with a 40x objective lens was used to observe smears (Marcondes et al., 2002). 16

17 SUPPLEMENTAL REFERENCES Adams, J.C. (1981). Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem. 6, 775. Gimpl, G., and Fahrenholz, F. (2001). The oxytocin receptor system: structure, function, and regulation. Physiol. Rev. 81, Hsu, S.-F., and Jackson, M.B. (1996). Rapid exocytosis and endocytosis in nerve terminals of the rat posterior pituitary. J. Physiol. 494, Klyachko, V.A., and Jackson, M.B. (2002). Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals. Nature 418, Marcondes F.K., Bianchi F.J., and Tanno A.P. (2002). Determination of the estrous cycle phases of rats: some helpful considerations. Braz. J. Biol. 62, Paxinos, G. and Watson, C. (1998). The Rat Brain in Stereo Coordinates. 4 th (Academic Press). edn Swanson, L. W., and Petrovich, G. D. (1998). What is the amygdala? Trends Neurosci. 21, Wittkowski, W. (1998). Tanycytes and pituicytes: morphological and functional aspects of neuroglial interaction. Microsc. Res. Tech 41,