Lipopolysaccharide-induced sickness behaviour evaluated in different models of anxiety and innate fear in rats.

The fact that there is a complex and bidirectional communication between the immune and nervous systems has been well demonstrated. Lipopolysaccharide (LPS), a component of gram-negative bacteria, is widely used to systematically stimulate the immune system and generate profound physiological and behavioural changes, also known as 'sickness behaviour' (e.g. anhedonia, lethargy, loss of appetite, anxiety, sleepiness). Different ethological tools have been used to analyse the behavioural modifications induced by LPS; however, many researchers analysed only individual tests, a single LPS dose or a unique ethological parameter, thus leading to disagreements regarding the data. In the present study, we investigated the effects of different doses of LPS (10, 50, 200 and 500 µg/kg, i.p.) in young male Wistar rats (weighing 180-200 g; 8-9 weeks old) on the ethological and spatiotemporal parameters of the elevated plus maze, light-dark box, elevated T maze, open-field tests and emission of ultrasound vocalizations. There was a dose-dependent increase in anxiety-like behaviours caused by LPS, forming an inverted U curve peaked at LPS 200 µg/kg dose. However, these anxiety-like behaviours were detected only by complementary ethological analysis (stretching, grooming, immobility responses and alarm calls), and these reactions seem to be a very sensitive tool in assessing the first signs of sickness behaviour. In summary, the present work clearly showed that there are resting and alertness reactions induced by opposite neuroimmune mechanisms (neuroimmune bias) that could lead to anxiety behaviours, suggesting that misunderstanding data could occur when only few ethological variables or single doses of LPS are analysed. Finally, it is hypothesized that this bias is an evolutionary tool that increases animals' security while the body recovers from a systemic infection.

The unconditioned fear produced by morphine withdrawal is regulated by mu- and kappa-opioid receptors in the midbrain tectum.

We have recently shown that morphine withdrawal sensitizes the neural substrates of fear in the midbrain tectum structures--the dorsal periaqueductal gray (dPAG) and inferior colliculus (IC). In the present study, we investigated the role of mu- and kappa-opioid receptors in the mediation of these effects. Periadolescent rats chronically treated with morphine (10 mg/kg; s.c.) twice daily for 10 days were implanted with an electrode glued to a guide-cannula into the dPAG or the IC. Forty-eight hours after the interruption of this treatment, the effects of intra-dPAG or intra-IC microinjections of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO; 0.6 and 1 nmol/0.2 microl)--a selective mu-receptor agonist--or nor-binaltorphimine (BNI; 2.5 and 5 microg/0.2 microl)--a selective kappa-receptor antagonist with tardive action--on the freezing and escape thresholds determined by electrical stimulation of the dPAG and the IC were examined. For both structures, morphine withdrawal produced pro-aversive effects. DAMGO and BNI had antiaversive effects when injected into the dPAG and IC of non-dependent rats. In morphine-withdrawn rats, only BNI continued to promote antiaversive effects in both structures. Whereas DAMGO lost its antiaversive efficacy when injected into the dPAG, only its highest dose promoted antiaversive effects in the IC of morphine-withdrawn rats, suggesting the development of an apparent tolerance. Thus, the enhanced reactivity of the midbrain tectum in morphine-withdrawn periadolescent rats may be due, at least partially, to an impairment of the inhibitory influence of mechanisms mediated by mu-receptors on the neural substrates of fear in this region.

GABA and opioid mechanisms of the central amygdala underlie the withdrawal-potentiated startle from acute morphine.

Anxiety is an affective symptom common to withdrawal from acute or chronic opiate treatment. Although the potentiation of the acoustic startle reflex has been proposed as an index of increased anxiety, there are variable effects of the opiate withdrawal on the startle reflex in chronic dependence models. On the other hand, withdrawal from acute morphine treatment consistently potentiates the acoustic startle reflex, a response that seems to be mediated by the central nucleus of the amygdala (CeA). However, the underlying neurochemical mechanisms have not been elucidated yet. In the present study, we firstly made a comparison between the effects of the withdrawal from both acute and chronic treatments with morphine on the motor activity and the anxiety-like behavior of rats tested in two experimental models, the acoustic startle reflex and the open-field tests. Our second objective was to investigate the role of GABAergic and opioid mechanisms of the CeA in the modulation of the withdrawal-potentiated startle as a measure of anxiety induced by morphine withdrawal. For the production of chronic dependence, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of this treatment, independent groups were probed in the startle reflex and open-field tests. For the acute dependence model, groups of rats were tested in the open field and startle tests under control conditions and under withdrawal from a single injection of morphine (10 mg/kg; s.c.) precipitated by naltrexone injections (0.1 mg/kg; s.c.). The results obtained showed that withdrawal from chronic and acute morphine treatments produced anxiety-like behavior in the open field test, although the anxiogenic-like effects could not be dissociated from the motor effects in the acute dependence model. On the other hand, only the withdrawal from acute morphine treatment significantly potentiated the startle response. Next, we examined the effects of intra-CeA microinjections of muscimol-a GABA(A) receptors agonist-and DAMGO-a mu-opioid receptors agonist-on the potentiated startle induced by acute morphine withdrawal. The results obtained showed that intra-CeA injections of muscimol (1 nmol) and DAMGO (0.5 and 1 nmol) significantly inhibited this response. These findings suggest that the acute dependence model is more suitable to study the aversive effects of morphine withdrawal on the acoustic startle response than the chronic opiate dependence model. Besides, mechanisms mediated by mu- and GABA(A)-receptors in the CeA appear to exert an inhibitory influence on the anxiety-like behavior induced by withdrawal from acute morphine treatment.

Involvement of the midbrain tectum in the unconditioned fear promoted by morphine withdrawal.

