Microinjection Of Carbachol Research Articles

Involvement of dopaminergic system in the dentate gyrus of the hippocampus in modulating the orofacial pain-related behaviors in the rats.

Chemical stimulation of the lateral hypothalamus (LH) induces analgesia by forming neural circuitries with multiple brain regions. The involvement of hippocampal dopaminergic receptors in the LH stimulation-induced antinociception in specific pain models in animals has been documented. However, because the neural circuitries involved in the mediation of orofacial pain are not the same as those that mediate the other types of pain, the present study aims to detect the role of dopamine receptors within the dentate gyrus (DG) in the antinociceptive responses induced by LH stimulation in an animal model of orofacial pain. Male Wistar rats (220-250 g) were implanted with two separate cannulae into the LH and DG on the same side. D1- or D2-like dopamine receptor antagonist, SCH23390, or sulpiride (0.25, 1, and 4 μg) were microinjected into the DG, five minutes before intra-LH injection of carbachol (250 nM). The animals were then injected with formalin 1% (50 μL; sc) into the upper lip lateral to the nose and subjected to the orofacial formalin test. Intra-DG administration of SCH23390 or sulpiride attenuated the antinociceptive responses induced by intra-LH microinjection of carbachol during the orofacial formalin test. The findings of the current study suggest that chemical stimulation of the LH modulates orofacial pain, possibly through activation of the DG dopaminergic neurons. Due to the high incidence and prevalence of orofacial pain in the general population, understanding how such neuronal circuitry modulates nociceptive processing will advance the search for novel therapeutics.

Involvement of Dopaminergic System in the Dentate Gyrus of the Hippocampus in Modulating the Orofacial Pain-Related Behaviors in the Rats

Chemical stimulation of the lateral hypothalamus (LH) induces analgesia by formation neural circuitries with multiple brain regions. The involvement of hippocampal dopaminergic receptors in the LH stimulation-induced antinociception in certain animal models of pain has been documented, however, considering the fact that the neural circuitries involved in the mediation of orofacial pain are not the same as those that mediate the other types of pain, the present study aims to detect the role of dopamine receptors within the dentate gyrus (DG) in the antinociceptive responses induced by LH stimulation in an animal model of orofacial pain. Male Wistar rats (220-250 g) were implanted with two separate cannulae into the LH and DG on the same side. D1- or D2-like dopamine receptor antagonist, SCH23390 or Sulpiride (0.25, 1, and 4 μg) were microinjected into the DG, five minutes prior to intra-LH injection of carbachol (250 nM). The animals were then injected with formalin 1% (50 μl; sc) into the upper lip lateral to the nose and subjected to the orofacial formalin test. Intra-DG administration of SCH23390 or Sulpiride attenuated the antinociceptive responses induced by intra-LH microinjection of carbachol during orofacial formalin test. The findings of the current study suggest that chemical stimulation of the LH modulates the orofacial pain possibly through activation of the DG dopaminergic neurons. Due to the high incidence and prevalence of orofacial pain in the general population, understanding how such neuronal circuitry modulates nociceptive processing will advance the search for novel therapeutics.

Neuromediators and defensive responses including tonic immobility (TI): Brain areas and circuits involved.

Serotonin, acetylcholine and GABA are the neuromediators most involved in tonic immobility (TI). TI duration, in fact, decreases in rabbits following systemic serotonin administration and in guinea pigs following serotonin microinjection administration into the amygdala owing to the activation of fear-related GABAergic inhibitory mechanisms. On the other hand, repeated TI inductions in rabbits and guinea pigs reduce brain serotonin turnover in several brain areas. Microinjections of the acetylcholine agonist carbachol into amygdala, hypothalamus and PAG increase TI duration and reduces other defensive responses to threatening stimuli in several animal species. The cholinergic and serotonergic systems exert different effects on TI in different regions of the PAG according to the receptors stimulated. Their combined action activates opioid-GABAergic neurons ultimately affecting TI duration. Mammals TI and human cataplexy are innate responses induced by different stimuli, although both characterized by deficiency in orexin, reduced muscle tone, normal jerk reflexes and preserved consciousness.

Role of the Orexinergic System Within the Ventral Tegmental Area in the Development of Sensitization to Morphine Induced by Lateral Hypothalamus Stimulation.

