Cannabidiol in the prelimbic cortex modulates the comorbid condition between the chronic neuropathic pain and depression-like behaviour in rats: The role of medial prefrontal cortex 5-HT1A and CB1 receptors.

The prelimbic division (PrL) of the medial prefrontal cortex (mPFC) is a cerebral division that is putatively implicated in the chronic pain and depression. We investigated the activity of PrL cortex neurons in Wistar rats that underwent chronic constriction injury (CCI) of sciatic nerve and were further subjected to the forced swimming (FS) test and mechanical allodynia (by von Frey test). The effect of blockade of synapses with cobalt chloride (CoCl2), and the treatment of the PrL cortex with cannabidiol (CBD), the CB1 receptor antagonist AM251 and the 5-HT1A receptor antagonist WAY-100635 were also investigated. Our results showed that CoCl2 decreased the time spent immobile during the FS test but did not alter mechanical allodynia. CBD (at 15, 30 and 60 nmol) in the PrL cortex also decreased the frequency and duration of immobility; however, only the dose of 30 nmol of CBD attenuated mechanical allodynia in rats with chronic NP. AM251 and WAY-100635 in the PrL cortex attenuated the antidepressive and analgesic effect caused by CBD but did not alter the immobility and the mechanical allodynia when administered alone. These data show that the PrL cortex is part of the neural substrate underlying the comorbidity between NP and depression. Also, the previous blockade of CB1 cannabinoid receptors and 5-HT1A serotonergic receptors in the PrL cortex attenuated the antidepressive and analgesics effect of the CBD. They also suggest that CBD could be a potential medicine for the treatment of depressive and pain symptoms in patients with chronic NP/depression comorbidity.

Both serotonergic and noradrenergic systems modulate the development of tolerance to chronic stress in rats with lesions of the serotonergic neurons of the median raphe nucleus.

Acute exposure to stress induces significant behavioural changes, while repeated exposure to the same stressor leads to the development of tolerance to stress. The development of tolerance appears to involve the serotonergic projections from the Median Raphe Nucleus (MnRN) to the dorsal Hippocampus (dH), since rats with lesions of this pathway does not develop tolerance to stress. Previous data from our laboratory showed that treatment with imipramine, a serotonin (5-HT) and noradrenaline (NA) reuptake inhibitor, lead to the development of tolerance. However, it remains to be elucidated whether such tolerance involves the participation of the noradrenergic system, apart from the serotonergic projections. Therefore, the aim of this work was to investigate the behavioural and neurochemical effects of chronic treatment with desipramine (NA reuptake inhibitor) or fluoxetine (5-HT reuptake inhibitor) in chronically stressed rats with lesions of the serotonergic neurons of the MnRN. Male Wistar rats with or without lesion in the MnRN were submitted or not to acute (2 h) or chronic restraint (2 h/seven days) stress and tested in the elevated pus maze (EPM). Treatment with fluoxetine, desipramine (10 mg/kg) or saline was performed twice daily (12-12 h interval), for 7 consecutive days. EPM test was conducted 24 h after the treatment. Fluoxetine attenuated the anxiogenic-induced effect of lesion in chronically restrained rats, without changing serotonin and noradrenaline levels in the hippocampus of lesioned rats. A similar profile was also observed after treatment with desipramine. These results suggest that both the serotonergic and the noradrenergic systems are involved in the development of tolerance to chronic stress. Additionally, the integrity of the serotonergic pathway of the MnRN-dH is not essential for the anxiolytic-like effects of these drugs.

Tolerance to repeated stress in rats with lesions of the serotoninergic neurons of the Median Raphe Nucleus and chronically treated with imipramine.

