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Cholinergic mechanism involved in the nociceptive modulation of dentate gyrus
Biochemical and Biophysical Research Communications 379 (2009) 975–979
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Cholinergic mechanism involved in the nociceptive modulation of dentate gyrus Runsheng Jiao a, Chunxiao Yang b, Ying zhang a, Manying Xu a,*, Xiaofang Yang a a b
Department of Physiology, Harbin Medical University, XueFu Road No. 194, NanGang District, Harbin 150081, Heilongjiang Province, PR China Department of Neurology of 2nd Affiliated Hospital, Harbin Medical University, Harbin 150081, PR China
a r t i c l e
i n f o
Article history: Received 28 December 2008 Available online 9 January 2009
Keywords: Dentate gyrus (DG) Nociceptive modulation Electrophysiology Acetylcholine (ACh) Pilocarpine Atropine
a b s t r a c t Acetylcholine (ACh) causes a wide variety of anti-nociceptive effects. The dentate gyrus (DG) region of the hippocampal formation (HF) has been demonstrated to be involved in nociceptive perception. However, the mechanisms underlying this anti-nociceptive role have not yet been elucidated in the cholinergic pain-related neurons of DG. The electrical activities of pain-related neurons of DG were recorded by a glass microelectrode. Two kinds of pain-related neurons were found: pain-excited neurons (PEN) and pain-inhibited neurons (PIN). The experimental protocol involved intra-DG administration of muscarinic cholinergic receptor (mAChR) agonist or antagonist. Intra-DG microinjection of 1 ll of ACh (0.2 lg/ll) or 1 ll of pilocarpine (0.4 lg/ll) decreased the discharge frequency of PEN and prolonged firing latency, but increased the discharge frequency of PIN and shortened PIN inhibitory duration (ID). Intra-DG administration of 1 ll of atropine (1.0 lg/ll) showed exactly the opposite effects. According to the above experimental results, we can presume that cholinergic pain-related neurons in DG are involved in the modulation of the nociceptive response by affecting the discharge of PEN and PIN. Ó 2009 Elsevier Inc. All rights reserved.
The hippocampal formation (HF), as part of the limbic system of the basal forebrain, plays a primary role in nociception. Noxious stimuli also modulate hippocampal neuronal activity and blood flow [1]. At barely detectable levels of nociception, regional blood flow was increased not only in the cortex, but also in the contralateral hippocampus and thalamus. In contrast, regional blood flow in the amygdala decreased [2]. Some scholars report that morphine, acupuncture, and acetylcholine (ACh) receptor [3] agonists can modulate hippocampal neuronal excitability. Multiple active neural compounds are involved, such as ACh, gamma-amino butyric acid (GABA), etc. The hippocampus receives cholinergic projections from the medial septal nucleus and Broca’s diagonal band, which terminate in the CA1, CA3, and dentate gyrus (DG) regions [4]. Endogenous ACh is probably released from the septal-hippocampal fasciculus and the perforating fibers of the entorhinal area, as well as from many sporadically distributed ACh local interneurons in the local neural circuit [5]. The central cholinergic system plays the crucial role; however, the roles of the individual cholinergic systems involved in these activities are not currently well understood [6]. HF is defined as the complex of six structures including the DG and hippocampus proprius. It was demonstrated in many experiments that the DG not only receives nociceptive afferents, but also plays an important role in nociceptive modulation [7–10]. * Corresponding author. Fax: +86 451 8669 7507. E-mail address: [email protected] (M. Xu). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.12.184
Nonetheless, the nociceptive process related to the electrical activities of pain-related neurons is still unclear and needs to be investigated further in DG. We studied this issue using one single glass microelectrode. In the current study, we investigated the role of muscarinic receptors in the DG of HF by measuring the electrical responses of pain-related neurons. For this purpose, we performed intra-DG injection of the exogenous muscarinic acetylcholine receptor (mAChR) agonist pilocarpine and ACh; atropine was used to block mAChR and thus the effects of endogenous ACh. Materials and methods Animals. Wistar rats (200–260 g, Animal Centre of Second Affiliated Hospital of Harbin Medical University. Certificated No. 09-21) were used in this study. The rats were divided into 4 groups (n = 10 per group): (1) control group: intra-DG administration of normal saline 1 ll, (2) pilocarpine group: intra-DG administration of 0.4 lg/ll pilocarpine, (3) atropine group: intra-DG administration of 1.0 lg/ll atropine, (4) ACh group: intra-DG administration of 0.2 lg/ll ACh. All injections were completed within 2 min via a microliter syringe driven by micro-injection pump (ZCZ-50, Zhejiang province, China). Neurosurgery and electrophysiological studies. The experiments were performed on Wistar rats, anaesthetized with urethane (1 g/kg, ip). The sciatic nerves were isolated. Two skull windows were opened and covered with liquid paraffin. Rats were fixed in the stereotaxic apparatus (SN-2, Narishige, Japan). After artificial
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ventilation was established, rats were paralyzed with tubocurarine chloride (1 mg/kg). Single-unit recordings were performed with glass microelectrode (tip extreme diameter: 0.5–1.0 lm, DC resistance 10–30 MX) filled with KCl (3 mol/L). The glass microelectrode was inserted with a micromanipulator (SM-21, Narishige, Japan) into the hippocampal DG (AP: 2.8–3.2 mm, R or L: 1.8– 2.2 mm, H: 3.0–3.5 mm) [11]. Another glass microelectrode filled with drugs was inserted with a micromanipulator (SM-11, Narishige, Japan) into the DG (AP: 3.0 mm, R or L: 2.0 mm, H: 3.2 mm) for drug administration. The electrical activity was amplified with a micro-electrical amplifier and recorded with the biological experimental system, while simultaneously monitored with an oscilloscope (VC-9, Nihon Kohden, Japan). Since the neural discharges were recorded by the biological experimental system, the electrical stimulation of sciatic nerves was performed through a twin pole stainless steel-shielded electrode as noxious stimulation (delay: 0 ms, interval: 5 ms, intensity: 5 mA; duration: 0.3 ms, train 5) (SEN-3301, Nihon Kohden, Japan). The discharge of each neuron