The Lateral Hypothalamus (LH) has long been known to implicate the addictive behaviors of drug abuse. The Ventral Tegmental Area (VTA) is a major area of the mesolimbic system that is strongly involved in developing morphine sensitization. The current study aimed to examine the role of intra-VTA orexin receptors in the LH stimulation-induced sensitization to the antinociceptive response of morphine. A total of 114 adult male Wistar rats underwent unilateral implantation of two separate cannulae in the LH and VTA using the stereotaxic apparatus. Intra-VTA administration of the Orexin-1 (OX1) and Orexin-2 (OX2) receptor antagonists, SB334867 and TCS OX2 29 (1, 3, and 10 nM/0.3 μL DMSO), respectively, was performed 5 min before concurrent microinjection of carbachol (250 nM/0.5 μL saline) into the LH and an ineffective dose of morphine (0.5 mg/kg; SC) during a 3-day sensitization period. After a 5-day free drug period, on the ninth day, for assessing the morphine sensitization, the nociceptive response was measured before and after morphine injection (1 mg/kg; SC) using the tail-flick test. The results revealed that the concurrent administration of carbachol (250 nM) and an ineffective dose of morphine significantly induced morphine sensitization. Besides, the blockade of OX1 and OX2 receptors within the VTA before intra-LH carbachol injection attenuated morphine sensitization. These findings suggest that LH stimulation potentiates the sensitization to morphine antinociceptive responses via affecting orexin receptors located in the VTA. However, OX1 receptors contribute more than OX2 receptors in the VTA to morphine sensitization in rats. LH stimulation enhances sensitization to the ineffective dose of morphineIntra-VTA OX1 receptor involves in morphine sensitization-induced by LH stimulationIntra-VTA OX2 receptor involves in morphine sensitization-induced by LH stimulation. Behavioral sensitization, such as sensitization to the antinociceptive response of drugs, which defines as an enhanced systemic reaction to the same dose of addictive drugs, occurs in response to continuous and intermittent administration of these drugs. The Lateral Hypothalamus (LH) sends the orexinergic projections to the various regions of the brain and stimulation of LH induces sensitization to the antinociceptive response of morphine. The Ventral tegmental area (VTA) is a region of the brain that is strongly involved in developing morphine sensitization and receives orexinergic projections of LH. The current study aimed to examine the role of orexin receptors within the VTA in the LH stimulation-induced sensitization to the antinociceptive response of morphine in rats. In this study orexin-1 (OX1) and orexin-2 (OX2) receptors within the VTA region were blocked using their antagonists. After five minutes chemical stimulation of LH was done using carbachol microinjection into this area and ineffective dose of morphine was injected subcutaneously. These interventions were done for three consecutive days as sensitization period. After a 5-day free drug period, on the ninth day, for assessing the morphine sensitization, the nociceptive response was measured. The results revealed that the concurrent administration of LH stimulation and an ineffective dose of morphine significantly induced morphine sensitization. Besides, the blockade of OX1 and OX2 receptors within the VTA before LH stimulation attenuated sensitization to the antinociceptive response of morphine. Therefore, the orexinergic system plays an important role in morphine sensitization and can be considered as one of the potential targets to increase the analgesic effect of morphine.

Orexin-2 receptor antagonism in the cornu ammonis 1 region of hippocampus prevented the antinociceptive responses induced by chemical stimulation of the lateral hypothalamus in the animal model of persistent pain.

Orexins are excitatory neuropeptides, mainly produced by neurons located in the lateral hypothalamus, which project to many brain areas. The orexinergic system plays a fundamental role in arousal, sleep/wakefulness, feeding, energy homeostasis, motivation, reward, stress and pain modulation. As a prominent part of the limbic system, the hippocampus has been involved in formalin-induced nociception modulation. Moreover, hippocampus regions express both orexin-1 (OX1) and orexin-2 (OX2) receptors. The present study investigated the role of OX2 receptors (OX2R) within the cornu ammonis 1 (CA1) region of the hippocampus in the mediation of lateral hypothalamus-induced antinociception. Fifty-three male Wistar rats were unilaterally implanted with two separate cannulae into the lateral hypothalamus and CA1. Animals were pretreated with intra-CA1 TCS OX2 29 as an OX2R antagonist before intra-lateral hypothalamus administration of carbachol (250 nM) as a muscarinic agonist for chemical stimulation of orexinergic neurons. Formalin test was used as an animal model of persistent pain, following intra-lateral hypothalamus carbachol microinjection. Results showed that the chemical stimulation of the lateral hypothalamus significantly attenuated formalin-evoked nociceptive behaviors during both phases of the formalin test, and administration of TCS OX2 29 into the CA1 blocked these antinociceptive responses in both phases, especially in the late phase. These findings suggest that OX2 receptors in the CA1 partially mediate the lateral hypothalamus-induced antinociceptive responses in persistent inflammatory pain.

