Experiment 1– Effects of the NMDA injection into the dlPAG in the acquisition of OFC.

<p>The experimental design used is outlined above the graph, where a vertical arrow shows the moment of the dlPAG infusion associated with amyl acetate odor during the conditioning session (10 min) in the conditioning box. Histograms represent the mean (+SEM) of the percentage of approach time (A), hide time (B), and head-out time (C) exhibited during OFC expression in an odor box. The hatched horizontal line represents the mean and the confidence limits (within 95%) for the familiarization session in the odor box. Subjects were grouped according to the different schedules of the dlPAG injection: PBS (n = 8), NMDA 25 pmol (n = 8), NMDA 50 pmol (n = 8) and NMDA 100 pmol (n = 8). CS1 represents the first-order CS exposure and CS2 represents the second-order context (no odor) exposure. *<i>p<0.05</i> and **<i>p<0.005</i> compared with the PBS control group (repeated measures ANOVA followed by Newman-Keuls test).</p

Summary diagram illustrating the dlPAG ascending projections to hypothalamic and thalamic targets influencing cortical-hippocampal-amygdalar circuits.

<p>Red lines indicate the dlPAG – medial hypothalamic defensive circuit – thalamic pathway, where we have shown that beta-adrenergic blockade of the dorsal premammillary nucleus impaired the acquisition of olfactory fear conditioning induced by the dlPAG-NMDA injection. Abbreviations: ACA – anterior cingulate area; AHN – anterior hypothalamic nucleus; AMv – anteromedial thalamic nucleus, ventral part; dlPAG – dorsolateral periaqueductal gray; HIP – hippocampal formation; IL – intralaminar thalamic nuclei; LA – lateral amygdalar nucleus; LD – lateral dorsal thalamic nucleus; PMd – dorsal premammillary nucleus; POR – postrhinal area;RE – nucleus reuniens; RSP – retrosplenial area; SGN – suprageniculate nucleus; SPFpl – subparafascicular nucleus thalamus, parvicelular part, lateral division; VMHdm – ventromedial hypothalamic nucleus, dorsomedial part.</p

Histological analysis of the injection site.

<p>A, B – Schematic plotting onto a standard drawing of <i>The rat brain in stereotaxic coordinates </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050361#pone.0050361-Paxinos1" target="_blank">[33]</a> showing the approximate location of the injection cannula tips centered in the dlPAG (A) and the PMd (B). Due to the large number of overlaps, the number of plotted points is lower than the number of subjects actually injected in these regions. C – Bright-field photomicrograph showing Evans blue-stained injection cannula placement in a representative animal that received NMDA 100 pmol into the dlPAG. D – Bright-field photomicrograph showing Evans blue-stained injection cannula placement in a representative animal that received ATE 40 nmol into the PMd region. Abbreviations: DR – dorsal nucleus raphé; III – oculomotor nucleus; PAGdl, dm, l – periaqueductal gray, dorsolateral, dorsomedial, and lateral parts; PMd – dorsal premammillary nucleus; PMv – ventral premammillary nucleus. Scale bars = 250 µm.</p

Freezing response during conditioning session.

<p>Freezing responses of rats following PBS (N = 8) or NMDA [25 (N = 8), 50 (N = 8) or 100 (N = 8) pmol] infusion in the dlPAG and placed in a conditioning box with an olfactory CS. Plotted values represent mean (+SEM) collapsed in two subsequent 5-min periods. *p<0.05, compared to PBS, during the first 5-min period. #p<0.05 compared to same group during the first 5-min period (repeated measures ANOVA followed by Newman-Keuls test).</p

Experiment 3– Effects of the duration of odor exposure during dlPAG-NMDA OFC acquisition.

<p>The experimental design used is outlined above the graph, where a vertical arrow shows the moment of the dlPAG infusion associated with amyl acetate odor during the conditioning session (10 min) in the conditioning box. Histograms represent the mean (+SEM) of the percentage of approach time (A), hide time (B), and head-out time (C). The hatched horizontal line represents the mean and the confidence limits (within 95%) for the familiarization session in the odor box. All subjects were microinjected with NMDA 100 pmol and grouped according to the time interval of AMYL odor exposure during the conditioning session: 5 min (NMDA 5 min, n = 9) or 10 min (NMDA 10 min, n = 8). A group microinjected with PBS (n = 8) paired with amyl acetate odor during 10 min during the conditioning session was considered as control. CS1 represents the first-order CS exposure and CS2 represents the second-order context (no odor) exposure. *<i>p<0.05</i> and <i>**p<0.005</i> compared with the PBS 10-min group (control group; repeated measures ANOVA followed by Newman-Keuls test).</p

Experiment 2 - Selectivity of the dlPAG NMDA injection in supporting OFC.

