The Dorsolateral Periaqueductal Gray and Its Role in Mediating Fear Learning to Life Threatening Events

The dorsolateral column of the periaqueductal gray (dlPAG) integrates aversive emotional experiences and represents an important site responding to life threatening situations, such as hypoxia, cardiac pain and predator threats. Previous studies have shown that the dorsal PAG also supports fear learning; and we have currently explored how the dlPAG influences associative learning. We have first shown that N-methyl-D-aspartate (NMDA) 100 pmol injection in the dlPAG works as a valuable unconditioned stimulus (US) for the acquisition of olfactory fear conditioning (OFC) using amyl acetate odor as conditioned stimulus (CS). Next, we revisited the ascending projections of the dlPAG to the thalamus and hypothalamus to reveal potential paths that could mediate associative learning during OFC. Accordingly, the most important ascending target of the dlPAG is the hypothalamic defensive circuit, and we were able to show that pharmacological inactivation using beta-adrenoceptor blockade of the dorsal premammillary nucleus, the main exit way for the hypothalamic defensive circuit to thalamo-cortical circuits involved in fear learning, impaired the acquisition of the OFC promoted by NMDA stimulation of the dlPAG. Moreover, our tracing study revealed multiple parallel paths from the dlPAG to several thalamic targets linked to cortical-hippocampal-amygdalar circuits involved in fear learning. Overall, the results point to a major role of the dlPAG in the mediation of aversive associative learning via ascending projections to the medial hypothalamic defensive circuit, and perhaps, to other thalamic targets, as well. These results provide interesting perspectives to understand how life threatening events impact on fear learning, and should be useful to understand pathological fear memory encoding in anxiety disorders.Brazilian public funding agencies CAPES (Coordenadoria de Aperfeicoamento de Pessoal de Nivel Superior)Brazilian public funding agencies CAPES (Coordenadoria de Aperfeicoamento de Pessoal de Nivel Superior)FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Dorsolateral periaqueductal gray stimulation prior to retrieval potentiates a contextual fear memory in rats

The association of a neutral context with an aversive stimulus, such as foot-shock, result in a contextual fear memory. A growing number of evidence have revealed that prior exposure to diverse threatening situations facilitates the encoding of fear memory during acquisition and such reports support the widespread notion that emotionally arousal results in stronger and long-lasting memories. However, few studies have investigated if a threatening experience can affect the recall and the persistence of such fear memory trace. To test the hypothesis that an emotionally negative experience could modify the retrieval of a memory and potentiate the expression of a fear memory, the present study used the chemical stimulation (microinjection of NMDA) of the dorsolateral periaqueductal gray matter (dlPAG) of rats in order to induce an aversive emotional state. Such stimulation was performed one day after a weak fear training protocol, and the fear expression was analyzed in subsequent re-exposures to the conditioned context. The results showed that the negative emotional state induced by the dlPAG stimulation enhanced the fear memory trace when this trace was reactivated one day after this aversive experience. Additionally, the potentiation of the fear response was contingent to the associated context since no potentiation was evident when NMDA-stimulated animals were subsequently placed in a non-associated context. Finally, the model suggests that the enhancement of fear responses is long-lasting since NMDA-treated animals performed a robust fear response six days after memory retrieval.Fil: Mochny, Cristiane R.. Universidade Federal de Santa Catarina; BrasilFil: Kincheski, Grasielle C.. Universidade Federal de Santa Catarina; BrasilFil: Molina, Víctor Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; ArgentinaFil: Carobrez, Antonio P.. Universidade Federal de Santa Catarina; Brasi

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

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 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

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 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