Modulation of Septo-Hippocampal Neural Responses in Anesthetized and Behaving Rats by Septal AMPA Receptor Mechanisms.

This study explored the effects of septal glutamatergic transmission on septal-hippocampal theta activity via intraseptal microinjection of antagonist at AMPA receptors (AMPAR). The current results showed that microinjection of AMPAR antagonist, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione, 20 μg/μl, 0.5 μl), evoked a decrease in the frequency of theta activity evoked by various means in anesthetized and behaving rat. Theta wave activity was induced on: (a) intraseptal microinjection of carbachol, an agonist at cholinergic receptors, (b) reticular stimulation, (c) exploration in novel open field (OF), and (d) hind paw (HP) injection of the algogen, formalin. The effect on frequency in the formalin test was observed in an early period on injection of formalin, which was novel to the animal, but not in the later more sustained phase of the formalin test. The effect of NBQX, being seen in both anesthetized and behaving animals, suggests that the modulation of theta wave frequency, including in novelty, is a function of AMPAR in MS. The effect of the antagonist on theta power was less apparent, being observed only in anesthetized animals. In addition to theta power and frequency, intraseptal NBQX also attenuated suppression of CA1 population spike (PS) induced by intraseptal carbachol, thus suggesting that septal glutamate neurotransmission is involved in the spectrum of MS-mediated network responses. Indeed, in the context of behavior, formalin injection induced an increase in the level of septal glutamate, while NBQX attenuated nociceptive behaviors. Notably, MS is involved in the modulation of formalin nociception. These findings suggest that AMPA receptors are a key modulator of septal physiological function.

Blockade of orexin receptors in the ventral tegmental area reduced the extinction period of the lateral hypothalamic-induced conditioned place preference in rats.

The orexinergic connection between the lateral hypothalamus (LH) and the ventral tegmental area (VTA) is involved in modulating the reward circuit. The conditioned place preference (CPP) can be induced by microinjection of carbachol, a cholinergic agonist, into the LH. The current research was conducted to understand whether intra-VTA orexin receptors (OXRs) could influence the duration of the extinction period or maintenance of the intra-LH carbachol-induced CPP. To this end, the rats unilaterally received intra-LH carbachol (250 nM) within a 3-day conditioning period. Animals that have already passed the conditioning test were unilaterally administered by intra-VTA microinjection of SB334867, an OX1R antagonist, or TCS OX2 29, an OX2R antagonist during the extinction phase of the LH stimulation-induced CPP. For the first time, our data indicated that daily intra-VTA administration of either SB334867 (30 nM) or TCS OX2 29 (10 and 30 nM) during the extinction period decreased the maintenance of intra-LH carbachol-induced CPP. In conclusion, OXRs in the VTA play crucial roles in the maintenance of reward processes.

Carbachol and Nicotine in Prefrontal Cortex Have Differential Effects on Sleep-Wake States.