Repeated exposure to aversive events leads to the development of tolerance to stress, which involves the serotonergic pathway originated in the Median Raphe Nucleus (MnRN) to the Dorsal Hippocampus (DH). However, it is not clear whether these lesion-induced deficits can be attenuated by treatment with antidepressants. Therefore, the aim of this work was to investigate the effects of chronic treatment with Imipramine (IMI) in rats with lesions in the MnRN and exposed to restraint stress. Male Wistar rats with or without neurochemical lesions of the MnRN serotonergic neurons with the neurotoxin 5,7-DHT were submitted to acute (2h) or chronic restraint (2h/day/seven consecutive days) and treated with saline (1 ml/kg) or imipramine (15 mg/kg) via intraperitoneal twice a day during the same period. In acutely restrained rats, stress occurred on the last day of treatment. Test in the elevated plus maze (EPM) was performed 24h later. After EPM test, animals were sacrificed and had their brains removed. Dorsal hippocampus and striatum were dissected and the levels of 5-HT and 5-hydroxy-indoleacetic acid (5-HIAA) measured by HPLC analysis. Our results showed that in control rats exposure to acute restraint stress decreased exploration of the open and enclose arms of the EPM, an effect that was attenuated by imipramine. In rats with 5,7-DHT lesions, acute restraint did not change the exploration of the EPM, independently of the treatment. On the other hand, when chronically restrained, saline treated rat with 5,7-DHT lesion showed a reduced exploration of the open arms of the EPM. This effect was attenuated by simultaneous treatment with imipramine. HPLC analysis showed significantly decreases on 5-HT and 5-HIAA levels in the hippocampus, but not in the striatum. These later results confirm that 5,7-DHT lesions of the MnRN had significant impact on the serotonergic projections to the dorsal hippocampus which seems to be essential for the development of tolerance to repeated stress in the absence of any pharmacological treatment.

Role of serotonin 1A receptors in the median raphe nucleus on the behavioral consequences of forced swim stress.

Despite the intense research on the neurobiology of stress, the role of serotonin (5-HT)1A receptors still remains to be elucidated. In the hippocampus, post-synaptic 5-HT1A receptors activation induces anxiolytic effects in animals previously exposed to stressful situations. However, little is known about somatodendritic 5-HT1A receptors in the median raphe nucleus (MRN). Therefore, the aim of this study was to investigate the role of 5-HT1A receptors located in the MRN in rats exposed to forced swim stress. After recovering from surgery, rats were forced to swim for 15 min in a cylinder. Intra-MRN injections of saline, 8-OH-DPAT (3 nmol/0.2 µL) and/or WAY-100635 (0.3 nmol/0.2 µL) were performed immediately before or after pre-exposure or 24 h later (immediately before test). Non-stressed rats received the same treatment 24 h or 10 min before test. Our data showed that 8-OH-DPAT increased latency to display immobility while decreasing time spent immobile in almost all experimental conditions. These effects were not prevented by previous treatment with WAY-100635. No effects of different treatments were described in non-stressed animals. Taken together, our data suggest that in addition to activation of 5-HT1A, 5-HT7 receptors may also be involved in the behavioural consequences of exposure to swim stress.

Antidepressant-like effects of NMDA-receptor antagonist injected into the dorsal hippocampus of rats.

Exposure to uncontrollable stressors causes behavioral changes that have been related to depressive states in humans. Poststress intrahippocampal administration of amino-7-phosphonoheptanoic acid (AP-7), a glutamate NMDA-receptor antagonist, attenuated the restraint-induced decreased exploration of an elevated plus maze 24 h later. The objective of the study was to test if this treatment would also attenuate the increased immobility seem in the forced swim test (FST) due to preexposition to this stressful situation. Male Wistar rats with cannulae aimed at the dorsal hippocampus were submitted to 15 min of forced swimming and tested 24 h later. They received bilateral intrahippocampal injections of AP-7 (10 nmol) either before or after the pretest swimming session or before the test. Poststress treatment increased latency to display the first episode of immobility and tended to reduce total immobility time. The drug was ineffective when given before stress or before test and in nonstressed animals. This suggests that glutamate NMDA receptors located in the dorsal hippocampus are involved in the behavioral changes observed in the FST.

Behavioral effects in the elevated plus maze of an NMDA antagonist injected into the dorsal hippocampus: influence of restraint stress.

The objective of the study was to investigate the influence of restraint stress on the effects of 2-amino-7-phosphonoheptanoic acid (AP7), an NMDA receptor antagonist, injected into the hippocampus of rats submitted to the elevated plus maze (EPM). Male Wistar rats with cannulas aimed to the dorsal hippocampus were forced immobilized for 2 h. Twenty four hours later they received bilateral injections of saline or AP7 (10 nmol/0.5 microl), and were tested in the EPM. In another experiment the animals received the treatment immediately before or after the restraint period, and were tested in the EPM 24 h later. AP7 had no effect in any anxiety measure in non-stressed rats. In stressed animals the drug increased the percentage of open arm entries when injected before the test in the EPM. When administered immediately after the restraint period, AP7 increased the percentage of time spent in the open arms and tended to do the same with the percentage of entries in these same arms. The results suggest that interference with hippocampal NMDA receptors modify the anxiogenic effect of restraint stress in an EPM.

