Capsaicin-Induced Changes in LTP in the Lateral Amygdala Are Mediated by TRPV1

The transient receptor potential vanilloid type 1 (TRPV1) channel is a well recognized polymodal signal detector that is activated by painful stimuli such as capsaicin. Here, we show that TRPV1 is expressed in the lateral nucleus of the amygdala (LA). Despite the fact that the central amygdala displays the highest neuronal density, the highest density of TRPV1 labeled neurons was found within the nuclei of the basolateral complex of the amygdala. Capsaicin specifically changed the magnitude of long-term potentiation (LTP) in the LA in brain slices of mice depending on the anesthetic (ether, isoflurane) used before euthanasia. After ether anesthesia, capsaicin had a suppressive effect on LA-LTP both in patch clamp and in extracellular recordings. The capsaicin-induced reduction of LTP was completely blocked by the nitric oxide synthase (NOS) inhibitor L-NAME and was absent in neuronal NOS as well as in TRPV1 deficient mice. The specific antagonist of cannabinoid receptor type 1 (CB1), AM 251, was also able to reduce the inhibitory effect of capsaicin on LA-LTP, suggesting that stimulation of TRPV1 provokes the generation of anandamide in the brain which seems to inhibit NO synthesis. After isoflurane anesthesia before euthanasia capsaicin caused a TRPV1-mediated increase in the magnitude of LA-LTP. Therefore, our results also indicate that the appropriate choice of the anesthetics used is an important consideration when brain plasticity and the action of endovanilloids will be evaluated. In summary, our results demonstrate that TRPV1 may be involved in the amygdala control of learning mechanisms

Capsaicin-induced changes in long-term potentiation in the lateral amygdala are mediated by TRPV1

In der vorliegenden Arbeit konnte erstmalig gezeigt werden, dass die LA-LTP – unter gleichen Ableitbedingungen – im coronalen Hirnschnitt geringer ist als im horizontalen. Im horizontalen Schnitt ist ferner bei Stimulation innerhalb der lateralen Amygdala die LTP im Vergleich zur EC-Reizung geringer. Immunhistochemisch konnte nachgewiesen werden, dass der TRPV1-Rezeptorkanal in der lateralen Amygdala stärker exprimiert wird als im zentralen Nucleus. Es konnte erstmalig gezeigt werden, dass der TRPV1-Rezeptor-Agonist Capsaicin dosisabhängig die HFS-induzierte LA-LTP sowohl im horizontalen als auch im coronalen Hirnschnitt inhibiert. Die Capsaicin-bedingte Hemmung der LA-LTP wird über die TRPV1-Stimulation vermittelt, da Capsazepin – ein TRPV1 -Rezeptor-Antagonist – die Capsaicin-vermittelte Inhibition blockiert. Der Capsaicin-Effekt ist hemisphärendifferent, jedoch nicht inputspezifisch, alters- oder geschlechtsabhängig. Innerhalb der lateralen Amygdala beeinflusst Capsaicin weder spontane inhibitorische oder exzitatorische postsynaptische Miniatur-Ströme (mIPSP, mEPSP) noch die Input-/Output-Kurve. Die Ergebnisse lassen vermuten, dass der Capsaicin-vermittelte Effekt nicht über GABA- Rezeptor vermittelt bzw. nicht durch Erhöhung der extrazellulären Kalzium- und Magnesium-Konzentrationen der ACSF moduliert wird. Die Capsaicin-vermittelte Inhibierung der LA-LTP wird durch den NOS-Inhibitor L-NAME blockiert und fehlt bei nNOS-Knockout-Mäusen. Der CB1-Rezeptor-Antagonisten AM251 reduziert den inhibitorischen Capsaicin-Effekt. Die Ergebnisse zeigen, dass Ursache der Capsaicin-vermittelten Hemmung der LA-LTP eine Aktivierung des CB1-Rezeptors und eine veränderte NO-Synthese sind. Zusammenfassend scheint die Wahl des Narkotikums bei Untersuchung des Einflusses des TRPV1-Rezeptorkanals auf plastische Prozesse von entscheidender Bedeutung zu sein. Die Ergebnisse der vorliegenden Arbeit bekräftigen die Vermutung, dass endogene Vanilloide durch TRPV1-Aktivierung die Schmerzverarbeitung und Lernmechanismen in der lateralen Amygdala beeinflussen könnten.In this study we show for the first time that the magnitude of HFS-induced LA- LTP is weaker in coronal slices than that in horizontal slices during the same conditions. In horizontal brain slice preparations the magnitude of LA-LTP is weaker when afferents within the LA are stimulated than by stimulation of the external capsule. In immunohistochemical studies we show that TRPV1 is expressed in the LA in a higher density than in the central nucleus. In this study we show for the first time that capsaicin – an agonist of TRPV1 – causes a dose-dependent decrease in HFS-induced LA-LTP in horizontal and coronal brain slice preparations by stimulation of external capsule fibers. The capsaicin-induced suppression of LA-LTP is mediated by the stimulation of TRPV1 receptors, since capsazepine – an antagonist of TRPV1 – blocks the capsaicin-induced effect. The suppressive effect of capsaicin differs between the hemispheres but is neither age- nor gender-dependent and does not show input-specificity. Within the LA, capsaicin does not change spontaneous miniature inhibitory or excitatory postsynaptic potentials (mIPSP, mEPSP). Input/output curves are unaltered after capsaicin application. Our results suggest that GABA receptors are not involved in mediating capsaicin-induced suppression of LA-LTP and the enhancement of Mg2+/Ca2+ in the ACSF has no influence on the suppressive effect, too. The capsaicin-induced suppression of LA-LTP is blocked by the nitric oxide synthetase inhibitor, L-NAME, and is absent in neuronal NOS deficient mice. The specific antagonist of cannabinoid receptor type 1, AM251, is also able to reduce the inhibitory effect of capsaicin on LA-LTP. Our results strongly support the assumption that the capsaicin-induced suppression of LA-LTP is caused by stimulation of the CB1 receptor and modulation of the NO system. Our results indicate that the method of anesthesia is an important consideration when brain plasticity and the action of endovanilloids will be evaluated. The present study strongly supports the idea that endogenous vanilloids can cause pain modulation mediated directly by TRPV1 receptors and that modulation of TRPV1 could affect learning mechanisms in the lateral amygdala