The role of the brainstem cholinergic system in the regulation of sleep-wake states has been studied extensively but relatively little is known about the role of cholinergic mechanisms in prefrontal cortex in the regulation of sleep-wake states. In a recent study, we showed that prefrontal cholinergic stimulation in anesthetized rat can reverse the traits associated with anesthesia and restore a wake-like state, thereby providing evidence for a causal role for prefrontal cholinergic mechanisms in modulating level of arousal. However, the effect of increase in prefrontal cholinergic tone on spontaneous sleep-wake states has yet to be demonstrated. Therefore, in this study, we tested the hypothesis that delivery of cholinergic agonists – carbachol or nicotine – into prefrontal cortex of rat during slow wave sleep (SWS) would produce behavioral arousal and increase the time spent in wake state. We show that unilateral microinjection (200 nL) of carbachol (1 mM) or nicotine (100 mM) into prefrontal cortex during SWS decreased the latency to the onset of wake state (p = 0.03 for carbachol, p = 0.03 for nicotine) and increased the latency to the onset of rapid eye movement sleep (p = 0.008 for carbachol, p = 0.006 for nicotine). Although the infusion of 1 mM carbachol increased the time spent in wake state (p = 0.01) and decreased the time spent in SWS (p = 0.01), infusion of 10 or 100 mM nicotine did not produce any statistically significant change in sleep-wake architecture. These data demonstrate a differential role of prefrontal cholinergic receptors in modulating spontaneous sleep-wake states.

Role of orexin receptors within the dentate gyrus in antinociception induced by chemical stimulation of the lateral hypothalamus in an animal model of inflammatory pain

Pain is a complex experience consisting of sensory, affective-motivational, and cognitive dimensions. Hence, identifying the multiple neural pathways subserving these functional aspects is a valuable task. The role of dentate gyrus (DG) as a relay station of neocortical afferents in the hippocampal formation (HF) in persistent pain is still controversial. The lateral hypothalamus (LH)-HF neural circuits are involved in numerous situations such as anxiety-like behavior, reward processing, feeding, orofacial as well as acute pain. Nonetheless, to our knowledge, the involvement of the LH-DG neural circuit in persistent pain has already remained unexplored. Adult male Wistar rats weighing 220–250 g were undergone stereotaxic surgery for unilateral implantation of two separate cannulae into the LH and DG. Intra-DG administration of the orexin-1 (OX1) and orexin-2 (OX2) receptor antagonists, SB334867 and TCS OX2 29, respectively, was performed 5 min before intra-LH microinjection of carbachol. Animals were then undergone the formalin test using 50 μl formalin injection (2.5%) into the plantar surface of the hind paw. Microinjection of SB334867 or TCS OX2 29 into the DG region attenuated the antinociceptive effect produced by carbachol microinjection into the LH. The preventive effect of SB334867 and TCS OX2 29 on intra-LH carbachol-induced antinociception was approximately equal in both early and late phases of formalin nociception. The results suggest a neural pathway from the LH to the DG, which contributes to the modulation of formalin-induced inflammatory pain through the recruitment of OX1 and OX2 receptors within the DG.

Role of Dopaminergic Receptors Within the Ventral Tegmental Area in Antinociception Induced by Chemical Stimulation of the Lateral Hypothalamus in an Animal Model of Orofacial Pain.

IntroductionThe ventral tegmental area (VTA), as one of the classical components of the brain reward circuitry, shares large neural networks with the pain processing system. We previously showed the role of VTA dopamine receptors in modulation of lateral hypothalamus (LH)-induced antinociception in acute pain conditions. However, considering the fact that the neural systems involved in the mediation of tonic pain are not the same as those that mediate phasic pain. In the present study, we aimed to examine the role of intra-VTA dopamine receptors in LH-induced antinociceptive responses during tonic orofacial pain conditions.MethodsMale Wistar rats weighing 230–250 g were implanted with two separate cannulae into the LH and VTA on the same side. Different solutions of carbachol (62.5, 125 and 250 nM), as a non-selective cholinergic receptor agonist that activates the LH projecting neurons, were microinjected into the LH. In the other groups, D1-like dopamine receptor antagonist, SCH-23390 (0.25, 1 and 4 µg/03 µL saline) or D2-like dopamine receptor antagonist, Sulpiride (0.25, 1 and 4 µg/0.3 µL DMSO 12%) were microinjected into VTA, 5 min prior intra-LH carbachol (250 nM), then subjected to orofacial formalin test. Intra-LH carbachol microinjection dose-dependently attenuated biphasic orofacial pain.ResultsIntra-VTA administration of SCH-23390 or Sulpiride dose-dependently decreased intra-LH carbachol-induced antinociception during both phases of orofacial formalin test with further effects in the late phase.DiscussionThe findings suggest that chemical stimulation of the LH by carbachol possibly activates the orexin projecting neurons and subsequently, the VTA dopaminergic neurons involved in the orofacial pain modulation. Detecting such neural circuitry offers an alternative approach in the development of more efficient therapies for such debilitating pain conditions.