was recorded three times every 2 min; a 30 min-recording was considered as a complete record. Definition of neurons. In our present experiment, the term ‘‘painrelated neurons” refers to PEN and PIN. Neurons that responded to noxious stimulation by increasing the discharge frequency were defined as PEN [12], while neurons that responded to a noxious stimulus by decreasing the discharge frequency were defined as PIN [13]. PEN showed very few spontaneous discharges before noxious stimulation. In contrast, spontaneous discharges were common in PIN, which were readily inhibited by the application of noxious stimulation. Net-increased value (NIV, Hz) refers to the differences in PEN or PIN between the average frequency of evoked discharges after noxious stimulation, and the average frequency of the discharges within 2 s prior to noxious stimulation. Inhibitory duration (ID, s) refers to the latency time from noxious stimulation to the appearance of the PIN redischarge. Histological identification. At the end of the experiment, pontamine sky blue was diffused out from the microelectrode with a negative direct current (30 l A, 15 min), to identify the tip position. Statistical analysis. Data were scanned into the computer with Powerlab/8 s (ADInstuments) and analyzed with Chart v5.3 software (Australian). All data were expressed as mean ± SEM, analyzed with SPSS 13.0 software. Statistical differences were evaluated by one-way ANOVA; SNK test was used for comparisons between two groups and statistical significances were determined at the level of p < 0.05. Results Effects of normal saline on the electrical activities of pain-related neurons in DG Administration of normal saline produced no significant effect on the electrical activities of PEN and PIN (Figs. 1A and 2A). Influence of pilocarpine on the electrical activities of pain-related neurons in DG The average NIV of PEN was 9.94 ± 0.69 Hz and the average latency was 0.29 ± 0.07 s. After intra-DG administration of pilocarpine, the NIV of PEN began to decrease and latency began to increase (Fig. 1B). This trend was clearest at 10 min after pilocarpine administration; NIV had decreased to 4.75 ± 0.70 Hz and latency increased to 0.78 ± 0.07 s. From 2 to 18 min after administration, the NIV and latency of PEN showed significant differences as compared with those before injection or those of the control group (p < 0.05, Fig. 3).
The average NIV of PIN was 4.69 ± 0.56 Hz and the ID was 0.75 ± 0.43 s. In PIN, the average NIV began to increase and ID began to shorten after injection of pilocarpine (Fig. 2B). These reactions were most obvious at 10 min after administration; the NIV increased to 2.31 ± 0.71 Hz and ID shortened to 0.58 ± 0.11 s. Measurements obtained from 0 to 16 min after injection differed significantly from those obtained at the same interval prior to injection, as well as in comparison to the control group (p < 0.05, Fig. 4). Effects of atropine on the electrical activities of pain-related neurons in DG Twelve minutes after intra-DG administration of atropine, the average NIV of PEN significantly increased from 9.99 ± 0.63 Hz to 13.53 ± 0.70 Hz, and the latency shortened from 0.29 ± 0.03 s to 0.11 ± 0.02 s (Fig. 1C). From 2 to 20 min after injection, the NIV and latency of PEN showed obvious changes, as compared with measurements before administration or in the control group (p < 0.05, Fig. 3). At the fourth minute after intra-DG administration of atropine, evoked PIN discharges began to change significantly. The average NIV decreased from 4.82 ± 0.45 Hz to the minimum value of 6.35 ± 0.41 Hz, and the ID increased from 0.77 ± 0.10 s to the maximum value of 1.00 ± 0.09 s (Fig. 2C): ID increased 29.87%, as compared with values obtained prior to administration. This tendency was most clear at 12 min after administration. From 2 to 22 min after injection, the NIV and ID of PIN showed obvious changes compared with values obtained before injection or in the control group (p < 0.05, Fig. 4). Influence of ACh on the electrical activities of pain-related neurons in DG Before injection of ACh, the average NIV of PEN was 9.99 ± 1.08 Hz and the latency was 0.27 ± 0.03 s. Soon after intraDG administration of ACh, the NIV of PEN began to decrease and latency began to increase (Fig. 1D). These effects peaked at 10 min after administration; NIV decreased to 3.62 ± 0.84 Hz, a reduction of 36.24%, compared with measurements obtained prior to administration. Latency increased to 0.94 ± 0.10 s. From 4 to 18 min after administration, the NIV and latency of PEN showed obvious changes, as compared with measurements obtained prior to drug administration or in the control group (p < 0.05, Fig. 3). Twenty-two minutes after administration, the NIV and latency of PEN returned to the values observed before treatment. Before injection of ACh, the average NIV of PIN was 4.58 ± 0.64 Hz and the average ID of PIN was 0.73 ± 0.05 s. Two minutes after injection of ACh, the average NIV began to increase and ID began to shorten (Fig. 2D). These trends peaked at 8 min after injection; the average NIV was 1.98 ± 0.66 Hz, an increase of 56.77% in comparison to measurements obtained prior to injection. The ID had declined to 0.50 ± 0.05 s by 6 min after injection. From 0 to 18 min after injection, the average NIV and ID showed obvious changes compared with measurements obtained prior to injection or in the control group (p < 0.05, Fig. 4). About 20 min after administration, the NIV and ID of PIN returned to the values observed before treatment. Discussion According to the responses of pain-related neurons to noxious stimulation of the sciatic nerve, pain-related neurons were classified as PEN or PIN. PEN and PIN exhibit contrasting responses to the same drug or stimulus, accounting for the effects of ACh, pilocarpine and atropine in modulating nociception. While the PEN/
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Fig. 1. Representative histograms of discharged PEN. (A) Intra-DG injection of normal saline did not significantly affect the electrical activities of PEN. (B) Intra-DG injection of pilocarpine decreased evoked PEN discharges. (C) Intra-DG injection of atropine increased evoked PEN discharge. (D) Intra-DG injection of acetylcholine decreased the electrical activity of PEN.", stimulus artifact; q, injection of normal saline; w, injection of pilocarpine; , injection of atropine; }, injection of acetylcholine; X, before injection; 0, 4,. . ., 28, time after injection (min).