<p>The experimental design used is outlined above the graph, where a vertical arrow shows the moment of the dlPAG infusion associated with amyl acetate odor during the conditioning session (10 min) in the conditioning box. Histograms represent the mean (+SEM) of the percentage of approach time (A), hide time (B), and head-out time (C). The hatched horizontal line represents the mean and the confidence limits (within 95%) for the familiarization session in the odor box. Subjects were grouped according to the different schedules of the dlPAG injection and training conditions during the acquisition phase: PBS/no odor (n = 8) - PBS infusion without amyl acetate odor pairing during conditioning; PBS/odor (n = 8) - PBS infusion with amyl acetate odor pairing during conditioning; NMDA/no odor (n = 8) - NMDA 100 pmol infusion without amyl acetate odor pairing during conditioning; NMDA/odor (n = 10) NMDA 100 pmol infusion with amyl acetate odor pairing during conditioning; and NMDA out/odor (n = 8) - NMDA 100 pmol infusion outside the dlPAG (in the midbrain reticular nucleus) with amyl acetate odor pairing during conditioning. CS1 represents the first-order CS exposure and CS2 represents the second-order context (no odor) exposure. *<i>p<0.05</i> and ***<i>p<0.0005</i> compared with the PBS/no odor group (repeated measures ANOVA followed by Newman-Keuls test).</p

Experiment 5– Effects of dorsal premammillary nucleus blockade on dlPAG-NMDA OFC acquisition.

<p>The experimental design used is outlined above the graph, where vertical arrows show when the animals received the PMd and dlPAG infusions and were placed in a conditioning box with the olfactory CS. Histograms represent the mean (+SEM) of the percentage of approach time (A), hide time (B), and head-out time (C). The hatched horizontal line represents the mean and the confidence limits (within 95%) for the familiarization session in the odor box. Subjects were first submitted to an infusion into the PMd with PBS (PBS group, n = 6) or atenolol 40 pmol (ATE 40 pmol group, n = 6), and, after 5 min, were microinjected with NMDA 100 pmol into the dlPAG paired with amyl acetate odor during 10 min in the conditioning session. CS1 represents the first-order CS exposure and CS2 represents the second-order context (no odor) exposure. *<i>p<0.05</i> compared with the PBS group (repeated measures ANOVA followed by Newman-Keuls test).</p

Percentage of rats exhibiting Flight or Jumping during the first min following PBS or NMDA infusion into dlPAG.

<p>Percentage of rats exhibiting Flight or Jumping during the first min following PBS or NMDA infusion into dlPAG.</p

Experiment 4– Projections of the dlPAG.

<p>Bright-field photomicrograph, to illustrate the appearance of a PHA-L injection site for a representative PHA-L injection localized in the dlPAG (experiment dlPAG# 4). Note the plexus of PHA-L labeled fibers in the contralateral dlPAG. Abbreviations: III – oculomotor nucleus; PAGdl, dm, l – periaqueductal gray, dorsolateral, dorsomedial, and lateral parts. Scale bar = 250 µm.</p

Experiment 4– Projections of the dlPAG.

<p>Dark-field photomicrographs showing the distribution pattern of PHA-L immunoreactive axons in the rostral nucleus reuniens (A), the intralaminar and lateral dorsal thalamic nuclei (B), the parvicellular subparafascicular, peripeduncular, suprageniculate and medial geniculate nuclei (C), and the anterior hypothalamic nucleus and subfornical region of the lateral hypothalamus (D). Abbreviations: 3 V – third ventricle; AD – anterodorsal nucleus thalamus; AHNc – anterior hypothalamic nucleus, central part; CL – central lateral nucleus thalamus; CM – central medial nucleus thalamus; fx – fornix; LD – lateral dorsal nucleus thalamus; LHAd – lateral hypothalamic area, dorsal region; LHAsf – lateral hypothalamic area, subfornical region; MD – mediodorsal nucleus thalamus; MGd, m, v – medial geniculate complex, dorsal, medial and ventral parts; MRN – midbrain reticular nucleus; PT – paratenial nucleus; PVH – paraventricular hypothalamic nucleus; PVT – paraventricular thalamic nucleus; RE – nucleus reuniens; SGN – suprageniculate nucleus; SPFpl – subparafascicular nucleus thalamus, parvicelular part, lateral division; VAL – ventral anterior-lateral complex thalamus; VMH – ventromedial hypothalamic nucleus. Scale bars = 200 µm.</p