Chemical stimulation of the lateral hypothalamus induces antiallodynic and anti-thermal hyperalgesic effects in animal model of neuropathic pain: Involvement of orexin receptors in the spinal cord

To date several circuities in supraspinal site of the central nervous system have been known to engage in pain modulation. Lateral hypothalamus (LH) is known as part of the circuit of pain modulation among supraspinal sites. Its role in several animal pain models has been well defined. In this study, we examined the role of spinal orexin receptors in antinociceptive response elicited by the LH stimulation in an animal model of neuropathic pain. Male Wistar rats were unilaterally implanted with a cannula into the LH and a catheter into the L4-L5 segments of the spinal cord followed by chronic constriction injury (CCI) surgery. Intra-LH microinjection of carbachol (500 nM; 0.5 μL) was done 5 min after intrathecal administration of the orexin receptor antagonists, SB-334867 or TCS OX2 29; control animals received DMSO. Mechanical allodynia and thermal hyperalgesia were evaluated using von Frey filaments and a thermal stimulus. The results showed that carbachol induces antiallodynic and anti-thermal hyperalgesic effects in a dose-dependent manner. The antiallodynic and anti-thermal hyperalgesic effects induced by intra-LH injection of carbachol were reversed by intrathecal administration of 10 μL–100 nM solutions of SB-334867 or TCS OX2 in neuropathic rats. However, solely intrathecal administration of both antagonists had no effect in neuropathic rats. There appears to be a neural pathway from the LH to the spinal cord, which potentially contributes to the modulation of neuropathic pain. The implications are that there may be novel therapeutic approaches for the treatment of people suffered from chronic neuropathic pain in clinic.

Involvement of Orexinergic System Within the Nucleus Accumbens in Pain Modulatory Role of the Lateral Hypothalamus in Orofacial Pain Model

Lateral hypothalamus (LH) contains a large population of orexinergic neurons. Many studies have investigated the function of these neurons and it is clear that they are involved in pain modulation. The nucleus accumbens (NAc) receives many orexinergic projections, and accumbal neurons express both receptors of orexin (OX1R and OX2R). In this study, we investigated the role of accumbal orexinergic receptors in the LH-induced antinociception during formalin-induced orofacial pain. Male adult Wistar rats weighing 230-250 g were used in this study. Cannulae were unilaterally implanted in their skull for microinjections. SB334867 (OX1 receptor antagonist) or TCS OX2 29 (OX2 receptor antagonist) at the doses of 3, 10 and 30 nM were injected into the NAc with/without intra-LH microinjection of carbachol (250 nM/rat). Carbachol was used for chemical stimulation of orexinergic neurons in the LH. Our results showed that intra-LH carbachol following injection of formalin into animals' upper lip reduced nociception in both phases of formalin test. SB334867 and TCS OX2 29 were able to reduce LH-induced antinociception in both phases. Although the highest dose of SB334867 and TCS OX2 29 (30 nM) was effective in both phases, the TCS OX2 29 but not SB334867 at the dose of 10 nM could not reduce the antinociceptive responses induced by LH stimulation during the first (early) phase. It suggests that contribution of accumbal orexinergic receptors in the first phase of formalin test is more than the second (late) phase, and these results provide further evidence for the involvement of orexinergic system in the modulation of inflammatory orofacial pain.

The contribution of orexin receptors within the ventral tegmental area to modulation of antinociception induced by chemical stimulation of the lateral hypothalamus in the animal model of orofacial pain in the rats.