Expression of neuronal nitric oxide synthase mRNA in stress-related brain areas after restraint in rats.

The objective of the present study was to investigate the expression of neuronal nitric oxide synthase (nNOS) mRNA in stress-related areas after restraint. Male Wistar rats (n=4-6/group) submitted to 2 h of restraint during one (acute) or seven (chronic) days were sacrificed 24 h after the last restraint period. In situ hybridisation was performed with oligonucleotide probes radiolabeled with (35)S. Acute restraint induced a significant increase in nNOS mRNA in the paraventricular hypothalamic nucleus (PVN), medial parvocellular part, dorsolateral periaqueductal grey (DLPAG) and medial amygdaloid nucleus, but not in the hippocampal formation. This effect persisted after chronic restraint in the PVN and DLPAG. These results suggest that restraint stress induces changes in gene expression of nNOS in areas related to stress reactions.

Restraint-induced hypoactivity in an elevated plus-maze.

Rodents submitted to restraint stress show decreased activity in an elevated plus-maze (EPM) 24 h later. The objective of the present study was to determine if a certain amount of time is needed after stress for the development of these changes. We also wanted to verify if behavioral tolerance of repeated daily restraint would be detectable in this model. Male Wistar rats were restrained for 2 h and tested in the EPM 1, 2, 24 or 48 h later. Another group of animals was immobilized daily for 2 h for 7 days, being tested in the EPM 24 h after the last restraint period. Restraint induced a significant decrease in the percent of entries and time spent in the open arms, as well as a decrease in the number of enclosed arm entries. The significant effect in the number of entries and the percentage of time spent in the open arms disappeared when the data were submitted to analysis of covariance using the number of enclosed arm entries as a covariate. This suggests that the restraint-induced hypoactivity influences the measures of open arm exploration. The modifications of restraint-induced hypoactivity are evident 24 or 48 h, but not 1 or 2 h, after stress. In addition, rats stressed daily for seven days became tolerant to this effect.

Restraint-induced hypoactivity in an elevated plus-maze

Rodents submitted to restraint stress show decreased activity in an elevated plus-maze (EPM) 24 h later. The objective of the present study was to determine if a certain amount of time is needed after stress for the development of these changes. We also wanted to verify if behavioral tolerance of repeated daily restraint would be detectable in this model. Male Wistar rats were restrained for 2 h and tested in the EPM 1, 2, 24 or 48 h later. Another group of animals was immobilized daily for 2 h for 7 days, being tested in the EPM 24 h after the last restraint period. Restraint induced a significant decrease in the percent of entries and time spent in the open arms, as well as a decrease in the number of enclosed arm entries. The significant effect in the number of entries and the percentage of time spent in the open arms disappeared when the data were submitted to analysis of covariance using the number of enclosed arm entries as a covariate. This suggests that the restraint-induced hypoactivity influences the measures of open arm exploration. The modifications of restraint-induced hypoactivity are evident 24 or 48 h, but not 1 or 2 h, after stress. In addition, rats stressed daily for seven days became tolerant to this effect

Pinealectomy attenuates the effect of restraint on plus maze exploration in rats.

To investigate a possible stress modulation role of the pineal gland, male Wistar albino rats (200-250 g) were submitted to pinealectomy and divided into four groups one week after surgery: i) sham-operated unrestrained animals (N = 14); ii) pinealectomized unrestrained animals (N = 22); iii) sham-operated animals submitted to 2 h of restraint (N = 52); iv) pinealectomized animals submitted to 2 h of restraint (N = 56). Twenty-four hours later the animals were tested in the elevated plus maze for 5 min. Pinealectomized rats submitted to restraint explored the open arms of the maze to a greater extent than sham-operated restrained rats (mean percent of open arm entries = 26.4 +/- 2.3 vs 18.0 +/- 2.1, mean percent of time spent in the open arms = 11.8 +/- 2.1 vs 6.8 +/- 1.2). Pinealectomized animals not submitted to restraint showed no difference in maze exploration when compared to sham-operated rats (mean percent of open arm entries = 29.3 +/- 3.8 vs 31.1 +/- 5.8, mean percent of time spent in the open arms = 8.8 +/- 1.8 vs 12.5 +/- 2.2). The results, therefore, suggest that the pineal gland may play a modulatory role in the behavioral consequences of stress.