Fig. 2. Representative histograms of discharged PIN. (A) Intra-DG injection of normal saline did not significantly affect the electrical activities of PIN. (B) Intra-DG injection of pilocarpine potentiated the electrical activity of PIN. (C) Intra-DG injection of atropine weakened PIN activity. (D) Intra-DG injection of acetylcholine potentiated PIN activity.", stimulus artifact; q, injection of saline; w, injection of pilocarpine; , injection of atropine; }, injection of acetylcholine; X, before injection; 0, 4, . . ., 28, time after injection (min).
Fig. 3. Influence of intra-DG injection of different substance on the NIV (A) and latency (B) of PEN in the DG. , injection of substance; X, before injection; 0, 4, . . ., 28, time after injection (min); values are given by means ± SEM. *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01, 4p < 0.05, 44p < 0.01 when compared with saline group.
PIN distinction is an important tool for nociceptive research [14], some researchers use the terms ‘‘on-” or ‘‘off-cell” to denote ex-
cited or inhibited neurons in the traditional nociceptive conducting pathway [15].
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Fig. 4. Influence of intra-DG injection of different substance on the NIV (A) and ID (B) of PIN in the DG. , injection of substance; X, before injection; 0, 4, . . ., 28, time after injection (min); values are given by means ± SEM. *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01, 4p < 0.05, 44p < 0.01 when compared with saline group.
The present study demonstrates that ACh or pilocarpine inhibited the evoked discharge of PEN and potentiated that of PIN, i.e., exhibiting the analgesic effect of ACh or pilocarpine. Atropine potentiated the evoked discharge of PEN and attenuated that of PIN, demonstrating facilitated nociceptive effects. The results revealed that cholinergic PEN and PIN exist in the DG of rats. Our study shows that M receptor agonists exert an anti-nociceptive effect in modulating pain; atropine excited PEN and inhibited PIN. Thus the muscarinic receptor antagonist plays a role in facilitated nociception. During the preliminary experiments, we used different dosages of ACh and pilocarpine and found that ACh and pilocarpine act in a dose-dependent manner. The dosages of ACh (0.2 lg/ll) or pilocarpine (0.4 lg/ll) used yielded significant effects and facilitated the recording of neuronal discharges. Surprisingly, the data curves obtained for the atropine group were not as distinct as those obtained for the ACh group or the pilocarpine group. We tried repeatedly to increase the dosage of atropine during the experiments. Nevertheless the small dosage of atropine had no effect on the electrical activities of PEN and PIN; higher doses are toxic to the central nervous system. Furthermore, noxious stimulus may evoke central cholinergic activation [16], weakening the effect of atropine as an ACh antagonist. Therefore we sought to use the smallest dosage possible of atropine. Furthermore, the molecular weights of these three drugs are unequal; a reasonable dosage should yield an obvious effect while maximizing the equimolarity of the various solutions. Previous studies, including a study performed in our lab, have shown that the (HF) is involved in nociception; nociceptive stimuli modify the electrical activity of the hippocampus [17] and are able to induce c-fos expression in this structure. Every region is connected by extensive fiber networks. Granular cells in the DG receive synapses from neurons of the entorhinal cortex through the perforating pathway. In turn, these granular cells send their axons into the CA3 area of the hippocampus via the mossy fiber pathway. In strata radiatum and oriens of the hippocampal CA1 area, the nerve terminals derive from axon collaterals of CA3 pyramidal cells. Aside from these major fiber connections, the dendritic shafts of various types of interneurons can build connections with the spines of principal cells. Because the HF integrates information from multiple connecting pathways, the various regions are intimately connected. The DG sub-region was found to be implicated in nociception. The disruption of neural activity in the DG reduces nociception. Local injection of anesthetic into the DG produces an anti-nociceptive effect in the formalin test [18]. Blocking neural transmission along the major afferent or efferent hippocampal pathways reduces noci-
ceptive behavior [19]. Microinjection of the NMDA receptor antagonist AP5 into DG served to attenuate nociceptive behaviors in both the acute and tonic phases of the formalin test [20]. Our present study indicates that the DG is involved in the modulation of nociception. ACh (an agonist of both mAChR and nAChR), pilocarpine (a selected mAChR agonist), and carbachol (a mAChR agonist) are known to exert anti-nociceptive effects. Pharmacological experiments have shown that the microinjection of ACh or carbachol into specific brainstem nuclei can produce anti-nociceptive effects and can be reversed by muscarinic receptor antagonists [20]. Some other types of drugs yield nociceptive effects mediated by ACh. It also has been demonstrated that endogenous ACh plays an important role in mediating the nociceptive effects of morphine and clonidine [21,22]. D2 antagonist prochlorperazine exerts an antinociceptive effect mediated by a central cholinergic mechanism [23]. In our present study, an anti-nociceptive effect was observed with injection of ACh or pilocarpine. This finding is in agreement with reports that mAChR agonists exert extensive anti-nociceptive effects in spinal and supraspinal nerves. Atropine, as an endogenous non-competitive antagonist of mAChR, usually exerts a nociceptive effect. Pretreatment with atropine antagonizes the anti-nociceptive effect of NSAIDs in spinal cord [24]; of prochlorperazine in i.c.v. [23]; of oxotremorine in spinal cord [25], and of