Involvement of the ventral tegmental area (VTA) and the lateral hypothalamus (LH) in the modulation of formalin-induced nociception is well documented. In this study, we investigated the role of orexin 1 (OX1) and orexin 2 (OX2) receptors within the VTA in modulation of the LH-induced antinociception during both phases of orofacial formalin test. Male adult Wistar rats weighing 230-250 g were unilaterally implanted with two stainless steel guide cannulae in the VTA and LH. In two separate supergroups, animals received SB334867 (OX1 receptor antagonist) or TCS OX2 29 (OX2 receptor antagonist), at the doses of 3, 10, and 30 nM/rat into the VTA before intra-LH microinjection of carbachol (250 nM/rat) as a nonselective cholinergic receptor agonist for chemical stimulation of orexinergic neurons in this region. Rats were subcutaneously injected with 1% formalin (50 µl; s) into the orofacial region, 5 min after intra-LH microinjection of carbachol or saline. The blockade of both orexin receptors in the VTA reduced intra-LH carbachol-induced antinociception. However, this effect was greater during the late phases of the orofacial formalin test. The blockade of the OX1 but not OX2 receptors in the VTA affect the pain-related behaviors during the early phase, and also, the contribution of OX2 receptor to modulate the LH-induced antinociceptive responses was greater than OX1 receptor during the late phase of orofacial formalin test. The results indicated the neural pathway projected from the LH to the VTA contributes to the modulation of formalin-induced orofacial pain. Orexinergic drugs might be considered as therapeutic agents for inflammatory pain treatment.

The Transition Between Slow-Wave Sleep and REM Sleep Constitutes an Independent Sleep Stage Organized by Cholinergic Mechanisms in the Rostrodorsal Pontine Tegmentum.

There is little information on either the transition state occurring between slow-wave sleep (SWS) and rapid eye movement (REM) sleep, as well as about its neurobiological bases. This transition state, which is known as the intermediate state (IS), is well-defined in rats but poorly characterized in cats. Previous studies in our laboratory demonstrated that cholinergic stimulation of the perilocus coeruleus α nucleus (PLCα) in the pontine tegmentum of cats induced two states: wakefulness with muscle atonia and a state of dissociated sleep we have called the SPGO state. The SPGO state has characteristics in common with the IS, such including the presence of ponto-geniculo-occipital waves (PGO) and EEG synchronization with δ wave reduction. Therefore, the aims of the present study were (1) to characterize the IS in the cat and, (2), to study the analogy between the SPGO and the different sleep stages showing PGO activity, including the IS. Polygraphic recordings of 10 cats were used. In seven cats carbachol microinjections (20–30 nL, 0.01–0.1 M) were delivered in the PLCα. In the different states, PGO waves were analyzed and power spectra obtained for the δ, θ, α, and β bands of the EEG from the frontal and occipital cortices, and for the θ hippocampal band. Statistical comparisons were made between the values obtained from the different states. The results indicate that the IS constitutes a state with characteristics that are distinct from both the preceding SWS and the following REM sleep, and that SPGO presents a high analogy with the IS. Therefore, the SPGO state induced by administering carbachol in the PLCα nucleus seems to be an expression of the physiological IS of the cat. Consequently, we propose that the PLCα region, besides being involved in the mechanisms of muscle atonia, may also be responsible for organizing the transition from SWS to REM sleep.

Role of orexin receptors within the dentate gyrus of the hippocampus in antinociception induced by chemical stimulation of the lateral hypothalamus in the tail-flick test as a model of acute pain in rats

The dentate gyrus (DG) as a part of the hippocampal formation is considered one of the brain areas involved in pain perception. The orexin neurons in the lateral hypothalamus (LH) also show connections to regions engaged in the circuitry of pain modulation and many studies have shown their potential to alter pain transmission through the nervous system. However, the role of orexin receptors (OXRs) of the DG in mediation of antinociceptive responses induced by the LH stimulation in rats has not yet been investigated. Hence, the present study was an attempt to examine the role of OXRs located within the DG in the pain modulation induced by chemical stimulation of the LH. Adult male Wistar rats weighing 220–250 g were unilaterally implanted with two separate cannulae into the LH and DG. Intra-DG administration of the orexin-1 receptor (OX1R) antagonist, SB334867 or the orexin-2 receptor (OX2R) antagonist, TCS OX2 29 was performed just 5 min before intra-LH carbachol microinjection. Antinociceptive effects were measured using the tail-flick apparatus. The results showed that OX1R and OX2R antagonists dose-dependently decreased the antinociceptive effect of carbachol. In addition, the effective dose of SB334867 was lesser than that of TCS OX2 29, which indicates the more prominent role of OX1R of the DG in carbachol-induced antinociception. None of the antagonists had any significant effect when administrated without intra-LH carbachol microinjection. It seems to be a neural circuit includes the LH and DG, which plays a major role in the neural basis of pain modulation.