Pinealectomy attenuates the effect of restraint on plus maze exploration in rats

To investigate a possible stress modulation role of the pineal gland, male Wistar albino rats (200-250 g) were submitted to pinealectomy and divided into four groups one week after surgery: i) sham-operated unrestrained animals (N = 14); ii) pinealectomized unrestrained animals (N = 22); iii) sham-operated animals submitted to 2 h of restraint (N = 52); iv) pinealectomized animals submitted to 2 h of restraint (N = 56). Twenty-four hours later the animals were tested in the elevated plus maze for 5 min. Pinealectomized rats submitted to restraint explored the open arms of the maze to a greater extent than sham-operated restrained rats (mean percent of open arm entries = 26.4 ñ 2.3 vs 18.0 ñ 2.1, mean percent of time spent in the open arms = 11.8 ñ 2.1 vs 6.8 ñ 1.2). Pinealectomized animals not submitted to restraint showed no difference in maze exploration when compared to sham-operated rats (mean percent of open arm entries = 29.3 ñ 3.8 vs 31.1 ñ 5.8, mean percent of time spent in the open arms = 8.8 ñ 1.8 vs 12.5 ñ 2.2). The results, therefore, suggest that the pineal gland may play a modulatory role in the behavioral consequences of stress.

Hippocampal 5-HT receptors and consolidation of stressful memories.

It has been suggested that postsynaptic 5-HT1A receptors in the hippocampus, innervated by 5-HT neurons localized in the median raphe nucleus, mediate adaptive or coping responses to aversive events and that dysfunction of this system is related to symptoms of depression. To test this hypothesis we investigated the expression of c-fos mRNA in animals submitted to immobilization stress. The results showed that c-fos mRNA expression is significantly increased in the dentate gyrus and CA1-CA3 regions of the hippocampus after 30 min of forced restraint, suggesting that this structure is activated during stress. To investigate the role of 5-HT neurotransmission in the hippocampus on adaptation to aversive events we immobilized rats for 2 h and tested them 24 h later in an elevated plus-maze. Our results showed that the previous restraint period decreases exploration of open arms in the maze. This effect was reversed by bilateral microinjection of zimelidine (20 and 100 nmol), a 5-HT re-uptake blocker, or 8-OH-DPAT (3 nmol), a 5-HT1A agonist, into the dorsal hippocampus immediately after restraint. These results are compatible with the idea that postsynaptic 5-HT1A receptors located in the hippocampus participate in the development of tolerance to aversive events.

Attenuation of behavioral consequences of immobilization stress by intra-hippocampal microinjection of zimelidine.

To investigate the role of hippocampal 5-HT neurotransmission on adaptation to aversive events, individually housed male Wistar rats (200-250 g) were immobilized for 2 h and tested 24 h later in an elevated plus-maze. Immediately after the restraint period they received bilateral microinjections into the dorsal hippocampus of either saline (0.5 microliters) or the nonselective 5-HT1 antagonist dl-propranolol (20 nmol/0.5 microliters). In a second experiment the first microinjection of saline or dl-propranolol was followed by a second microinjection of saline (0.5 microliters) or the 5-HT reuptake blocker zimelidine (20 nmol/0.5 microliters). Although dl-propranolol alone did not change exploration of the elevated plus-maze, it antagonized the increase in the percentage of open arm entries induced by zimelidine (26.0 +/- 4.1 vs 5.64 +/- 3.7 in controls). These results are compatible with the view that post-synaptic 5-HT1a receptors in the hippocampus mediate adaptive or coping responses to aversive events.

Attenuation of behavioral consequences of immobilization stress by intra-hippocampal microinjection of zimelidine

To investigate the role of hippocampal 5-HT neurotransmission on adaptation to aversive events, individually housed male Wistar rats (200-250 g) were immobilized for 2 h and tested 24 h later in an elevated plus-maze. Immediately after the restraint period they received bilateral microinjections into the dorsal hippocampus of either saline (0.5 microliters) or the nonselective 5-HT1 antagonist dl-propranolol (20 nmol/0.5 microliters). In a second experiment the first microinjection of saline or dl-propranolol was followed by a second microinjection of saline (0.5 microliters) or the 5-HT reuptake blocker zimelidine (20 nmol/0.5 microliters). Although dl-propranolol alone did not change exploration of the elevated plus-maze, it antagonized the increase in the percentage of open arm entries induced by zimelidine (26.0 +/- 4.1 vs 5.64 +/- 3.7 in controls). These results are compatible with the view that post-synaptic 5-HT1a receptors in the hippocampus mediate adaptive or coping responses to aversive events