carbachol in dorsal hippocampus [26]. These findings suggest that intrinsic cholinergic pathways represent an important modulating system in nociception. In our study, injection of the exogenous muscarinic receptor antagonist atropine can increase the NIV and shorten the latency of PEN, while decreasing NIV of PIN and prolonging ID of PIN. This effect was opposite to that of ACh and pilocarpine. This effect is mediated by the blockade of endogenous ACh due to atropine. While our results are similar to those of previous experiments, some controversial reports indicate that atropine can produce anti-nociception [27]. These discrepancies may result from different doses and sites of injection, or the various subtypes of mAChR found in different tissues. Several families of muscarinic ACh receptor are expressed in the HF; five distinct mAChR subtypes referred to as M1 to M5 are localized in separate layers of the HF [28]. Researcher used quantitative immunoprecipitations of the rat brain to demonstrate 36% M1, 33% M2 and 27% M4 expression in the hippocampus. Others found that the M1 muscarinic receptor subtype is critical to induce central cholinergic nociception in mice; M1 is widely expressed in the somata and dendrites of pyramidal neurons and in DG granule cells. Activation of the peripheral M2 receptor produces anti-nociception in vivo and inhibition of nociceptive activity in vitro. In HF, M3 expression is mainly enriched in the pyramidal neurons and the
R. Jiao et al. / Biochemical and Biophysical Research Communications 379 (2009) 975–979
outer third of the molecular layer of the DG. Gene knockout studies reveal that the functional activity of some M4 receptors requires the presence of M2 receptors [29]. The unique laminar and regional distributions of M1–M4 in the hippocampus reflect association of the receptor subtypes with particular afferents, as well as a special intrinsic connection with the M1 and M3 receptors expressed in principal neurons and the M2 and M4 receptors on interneurons. This may imply that each subtype plays a different role in excitatory and inhibitory modulation of the hippocampal circuits. In contrast, neurons in the spinal cord express primarily M2 and M3 [30]. Our experiments showed that intra-DG injection of pilocarpine can reinforce the anti-nociceptive pathway; this is in agreement with previous results that indicate that activation of ACh-M1 and/or ACh-M3 receptors mediate anti-nociceptive effects in central nervous system [31]. The current study suggests that synaptic release of ACh results in a complex and differential mAChR-mediated modulation of cellular excitability within the hippocampal interneuron population [5]. However, further investigation is required to determine which mAChR subtypes play a critical role in anti-nociceptive effects mediated by the DG. In conclusion, muscarinic cholinergic receptor agonists modulate nociception by inhibiting PEN activity and potentiating PIN activity. Intra-DG administration of atropine produces the opposite effect. Our present study confirms that a cholinergic-sensitive mechanism in the DG region of the HF modulates the processing of noxious information. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 30240058), the Science & Technology Agency in Heilongjiang, China (No. GC05C40607) and Science Foundation of Health Department in Heilongjiang, China (No. 2007-525). References [1] I.H. Lorenz, K. Egger, H. Schubert, C. Schnurer, W. Tiefenthaler, M. Hohlrieder, M.F. Schocke, C. Kremser, R. Esterhammer, A. Ischebeck, P.L. Moser, C. Kolbitsch, Lornoxicam characteristically modulates cerebral pain-processing in human volunteers: a functional magnetic resonance imaging study, Br. J. Anaesth. 100 (2008) 827–833. [2] S.W. Derbyshire, A.K. Jones, F. Gyulai, S. Clark, D. Townsend, L.L. Firestone, Pain processing during three levels of noxious stimulation produces differential patterns of central activity, Pain 73 (1997) 431–445. [3] X.F. Yang, Y. Xiao, M.Y. Xu, Both endogenous and exogenous ACh plays antinociceptive role in the hippocampus CA1 of rats, J. Neural. Transm. 115 (2008) 1–6. [4] W. Buno, C. Cabezas, D. Fernandez de Sevilla, Presynaptic muscarinic control of glutamatergic synaptic transmission, J. Mol. Neurosci. 30 (2006) 161–164. [5] H. Widmer, L. Ferrigan, C.H. Davies, S.R. Cobb, Evoked slow muscarinic acetylcholinergic synaptic potentials in rat hippocampal interneurons, Hippocampus 16 (2006) 617–628. [6] N. Kuczewski, E. Aztiria, D. Gautam, J. Wess, L. Domenici, Acetylcholine modulates cortical synaptic transmission via different muscarinic receptors, as studied with receptor knockout mice, J. Physiol. 566 (2005) 907–919. [7] M.B. Echeverry, F.S. Guimaraes, M.A. Oliveira, W.A. do Prado, E.A. Del Bel, Delayed stress-induced antinociceptive effect of nitric oxide synthase inhibition in the dentate gyrus of rats, Pharmacol. Biochem. Behav. 74 (2002) 149–156.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Cholinergic mechanism involved in the nociceptive modulation of dentate gyrus Runsheng Jiao a, Chunxiao Yang b, Ying zhang a, Manying Xu a,*, Xiaofang Yang a a b
Department of Physiology, Harbin Medical University, XueFu Road No. 194, NanGang District, Harbin 150081, Heilongjiang Province, PR China Department of Neurology of 2nd Affiliated Hospital, Harbin Medical University, Harbin 150081, PR China
a r t i c l e
i n f o
Article history: Received 28 December 2008 Available online 9 January 2009
Keywords: Dentate gyrus (DG) Nociceptive modulation Electrophysiology Acetylcholine (ACh) Pilocarpine Atropine