The antagonistic relationship between aversive and appetitive emotional states in rats as studied by pharmacologically-induced ultrasonic vocalization from the nucleus accumbens and lateral septum

Rats can emit 22-kHz or 50-kHz ultrasonic vocalizations (USVs) in negative, as well as positive contexts which index their emotional state. 22-kHz USVs are emitted during aversive contexts and can be initiated by activation of the ascending cholinergic pathways originating from the laterodorsal tegmental nucleus or initiated pharmacologically by injection of cholinergic agonists into target areas of these pathways (medial cholinoceptive vocalization strip). Conversely, 50-kHz USVs are emitted during positive pro-social contexts and can be initiated by stimulation of ascending dopaminergic pathways originating from the ventral tegmental area or by injection of dopamine agonists into target areas of these pathways (nucleus accumbens shell). Recently, we have shown an inhibitory effect a positive emotional state has on the emission of carbachol-induced 22-kHz USVs from the anterior hypothalamic/medial preoptic area. However, this structure is a fragment of that cholinoceptive vocalization strip. We wanted to examine if we could observe similar effect when the aversive state is induced from the lateral septum, the most rostral division of the cholinoceptive vocalization strip. The results supported previous findings. First, microinjection of the dopamine agonist R-(-)-apomorphine into the nucleus accumbens shell resulted in increased emission of frequency modulated (FM) 50-kHz USVs that are regarded as signals expressing a positive emotional state in rats. Second, FM 50-kHz USVs and not flat (F) 50-kHz USVs were able to decrease 22-kHz USVs induced by microinjections of carbachol into the lateral septum. This research provides further support to the hypothesis that the initiation of a positive emotional state functionally antagonizes initiation of a negative emotional state in rats.

Role of D1- and D2-like dopaminergic receptors in the nucleus accumbens in modulation of formalin-induced orofacial pain: Involvement of lateral hypothalamus

The role of dopaminergic system in modulation of formalin-induced orofacial nociception has been established. The present study aims to investigate the role of dopaminergic receptors in the nucleus accumbens (NAc) in modulation of nociceptive responses induced by formalin injection in the orofacial region. One hundred and six male Wistar rats were unilaterally implanted with two cannulae into the lateral hypothalamus (LH) and NAc. Intra-LH microinjection of carbachol, a cholinergic receptor agonist, was done 5min after intra-accumbal administration of different doses of SCH23390 (D1-like receptor antagonist) or sulpiride (D2-like receptor antagonist). After 5min, 50μl of 1% formalin was subcutaneously injected into the upper lip for inducing the orofacial pain. Carbachol alone dose-dependently reduced both phases of the formalin-induced orofacial pain. Intra-accumbal administration of SCH23390 (0.25, 1 and 4μg/0.5μl saline) or sulpiride (0.25, 1 and 4μg/0.5μl DMSO) before LH stimulation by carbachol (250nM/0.5μl saline) antagonized the antinociceptive responses during both phases of orofacial formalin test. The effects of D1- and D2-like receptor antagonism on the LH stimulation-induced antinociception were almost similar during the early phase. However, compared to D1-like receptor antagonism, D2-like receptor antagonism was a little more effective but not significant, at blocking the LH stimulation-induced antinociception during the late phase of formalin test. The findings revealed that there is a direct or indirect neural pathway from the LH to the NAc which is at least partially contributed to the modulation of formalin-induced orofacial nociception through recruitment of both dopaminergic receptors in this region.

Role of dorsal hippocampal orexin-1 receptors in modulation of antinociception induced by chemical stimulation of the lateral hypothalamus