Isoflurane anesthesia before euthanasia instead of ether caused a capsaicin-induced enhancement of LA-LTP in horizontal slices derived from adult mice.

<p>(A) HFS-induced LTP is increased in magnitude by 1 µM capsaicin in comparison with control. This increase can be blocked by the specific TRPV1 antagonist AMG9810. (B) Capsaicin-induced LTP enhancement is absent in TRPV1^−/− mice. Representative traces were recorded 5 min prior to tetanus (dashed lines) and 60 min after tetanus (solid lines). (C) Bar histogram of data points averaged 57 to 60 min after HFS and normalized with respect to baseline (mean ± SEM). Significant differences are indicated. *<i>p</i>≤0.05.</p

The capsaicin-induced reduction of LTP recorded in horizontal slices is blocked by TRPV1 antagonists and is absent in TRPV1 deficient mice.

<p>(A) Input-output curves as evoked by single stimuli applied at EC fibers. Drug-free control recordings (n = 15) did not differ from recordings made in capsaicin-treated slices (1 µM cap: n = 15; 10 µM cap: n = 8). (B) EC stimulation caused a stable LA-LTP in horizontal slices derived from adult male mice. Bath-applied capsaicin (cap) induced a dose-dependent reduction in the magnitude of LA-LTP. Data points represent averaged amplitudes (mean ± SEM) normalized with respect to baseline values. Representative traces were recorded 5 min prior to tetanus (dashed lines) and 60 min after tetanus (solid lines). (C) Capsazepine completely blocks the capsaicin-induced reduction of LTP only at a concentration of 50 µM. (D) The capsaicin-induced reduction of LA-LTP could be also blocked by the specific TRPV1 antagonist AMG9810. (E) Bar histogram of data points averaged 57 to 60 min after HFS and normalized with respect to baseline (mean ± SEM). Significant differences are indicated. *<i>p</i><0.05. (F) In TRPV1^−/− mice the inhibitory effect of 1 µM capsaicin was absent. HFS: high-frequency stimulation.</p

Properties of spontaneous PSCs in the absence and presence of TTX.