a b s t r a c t Acetylcholine (ACh) causes a wide variety of anti-nociceptive effects. The dentate gyrus (DG) region of the hippocampal formation (HF) has been demonstrated to be involved in nociceptive perception. However, the mechanisms underlying this anti-nociceptive role have not yet been elucidated in the cholinergic pain-related neurons of DG. The electrical activities of pain-related neurons of DG were recorded by a glass microelectrode. Two kinds of pain-related neurons were found: pain-excited neurons (PEN) and pain-inhibited neurons (PIN). The experimental protocol involved intra-DG administration of muscarinic cholinergic receptor (mAChR) agonist or antagonist. Intra-DG microinjection of 1 ll of ACh (0.2 lg/ll) or 1 ll of pilocarpine (0.4 lg/ll) decreased the discharge frequency of PEN and prolonged firing latency, but increased the discharge frequency of PIN and shortened PIN inhibitory duration (ID). Intra-DG administration of 1 ll of atropine (1.0 lg/ll) showed exactly the opposite effects. According to the above experimental results, we can presume that cholinergic pain-related neurons in DG are involved in the modulation of the nociceptive response by affecting the discharge of PEN and PIN. Ó 2009 Elsevier Inc. All rights reserved.
The hippocampal formation (HF), as part of the limbic system of the basal forebrain, plays a primary role in nociception. Noxious stimuli also modulate hippocampal neuronal activity and blood flow [1]. At barely detectable levels of nociception, regional blood flow was increased not only in the cortex, but also in the contralateral hippocampus and thalamus. In contrast, regional blood flow in the amygdala decreased [2]. Some scholars report that morphine, acupuncture, and acetylcholine (ACh) receptor [3] agonists can modulate hippocampal neuronal excitability. Multiple active neural compounds are involved, such as ACh, gamma-amino butyric acid (GABA), etc. The hippocampus receives cholinergic projections from the medial septal nucleus and Broca’s diagonal band, which terminate in the CA1, CA3, and dentate gyrus (DG) regions [4]. Endogenous ACh is probably released from the septal-hippocampal fasciculus and the perforating fibers of the entorhinal area, as well as from many sporadically distributed ACh local interneurons in the local neural circuit [5]. The central cholinergic system plays the crucial role; however, the roles of the individual cholinergic systems involved in these activities are not currently well understood [6]. HF is defined as the complex of six structures including the DG and hippocampus proprius. It was demonstrated in many experiments that the DG not only receives nociceptive afferents, but also plays an important role in nociceptive modulation [7–10]. * Corresponding author. Fax: +86 451 8669 7507. E-mail address: [email protected] (M. Xu). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.12.184
Nonetheless, the nociceptive process related to the electrical activities of pain-related neurons is still unclear and needs to be investigated further in DG. We studied this issue using one single glass microelectrode. In the current study, we investigated the role of muscarinic receptors in the DG of HF by measuring the electrical responses of pain-related neurons. For this purpose, we performed intra-DG injection of the exogenous muscarinic acetylcholine receptor (mAChR) agonist pilocarpine and ACh; atropine was used to block mAChR and thus the effects of endogenous ACh. Materials and methods Animals. Wistar rats (200–260 g, Animal Centre of Second Affiliated Hospital of Harbin Medical University. Certificated No. 09-21) were used in this study. The rats were divided into 4 groups (n = 10 per group): (1) control group: intra-DG administration of normal saline 1 ll, (2) pilocarpine group: intra-DG administration of 0.4 lg/ll pilocarpine, (3) atropine group: intra-DG administration of 1.0 lg/ll atropine, (4) ACh group: intra-DG administration of 0.2 lg/ll ACh. All injections were completed within 2 min via a microliter syringe driven by micro-injection pump (ZCZ-50, Zhejiang province, China). Neurosurgery and electrophysiological studies. The experiments were performed on Wistar rats, anaesthetized with urethane (1 g/kg, ip). The sciatic nerves were isolated. Two skull windows were opened and covered with liquid paraffin. Rats were fixed in the stereotaxic apparatus (SN-2, Narishige, Japan). After artificial
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ventilation was established, rats were paralyzed with tubocurarine chloride (1 mg/kg). Single-unit recordings were performed with glass microelectrode (tip extreme diameter: 0.5–1.0 lm, DC resistance 10–30 MX) filled with KCl (3 mol/L). The glass microelectrode was inserted with a micromanipulator (SM-21, Narishige, Japan) into the hippocampal DG (AP: 2.8–3.2 mm, R or L: 1.8– 2.2 mm, H: 3.0–3.5 mm) [11]. Another glass microelectrode filled with drugs was inserted with a micromanipulator (SM-11, Narishige, Japan) into the DG (AP: 3.0 mm, R or L: 2.0 mm, H: 3.2 mm) for drug administration. The electrical activity was amplified with a micro-electrical amplifier and recorded with the biological experimental system, while simultaneously monitored with an oscilloscope (VC-9, Nihon Kohden, Japan). Since the neural discharges were recorded by the biological experimental system, the electrical stimulation of sciatic nerves was performed through a twin pole stainless steel-shielded electrode as noxious stimulation (delay: 0 ms, interval: 5 ms, intensity: 5 mA; duration: 0.3 ms, train 5) (SEN-3301, Nihon Kohden, Japan). The discharge of each neuron was recorded three times every 2 min; a 30 min-recording was considered as a complete record. Definition of neurons. In our present experiment, the term ‘‘painrelated neurons” refers to PEN and PIN. Neurons that responded to noxious stimulation by increasing the discharge frequency were defined as PEN [12], while neurons that responded to a noxious stimulus by decreasing the discharge frequency were defined as PIN [13]. PEN showed very few spontaneous discharges before noxious stimulation. In contrast, spontaneous discharges were common in PIN, which were readily inhibited by the application