The midbrain tectum structures, dorsal periaqueductal gray (dPAG) and inferior colliculus (IC), are involved in the organization of fear and anxiety states during the exposure to dangerous stimuli. Since opiate withdrawal is associated with increased anxiety in both humans and animals, this study aimed to investigate the possible sensitization of the neural substrates of fear in the midbrain tectum and its influence on the morphine withdrawal-induced anxiety. For the production of drug withdrawal, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of the chronic treatment, independent groups were probed in the elevated plus-maze and open-field tests. Additional groups of animals were implanted with a bipolar electrode into the dPAG or the IC and submitted to the electrical stimulation of these structures for the determination of the freezing and escape thresholds after 48 h of withdrawal. Our results showed that the morphine withdrawal promoted clear-cut levels of anxiety without the somatic signs of opiate withdrawal. Moreover, morphine-withdrawn rats had an increase in the reactivity to the electrical stimulation of the dPAG and the IC. These findings suggest that the increased anxiety induced by morphine withdrawal is associated with the sensitization of the neural substrates of fear in the dPAG and the IC. So, the present results give support to the hypothesis that withdrawal from chronic treatment with morphine leads to fear states possibly engendered by activation of the dPAG and IC, regardless of the production of somatic symptoms.

Anxiogenic effects of activation of NK-1 receptors of the dorsal periaqueductal gray as assessed by the elevated plus-maze, ultrasound vocalizations and tail-flick tests.

Ultrasound vocalizations (USVs) known as 22kHz are usual components of the defensive responses of rats exposed to threatening conditions. The amount of emission of 22kHz USVs depends on the intensity of the aversive stimuli. While moderate fear causes an anxiolytic-sensitive enhancement of the defensive responses, high fear tended to reduce the defensive performance of the animals to aversive stimuli. The dorsal periaqueductal gray (dPAG) is an important vocal center and a crucial structure for the expression of defensive responses. Substance P (SP) is involved in the modulation of the defensive response at this midbrain level, but the type of neurokinin receptors involved in this action is not completely understood. In this study we examined whether local injections of the selective NK-1 agonist SAR-MET-SP (10-100 pmol/0.2microL) into the dPAG (i) cause anxiogenic effects in the elevated plus-maze (EPM) (Exp. I), (ii) influence the novelty-induced 22kHz USVs recorded within the frequency range of 20-26kHz (Exp. II) and (iii) change the nociceptive reactivity to heat applied to the rat's tail (Exp III). The data obtained showed that SAR-MET-SP elicited significant "anxiety-like" behaviors, as revealed by the decrease in the number of entries into and time spent onto the open arms of the EPM. These anxiogenic effects were accompanied with antinociception and disruption of the novelty-induced increase in the number and duration of 22kHz USVs. These findings are in agreement with the notion that NK-1 receptors of the dPAG may be an important neurochemical target for new selective drugs aimed at the control of pathological anxiety states.

Role of amygdala in conditioned and unconditioned fear generated in the periaqueductal gray.

The amygdala and ventral portion of the periaqueductal gray (vPAG) are crucial for the expression of the contextual freezing behavior. However, it is still unclear whether the amygdala also plays a role in defensive behaviors induced by electrical stimulation of the dorsal periaqueductal gray (dPAG). In the present study, rats were implanted with electrodes into dPAG for determination of the thresholds for freezing and escape responses before and after sham or electrolytic lesions in the amygdala. Animals were then submitted to a context fear conditioning procedure. Amygdala lesions disrupted contextual freezing but did not affect defensive behaviors induced by dPAG electrical stimulation. These results indicate that contextual and unconditioned freezing behaviors are mediated by independent neural circuits.

Defense reaction mediated by NMDA mechanisms in the inferior colliculus is modulated by GABAergic nigro-collicular pathways.

Electrical stimulation of the inferior colliculus (IC) causes a behavioral activation together with autonomic responses similar to fear reactions to threatening situations. GABAergic mechanisms exert a tonic inhibitory control on the neural substrates of aversion in the IC insofar as local injections of GABA agonists or antagonists inhibit or mimic these defensive behaviors, respectively. Recently, we have shown that systemic injections of the GABA-A receptor agonist muscimol unexpectedly enhanced the freezing and escape responses provoked by gradual increases in the intensity of the electrical stimulation of the IC. Taking into account that the neural circuits mediated by excitatory amino acids (EAA) in the IC may be responsible for the integration of fear states, in the present study we examined whether the defensive behavior induced by local injections of NMDA into the IC is influenced by prior treatment with systemic muscimol and also whether this GABAergic control could be exerted by GABAergic fibers that project to the inferior colliculus from the substantia nigra pars reticulata (SNpr). Rats were implanted with two guide-cannulae aimed at the IC and SNpr through which drug microinfusions with glass micropipette could be made with reduced brain damage. One week after surgery, the animals received either NMDA (7 nmol/0.2 microl) or saline into the IC and were placed into the middle of an enclosure where behavioral responses such as freezing, crossings, jumping, rearing, and turnings could be measured as an indirect index of unconditioned fear. These animals were pretreated either with saline or muscimol (0.5 mg/kg, IP) or with brain injections of saline or muscimol (1 nmol/0.2 ìl into SNpr). NMDA applied into the IC produced a behavioral activation with significant increases in all behavioral measures. IP injections of muscimol or into the SNpr enhanced the defense reaction caused by microinjections of NMDA into the IC. These findings give support to the idea that unconditioned defensive responses generated in the IC may be mediated by NMDA mechanisms. Additionally, a reduction of the inhibitory control exerted by nigrocollicular GABAergic neurons seems to be responsible for the unexpected pro-aversive action of systemic injections of muscimol on the neural substrates of aversion mediated by NMDA in the IC.