<p>(A) Continuous recording shows spontaneous EPSCs in the presence of TTX (mEPSCs) from a LA projection neuron under control conditions and 10 min after bath application of capsaicin (1 µM), V_m = −70 mV. (B) Continuous recording shows spontaneous IPSCs in the presence of TTX (mIPSCs) from another LA projection neuron under control conditions and 10 min after bath application of capsaicin (1 µM), V_m = 0 mV. (C) Summary data shows mean frequency of mEPSCs, mIPSCs and sIPSCs under control conditions and in the presence of capsaicin, SEM is indicated by error bars. (D) Amplitude distributions of mIPSCs recorded in under control conditions (black) and in the presence of 1 µM capsaicin (grey). Superimposed lines show cumulative amplitude histograms (p>0.05, Kolmogoroff-Smirnov test). (E) Continuous recording shows spontaneous IPSCs in the absence of TTX (sIPSCs) under control conditions and 10 min after bath application of capsaicin (1 µM), V_m = −70 mV. (F) Amplitude distributions of sIPSCs recorded in under control conditions (black) and in the presence of 1 µM capsaicin (grey). Superimposed lines show cumulative amplitude histograms.</p

TRPV1 protein expression within the amygdala.

<p>(A–C) These graphs illustrate the densities of cell nuclei (visualized with DAPI), neurons (visualized with NeuroTrace), cells expressing TRPV1 (visualized by immunohistochemistry) as well as the densities of neuronal and non-neuronal cells expressing TRPV1 in the lateral nucleus (A), basolateral (B) and central nucleus (C) of the amygdala. (D) The numerical neuronal density of TRPV1 immunopositive neurons in the basolateral amygdala (LA, BL) is significantly higher than in the CE, as determined by ANOVA followed by a Tukey's posthoc test (ns: non-significant; **: p≤0.01; ***: p≤0.001). (E–G) Example of a triple-stained section (DAPI, NeuroTrace and anti-TRPV1; lateral nucleus of the amygdala). NeuroTrace was used to visualize neuronal cells (in green, E). In the same section, binding of TRPV1 antibodies was visualized with Cy5 (in red, F). Figure G displays the merged images of E and F (in each case DAPI (in blue) was used to visualize cell nuclei).</p

NO is involved in the mediation of the suppressive effect of capsaicin on LA-LTP.

<p>(A) The application of the unspecific NOS antagonist L-NAME caused a reduction of LA-LTP and blocked the capsaicin-induced suppression of LA-LTP. (B) In nNOS deficient mice the suppressive effect of capsaicin on LA-LTP is again absent. (C) In wild-type mice the decrease in LA-LTP induced by capsaicin was reproduced. Representative traces were recorded 5 min prior to tetanus (dashed lines) and 60 min after tetanus (solid lines) in the LA. (D) Bar histogram of data points averaged 57 to 60 min after HFS and normalized with respect to baseline (mean ± SEM). Significant differences are indicated. *<i>p</i><0.05. Recordings were done in horizontal slices derived from adult mice.</p

EC-induced LTP is also suppressed by capsaicin in coronal brain slices.

<p>This suppression cannot be blocked in the presence of 10 µM SR95531. (A) In extracellular recordings the magnitude of LA-LTP obtained in coronal slices derived from juvenile mice is lower than that in horizontal slices. Nevertheless, capsaicin reduced LA-LTP in both instances. (B) In patch clamp recordings the complete blockade of GABA_A receptors did not change the inhibitory effect of capsaicin on LA-LTP obtained in coronal slices derived from adult mice. Representative traces were recorded 5 min prior to tetanus (dashed lines) and 60 min and 30 min, respectively, after tetanus (solid lines) in the lateral nucleus of the amygdala.</p

The CB1 receptor is involved in the mediation of capsaicin-induced inhibition of LA-LTP.

<p>(A) The CB1 receptor antagonist AM251 (2.5 µM) evoked a reduction of LA-LTP in comparison with controls. This reduction was not enhanced by capsaicin. In contrast, the co-administration of AM251 and capsaicin (1, 10 µM) caused an increase in the magnitude of HFS-induced LA-LTP. (B) Bar histogram of data points averaged 57 to 60 min after HFS and normalized with respect to baseline (mean ± SEM). Significant differences are indicated. *<i>p</i><0.05, **p<0.01. (C) Anandamide (1 µM, 10 µM) provoked a significant suppression of LA-LTP. Representative traces were recorded 5 min prior to tetanus (dashed lines) and 60 min after tetanus (solid lines). Data were obtained by using horizontal slices derived from adult mice.</p