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Basal forebrain cholinergic neurons modulate the activation of cortical neurons by several stimuli such as fear and anxiety. However, the role of the muscarinic receptor in the medial prefrontal cortex (MPFC) in the modulation of the conditioned emotional response (CER) evoked in the model contextual conditioned fear remains unclear. The objective of this study is to test the hypothesis that inhibition of the muscarinic receptor in ventral MPFC modulates CER observed during animal’s re-exposure to the aversive context. Rats implanted with cannulae aimed at the prelimbic (PL) or the infralimbic (IL) were submitted to a high-intensity contextual fear conditioning protocol. Before the test session, they received microinjections of the hemicholinium (choline reuptake blocker), atropine (muscarinic antagonist), J104129 fumarate (M1-M3 muscarinic antagonists), pirenzepine (M1 muscarinic antagonist), neostigmine (inhibitor acetylcholinesterase enzyme), or the systemic administration of the FG7142 (inverse benzodiazepine agonist). Additional independent groups received the neostigmine or FG7142 before the ineffective doses of J104129 fumarate in the low-intensity protocol of contextual fear conditioning. In the high-intensity protocol, the administration of hemicholinium (1 nmol), atropine (0.06–6 nmol), J104129 fumarate (6 nmol), or pirenzepine (6 nmol) attenuated the expression of CER in rats. However, in the low-intensity protocol, only J10129 fumarate (0.06 nmol) reduced the expression of the CER. Finally, neostigmine (0.1–1 nmol) or FG7142 (8 mg/Kg) increased CER expression, an effect inhibited by the low dose of the J10129 fumarate. These results indicated that the blockade of M3 muscarinic receptor in the vMPFC attenuates the CER expression.

The periaqueductal gray (PAG) columns have been implicated in controlling stress responses through corticotropin-releasing factor (CRF), which is a neuropeptide with a prominent role in the etiology of fear- and anxiety-related psychopathologies. Several studies have investigated the involvement of dorsal PAG (dPAG) CRF mechanisms in models of unconditioned fear. However, less is known about the role of this neurotransmission in the expression of conditioned fear memories in the dPAG and ventrolateral PAG (vlPAG) columns. We assessed the effects of ovine CRF (oCRF 0.25 and 1.0μg/0.2μL) locally administered into the dPAG and vlPAG on behavioral (fear-potentiated startle and freezing) and autonomic (arterial pressure and heart rate) responses in rats subjected to contextual fear conditioning. The lower dose injected into the columns promoted proaversive effects, enhanced contextual freezing, increased the blood pressure and heart rate and decreased tail temperature. The lower dose of oCRF into the vlPAG, but not into the dPAG, produced a pronounced enhancement of the fear-potentiated startle response. The results imply that the PAG is a heterogeneous structure that is involved in the coordination of distinct behaviors and autonomic control, suggest PAG involvement in the expression of contextual fear memory as well as implicate the CRF as an important modulator of the neural substrates of fear in the PAG.

Neural reflex mechanisms, such as the baroreflex, are involved in the regulation of cardiovascular system activity. Previous results from our group (Resstel LB, Correa FM. Medial prefrontal cortex NMDA receptors and nitric oxide modulate the parasympathetic component of the baroreflex. Eur J Neurosci 23: 481–488, 2006) have shown that glutamatergic synapses in the ventral portion of the medial prefrontal cortex (vMPFC) modulate baroreflex activity. Moreover, glutamatergic neurotransmission in the vMPFC can be modulated by the endocannabinoids system (eCBs), particularly the endocannabinoid anandamide, through presynaptic CB1receptor activation. Therefore, in the present study, we investigated eCBs receptors that are present in the vMPFC, and more specifically whether CB1receptors modulate baroreflex activity. We found that bilateral microinjection of the CB1receptor antagonist AM251 (100 or 300 pmol/200 nl) into the vMPFC increased baroreflex activity in unanesthetized rats. Moreover, bilateral microinjection of either the anandamide transporter inhibitor AM404 (100 pmol/200 nl) or the inhibitor of the enzyme fatty acid amide hydrolase that degrades anandamide, URB597 (100 pmol/200 nl), into the MPFC decreased baroreflex activity. Finally, pretreatment of the vMPFC with an ineffective dose of AM251 (10 pmol/200 nl) was able to block baroreflex effects of both AM404 and URB597. Taken together, our results support the view that the eCBs in the vMPFC is involved in the modulation of baroreflex activity through the activation of CB1receptors, which modulate local glutamate release.

The prelimbic (PL) medial prefrontal cortex is a brain region highly involved in the control of emotional responses, being modulated by several neurotransmitter systems, including the cholinergic and endocannabinoid. Activation of muscarinic type 1 (M1) receptors in the brain induces retrograde suppression of inhibition through the induction of endocannabinoid release, which, in turn, activates cannabinoid type 1 (CB1) receptors. No study so far, however, has been conducted to investigate if the cholinergic and endocannabinoid systems interact in the PL to modulate anxiety-related behaviors. Thus, the present work aimed at verifying if intra-PL administration of neostigmine, an acetylcholinesterase inhibitor, would produce changes in anxiety-like behavior and if these effects are mediated by M1 and CB1 receptor activation. Independent groups of animals received bilateral injections of vehicle, the M1 receptor antagonist pirenzepine (0.06, 0.6, and 6 nmol), the CB1 receptor antagonist AM251 (0.1 nmol), or the fatty acid amide hydrolase (FAAH) enzyme inhibitor URB597 (1, 3, and 10 pmol), followed by vehicle or neostigmine (0.01, 0.1, and 1 nmol), and were submitted to the elevated plus-maze (EPM) test. Neostigmine (1 nmol) decreased open arm exploration of the maze. This anxiogenic-like effect was reproduced in another anxiety-related animal model, the light-dark box. Previous injection of pirenzepine or AM251 abolished this response in the EPM, whereas URB597 had no effect. These results suggest that CB1 and M1 receptors interact in the PL to control anxiety-like behaviors.

Leaf extract of Centella asiatica has been used as an alternative medicine for memory improvement in the Indian Ayurvedic system of medicine for a long time. Although several studies have revealed its effect in ameliorating the cognitive impairment in rat models of Alzheimer's disease, the molecular mechanism of C. asiatica on neuroprotection still remains unexplained. In this study, we investigated the effects of C. asiatica water extract on activity of subtypes of phospholipase A2 (PLA2) in primary cultures of rat cortical neurons and quantified by HPLC a possible molecule responsible for the activity. The cPLA2 and sPLA2 activities were inhibited in vitro by asiaticoside present in the water extract of C. asiatica. This extract may be a candidate for the treatment of neurodegenerative processes because of its pharmacological activity in the brain and its low toxicity, as attested by its long popular use as a natural product.