The Hippocampus has a role not only in nociception but also in modulation of pain perception. In addition, orexinergic neurons present in the lateral hypothalamus (LH) have a recognized role in pain modulation. The presence of orexinergic projections from the lateral hypothalamus (LH) to the dorsal hippocampal Cornu Ammonis 1 (CA1) region raises the question of whether pain modulatory role of LH is mediated through the CA1. To elucidate the interactions between the LH and neural substrates involved in modulation of formalin-induced nociception, the study aimed to test the pain modulatory role of CA1 orexin receptors in the formalin test. Seventy-one male Wistar rats were unilaterally implanted with two cannulae above the LH and CA1. In the treatment groups, intra-CA1 administration of SB-334867, as an orexin-1 receptor antagonist, was performed 5min before intra-LH microinjection of carbachol, as a cholinergic receptor agonist. In dimethyl sulfoxide (DMSO)-control group, DMSO and saline as well as in carbachol-control group, DMSO and carbachol were microinjected into the CA1 and LH, respectively. In all rats, the procedure was followed by subcutaneous injection of formalin after 5-min time interval. Carbachol reduced both phases of formalin-induced nociception. Intra-CA1 administration of SB-334867 antagonized the LH-induced analgesia during both phases in a dose-dependent manner. It seems that the blockade of orexin-1 receptors has more effects on reduction of antinociception during the late phase compared to the early phase. Pain modulatory role of orexinergic system in the formalin test through a neural pathway from the LH to CA1 provides the evidence that orexins can be useful therapeutic agents for chronic pain treatment.

Blockade of the orexin receptors in the CA1 region of hippocampus decreased the lateral hypothalamic-induced antinociceptive responses in the model of orofacial formalin test in the rats

The role of hippocampus and lateral hypothalamus (LH) in modulation of formalin-induced nociception has been established. The present study aims to examine the role of orexin receptors in the Cornu Ammonis 1 (CA1) region of hippocampus in modulation of the LH-induced antinociception in the orofacial formalin test. Male Wistar rats were unilaterally implanted with two cannulae into the LH and CA1. Intra-LH microinjection of carbachol was done 5min after intra-CA1 administration of SB-334867 (OX1R antagonist) or TCS OX2 29 (OX2R antagonist). After 5min, 50μl of 1% formalin was subcutaneously injected into the upper lip for inducing the nociceptive behaviors. Solely intra-LH administration of carbachol reduced early and late phases of formalin-induced orofacial nociception in a dose-dependent manner. The antinociception evoked by intra-LH injection of carbachol (0.5μl of 250nM carbachol) was antagonized by intra-CA1 administration of 0.5μl of 3, 10 and 30nM solutions of SB-334867 or TCS OX2 29 during the early and late phases of orofacial formalin test. This effect was more remarkable during the late phase in comparison to the early phase. In addition, anti-analgesic effect of SB-334867 was more than TCS OX2 29 during the early and late phases. The results suggest the interpretation that a neural pathway from the LH to the CA1 probably contributes to the modulation of formalin-induced orofacial nociception through recruitment of both CA1 orexin receptors. Clinical studies are recommended to study the probable effectiveness of orexinergic system in modulation of the orofacial nociceptive responses.

Cholinergic/opioid interaction in anterior cingulate cortex reduces the nociceptive response of vocalization in guinea pigs

The anterior cingulate cortex (ACC) is crucial in the modulation of the sensory, affective and cognitive aspects of nociceptive processing. Also, it participates in the planning and execution of behavioral responses evoked by nociceptive stimuli via descending projections to the brainstem. In laboratory animals nociceptive experimental tests evaluate behavioral responses that preferentially express the sensory-discriminative or affective-motivational component of pain. The objective of this study was to investigate the participation of opioid and cholinergic neurotransmission in the ACC on different nociceptive responses in guinea pigs. We used nociceptive tests of formalin and vocalization evoked by peripheral noxious stimuli (electric shock) to evaluate the behavioral expression of the sensory-discriminative and affective motivational components, respectively. We verified that the microinjection of morphine (4.4nmol) in the ACC of guinea pigs promotes antinociception in the two experimental tests investigated. This effect is blocked by prior microinjection of naloxone (2.7nmol). On the other hand, the microinjection of carbachol (2.7nmol) in the ACC induces antinociception only in the vocalization test. This effect was prevented by prior microinjection of atropine (0.7nmol) and naloxone (2.7nmol). In fact, the blockade of µ-opioids receptors with naloxone in ACC prevented the antinociceptive effect of carbachol in the vocalization test. Accordingly, we suggest that the antinociception promoted by carbachol was mediated by the activation of muscarinic receptors on local ACC opioid interneurons. The release of endogenous opioids seems to inhibited the expression of the behavioral response of vocalization. Therefore, we verified that the antinociceptive effect of morphine microinjection in ACC is broader and more robust than that promoted by carbachol.