of noxious stimulation. Net-increased value (NIV, Hz) refers to the differences in PEN or PIN between the average frequency of evoked discharges after noxious stimulation, and the average frequency of the discharges within 2 s prior to noxious stimulation. Inhibitory duration (ID, s) refers to the latency time from noxious stimulation to the appearance of the PIN redischarge. Histological identification. At the end of the experiment, pontamine sky blue was diffused out from the microelectrode with a negative direct current (30 l A, 15 min), to identify the tip position. Statistical analysis. Data were scanned into the computer with Powerlab/8 s (ADInstuments) and analyzed with Chart v5.3 software (Australian). All data were expressed as mean ± SEM, analyzed with SPSS 13.0 software. Statistical differences were evaluated by one-way ANOVA; SNK test was used for comparisons between two groups and statistical significances were determined at the level of p < 0.05. Results Effects of normal saline on the electrical activities of pain-related neurons in DG Administration of normal saline produced no significant effect on the electrical activities of PEN and PIN (Figs. 1A and 2A). Influence of pilocarpine on the electrical activities of pain-related neurons in DG The average NIV of PEN was 9.94 ± 0.69 Hz and the average latency was 0.29 ± 0.07 s. After intra-DG administration of pilocarpine, the NIV of PEN began to decrease and latency began to increase (Fig. 1B). This trend was clearest at 10 min after pilocarpine administration; NIV had decreased to 4.75 ± 0.70 Hz and latency increased to 0.78 ± 0.07 s. From 2 to 18 min after administration, the NIV and latency of PEN showed significant differences as compared with those before injection or those of the control group (p < 0.05, Fig. 3).
The average NIV of PIN was 4.69 ± 0.56 Hz and the ID was 0.75 ± 0.43 s. In PIN, the average NIV began to increase and ID began to shorten after injection of pilocarpine (Fig. 2B). These reactions were most obvious at 10 min after administration; the NIV increased to 2.31 ± 0.71 Hz and ID shortened to 0.58 ± 0.11 s. Measurements obtained from 0 to 16 min after injection differed significantly from those obtained at the same interval prior to injection, as well as in comparison to the control group (p < 0.05, Fig. 4). Effects of atropine on the electrical activities of pain-related neurons in DG Twelve minutes after intra-DG administration of atropine, the average NIV of PEN significantly increased from 9.99 ± 0.63 Hz to 13.53 ± 0.70 Hz, and the latency shortened from 0.29 ± 0.03 s to 0.11 ± 0.02 s (Fig. 1C). From 2 to 20 min after injection, the NIV and latency of PEN showed obvious changes, as compared with measurements before administration or in the control group (p < 0.05, Fig. 3). At the fourth minute after intra-DG administration of atropine, evoked PIN discharges began to change significantly. The average NIV decreased from 4.82 ± 0.45 Hz to the minimum value of 6.35 ± 0.41 Hz, and the ID increased from 0.77 ± 0.10 s to the maximum value of 1.00 ± 0.09 s (Fig. 2C): ID increased 29.87%, as compared with values obtained prior to administration. This tendency was most clear at 12 min after administration. From 2 to 22 min after injection, the NIV and ID of PIN showed obvious changes compared with values obtained before injection or in the control group (p < 0.05, Fig. 4). Influence of ACh on the electrical activities of pain-related neurons in DG Before injection of ACh, the average NIV of PEN was 9.99 ± 1.08 Hz and the latency was 0.27 ± 0.03 s. Soon after intraDG administration of ACh, the NIV of PEN began to decrease and latency began to increase (Fig. 1D). These effects peaked at 10 min after administration; NIV decreased to 3.62 ± 0.84 Hz, a reduction of 36.24%, compared with measurements obtained prior to administration. Latency increased to 0.94 ± 0.10 s. From 4 to 18 min after administration, the NIV and latency of PEN showed obvious changes, as compared with measurements obtained prior to drug administration or in the control group (p < 0.05, Fig. 3). Twenty-two minutes after administration, the NIV and latency of PEN returned to the values observed before treatment. Before injection of ACh, the average NIV of PIN was 4.58 ± 0.64 Hz and the average ID of PIN was 0.73 ± 0.05 s. Two minutes after injection of ACh, the average NIV began to increase and ID began to shorten (Fig. 2D). These trends peaked at 8 min after injection; the average NIV was 1.98 ± 0.66 Hz, an increase of 56.77% in comparison to measurements obtained prior to injection. The ID had declined to 0.50 ± 0.05 s by 6 min after injection. From 0 to 18 min after injection, the average NIV and ID showed obvious changes compared with measurements obtained prior to injection or in the control group (p < 0.05, Fig. 4). About 20 min after administration, the NIV and ID of PIN returned to the values observed before treatment. Discussion According to the responses of pain-related neurons to noxious stimulation of the sciatic nerve, pain-related neurons were classified as PEN or PIN. PEN and PIN exhibit contrasting responses to the same drug or stimulus, accounting for the effects of ACh, pilocarpine and atropine in modulating nociception. While the PEN/
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Fig. 1. Representative histograms of discharged PEN. (A) Intra-DG injection of normal saline did not significantly affect the electrical activities of PEN. (B) Intra-DG injection of pilocarpine decreased evoked PEN discharges. (C) Intra-DG injection of atropine increased evoked PEN discharge. (D) Intra-DG injection of acetylcholine decreased the electrical activity of PEN.", stimulus artifact; q, injection of normal saline; w, injection of pilocarpine; , injection of atropine; }, injection of acetylcholine; X, before injection; 0, 4,. . ., 28, time after injection (min).
Fig. 2. Representative histograms of discharged PIN. (A) Intra-DG injection of normal saline did not significantly affect the electrical activities of PIN. (B) Intra-DG injection of pilocarpine potentiated the electrical activity of PIN. (C) Intra-DG injection of atropine weakened PIN activity. (D) Intra-DG injection of acetylcholine potentiated PIN activity.", stimulus artifact; q, injection of saline; w, injection of pilocarpine; , injection of atropine; }, injection of acetylcholine; X, before injection; 0, 4, . . ., 28, time after injection (min).
Fig. 3. Influence of intra-DG injection of different substance on the NIV (A) and latency (B) of PEN in the DG. , injection of substance; X, before injection; 0, 4, . . ., 28, time after injection (min); values are given by means ± SEM. *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01, 4p < 0.05, 44p < 0.01 when compared with saline group.
PIN distinction is an important tool for nociceptive research [14], some researchers use the terms ‘‘on-” or ‘‘off-cell” to denote ex-
cited or inhibited neurons in the traditional nociceptive conducting pathway [15].
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Fig. 4. Influence of intra-DG injection of different substance on the NIV (A) and ID (B) of PIN in the DG. , injection of substance; X, before injection; 0, 4, . . ., 28, time after injection (min); values are given by means ± SEM. *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01, 4p < 0.05, 44p < 0.01 when compared with saline group.
The present study demonstrates that ACh or pilocarpine inhibited the evoked discharge of PEN and potentiated that of PIN, i.e., exhibiting the analgesic effect of ACh or pilocarpine. Atropine potentiated the evoked discharge of PEN and attenuated that of PIN, demonstrating facilitated nociceptive effects. The results revealed that cholinergic PEN and PIN exist in the DG of rats. Our study shows that M receptor agonists exert an anti-nociceptive effect in modulating pain; atropine excited PEN and inhibited PIN. Thus the muscarinic receptor antagonist plays a role in facilitated nociception. During the preliminary experiments, we used different dosages of ACh and pilocarpine and found that ACh and pilocarpine act in a dose-dependent manner. The dosages of ACh (0.2 lg/ll) or pilocarpine (0.4 lg/ll) used yielded significant effects and facilitated the recording of neuronal discharges. Surprisingly, the data curves obtained for the atropine group were not as distinct as those obtained for the ACh group or the pilocarpine group. We tried repeatedly to increase the dosage of atropine during the experiments. Nevertheless the small dosage of atropine had no effect on the electrical activities of PEN and PIN; higher doses are toxic to the central nervous system. Furthermore, noxious stimulus may evoke central cholinergic activation [16], weakening the effect of atropine as an ACh antagonist. Therefore we sought to use the smallest dosage possible of atropine. Furthermore, the molecular weights of these three drugs are unequal; a reasonable dosage should yield an obvious effect while maximizing the equimolarity of the various solutions. Previous studies, including a study performed in our lab, have shown that the (HF) is involved in nociception; nociceptive stimuli modify the electrical activity of the hippocampus [17] and are able to induce c-fos expression in this structure. Every region is connected by extensive fiber networks. Granular cells in the DG receive synapses from neurons of the entorhinal cortex through the perforating pathway. In turn, these granular cells send their axons into the CA3 area of the hippocampus via the mossy fiber pathway. In strata radiatum and oriens of the hippocampal CA1 area, the nerve terminals derive from axon collaterals of CA3 pyramidal cells. Aside from these major fiber connections, the dendritic shafts of various types of interneurons can build connections with the spines of principal cells. Because the HF integrates information from multiple connecting pathways, the various regions are intimately connected. The DG sub-region was found to be implicated in nociception. The disruption of neural activity in the DG reduces nociception. Local injection of anesthetic into the DG produces an anti-nociceptive effect in the formalin test [18]. Blocking neural transmission along the major afferent or efferent hippocampal pathways reduces noci-
ceptive behavior [19]. Microinjection of the NMDA receptor antagonist AP5 into DG served to attenuate nociceptive behaviors in both the acute and tonic phases of the formalin test [20]. Our present study indicates that the DG is involved in the modulation of nociception. ACh (an agonist of both mAChR and nAChR), pilocarpine (a selected mAChR agonist), and carbachol (a mAChR agonist) are known to exert anti-nociceptive effects. Pharmacological experiments have shown that the microinjection of ACh or carbachol into specific brainstem nuclei can produce anti-nociceptive effects and can be reversed by muscarinic receptor antagonists [20]. Some other types of drugs yield nociceptive effects mediated by ACh. It also has been demonstrated that endogenous ACh plays an important role in mediating the nociceptive effects of morphine and clonidine [21,22]. D2 antagonist prochlorperazine exerts an antinociceptive effect mediated by a central cholinergic mechanism [23]. In our present study, an anti-nociceptive effect was observed with injection of ACh or pilocarpine. This finding is in agreement with reports that mAChR agonists exert extensive anti-nociceptive effects in spinal and supraspinal nerves. Atropine, as an endogenous non-competitive antagonist of mAChR, usually exerts a nociceptive effect. Pretreatment with atropine antagonizes the anti-nociceptive effect of NSAIDs in spinal cord [24]; of prochlorperazine in i.c.v. [23]; of oxotremorine in spinal cord [25], and of carbachol in dorsal hippocampus [26]. These findings suggest that intrinsic cholinergic pathways represent an important modulating system in nociception. In our study, injection of the exogenous muscarinic receptor antagonist atropine can increase the NIV and shorten the latency of PEN, while decreasing NIV of PIN and prolonging ID of PIN. This effect was opposite to that of ACh and pilocarpine. This effect is mediated by the blockade of endogenous ACh due to atropine. While our results are similar to those of previous experiments, some controversial reports indicate that atropine can produce anti-nociception [27]. These discrepancies may result from different doses and sites of injection, or the various subtypes of mAChR found in different tissues. Several families of muscarinic ACh receptor are expressed in the HF; five distinct mAChR subtypes referred to as M1 to M5 are localized in separate layers of the HF [28]. Researcher used quantitative immunoprecipitations of the rat brain to demonstrate 36% M1, 33% M2 and 27% M4 expression in the hippocampus. Others found that the M1 muscarinic receptor subtype is critical to induce central cholinergic nociception in mice; M1 is widely expressed in the somata and dendrites of pyramidal neurons and in DG granule cells. Activation of the peripheral M2 receptor produces anti-nociception in vivo and inhibition of nociceptive activity in vitro. In HF, M3 expression is mainly enriched in the pyramidal neurons and the
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outer third of the molecular layer of the DG. Gene knockout studies reveal that the functional activity of some M4 receptors requires the presence of M2 receptors [29]. The unique laminar and regional distributions of M1–M4 in the hippocampus reflect association of the receptor subtypes with particular afferents, as well as a special intrinsic connection with the M1 and M3 receptors expressed in principal neurons and the M2 and M4 receptors on interneurons. This may imply that each subtype plays a different role in excitatory and inhibitory modulation of the hippocampal circuits. In contrast, neurons in the spinal cord express primarily M2 and M3 [30]. Our experiments showed that intra-DG injection of pilocarpine can reinforce the anti-nociceptive pathway; this is in agreement with previous results that indicate that activation of ACh-M1 and/or ACh-M3 receptors mediate anti-nociceptive effects in central nervous system [31]. The current study suggests that synaptic release of ACh results in a complex and differential mAChR-mediated modulation of cellular excitability within the hippocampal interneuron population [5]. However, further investigation is required to determine which mAChR subtypes play a critical role in anti-nociceptive effects mediated by the DG. In conclusion, muscarinic cholinergic receptor agonists modulate nociception by inhibiting PEN activity and potentiating PIN activity. Intra-DG administration of atropine produces the opposite effect. Our present study confirms that a cholinergic-sensitive mechanism in the DG region of the HF modulates the processing of noxious information. Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 30240058), the Science & Technology Agency in Heilongjiang, China (No. GC05C40607) and Science Foundation of Health Department in Heilongjiang, China (No. 2007-525). References [1] I.H. Lorenz, K. Egger, H. Schubert, C. Schnurer, W. Tiefenthaler, M. Hohlrieder, M.F. Schocke, C. Kremser, R. Esterhammer, A. Ischebeck, P.L. Moser, C. Kolbitsch, Lornoxicam characteristically modulates cerebral pain-processing in human volunteers: a functional magnetic resonance imaging study, Br. J. Anaesth. 100 (2008) 827–833. [2] S.W. Derbyshire, A.K. Jones, F. Gyulai, S. Clark, D. Townsend, L.L. Firestone, Pain processing during three levels of noxious stimulation produces differential patterns of central activity, Pain 73 (1997) 431–445. [3] X.F. Yang, Y. Xiao, M.Y. Xu, Both endogenous and exogenous ACh plays antinociceptive role in the hippocampus CA1 of rats, J. Neural. Transm. 115 (2008) 1–6. [4] W. Buno, C. Cabezas, D. Fernandez de Sevilla, Presynaptic muscarinic control of glutamatergic synaptic transmission, J. Mol. Neurosci. 30 (2006) 161–164. [5] H. Widmer, L. Ferrigan, C.H. Davies, S.R. Cobb, Evoked slow muscarinic acetylcholinergic synaptic potentials in rat hippocampal interneurons, Hippocampus 16 (2006) 617–628. [6] N. Kuczewski, E. Aztiria, D. Gautam, J. Wess, L. Domenici, Acetylcholine modulates cortical synaptic transmission via different muscarinic receptors, as studied with receptor knockout mice, J. Physiol. 566 (2005) 907–919. [7] M.B. Echeverry, F.S. Guimaraes, M.A. Oliveira, W.A. do Prado, E.A. Del Bel, Delayed stress-induced antinociceptive effect of nitric oxide synthase inhibition in the dentate gyrus of rats, Pharmacol. Biochem. Behav. 74 (2002) 149–156.
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