Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Δ⁹-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Δ⁹-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.     

The Interaction Between Intrathecal Administration of Low Doses of Palmitoylethanolamide and AM251 in Formalin-Induced Pain Related Behavior and Spinal Cord IL1-β Expression in Rats

Most of the modulating effects of cannabinoids on pain are through putative cannabinoid CB1 and CB2 receptors. However, the involvement of other receptors is also suggested. Cannabinoid compounds with analgesic activity such as palmitoylethanolamide (PEA) show low affinity to CB1 and CB2 receptors, yet selectively activate GPR55 receptors. The objective of the present study was to evaluate the possible role of spinal CB1 and GPR55 receptors on antinociceptive activity of PEA in formalin test as well as in the spinal expression of IL1-β in rat. Intrathecal (i.t.) administration of PEA (1, 10 μg) significantly decreased both pain-related scores in formalin test and IL1-β expression in rat spinal cord. Pretreatment of rats with low doses of CB1 receptor antagonist/GPR55 receptor agonist AM251 (10, 100 ng; i.t.), did not attenuated the effect of PEA, yet even significantly increased the effect of PEA on IL1-β expression in rat spinal cord. Interestingly, i.t. administration of low doses of AM251 per se significantly decreased both pain related behavior and spinal IL1-β expression in formalin test. These findings suggest the possible involvement of receptors other than CB1 receptors in spinal pain pathways, such as GPR55, in pain modulating activity of cannabinoids.     

Heterogeneity of neuronal nicotinic acetylcholine receptors: Structural and functional aspects

Decay kinetics of the postsynaptic excitatory currents (EPSC), distribution of the antibodies specific to different α-subunits of neuronal nicotinic acetylcholine receptors (nAChR), and the effects of these antibodies on ACh-induced membrane currents were studied in neurons of different autonomic ganglia of rats. It was shown that α3-, α5- and α7-subunits were present in all studied cultured neurons of the rat superior cervical ganglion (SCG), while the α4-subunit was present only in about half of the neurons; this α-subunit distribution differed from that in cultured intracardial neurons of rats. Two nAChR populations were found in rat SCG neurons, and a series of nAChR populations were found in murine superior mesenteric ganglion neurons; they differed in kinetics of their ion channel activity, voltage dependence and the rate of their open channel blockade. The possible functional role of neuronal nAChR heterogeneity is discussed.     

Gz mediates the long-lasting desensitization of brain CB1 receptors and is essential for cross-tolerance with morphine

Background

Although the systemic administration of cannabinoids produces antinociception, their chronic use leads to analgesic tolerance as well as cross-tolerance to morphine. These effects are mediated by cannabinoids binding to peripheral, spinal and supraspinal CB1 and CB2 receptors, making it difficult to determine the relevance of each receptor type to these phenomena. However, in the brain, the CB1 receptors (CB1Rs) are expressed at high levels in neurons, whereas the expression of CB2Rs is marginal. Thus, CB1Rs mediate the effects of smoked cannabis and are also implicated in emotional behaviors. We have analyzed the production of supraspinal analgesia and the development of tolerance at CB1Rs by the direct injection of a series of cannabinoids into the brain. The influence of the activation of CB1Rs on supraspinal analgesia evoked by morphine was also evaluated.

Results

Intracerebroventricular (icv) administration of cannabinoid receptor agonists, WIN55,212-2, ACEA or methanandamide, generated a dose-dependent analgesia. Notably, a single administration of these compounds brought about profound analgesic tolerance that lasted for more than 14 days. This decrease in the effect of cannabinoid receptor agonists was not mediated by depletion of CB1Rs or the loss of regulated G proteins, but, nevertheless, it was accompanied by reduced morphine analgesia. On the other hand, acute morphine administration produced tolerance that lasted only 3 days and did not affect the CB1R. We found that both neural mu-opioid receptors (MORs) and CB1Rs interact with the HINT1-RGSZ module, thereby regulating pertussis toxin-insensitive Gz proteins. In mice with reduced levels of these Gz proteins, the CB1R agonists produced no such desensitization or morphine cross-tolerance. On the other hand, experimental enhancement of Gz signaling enabled an acute icv administration of morphine to produce a long-lasting tolerance at MORs that persisted for more than 2 weeks, and it also impaired the analgesic effects of cannabinoids.

Conclusion

In the brain, cannabinoids can produce analgesic tolerance that is not associated with the loss of surface CB1Rs or their uncoupling from regulated transduction. Neural specific Gz proteins are essential mediators of the analgesic effects of supraspinal CB1R agonists and morphine. These Gz proteins are also responsible for the long-term analgesic tolerance produced by single doses of these agonists, as well as for the cross-tolerance between CB1Rs and MORs.     

Cannabinoids Inhibit Acid-Sensing Ion Channel Currents in Rat Dorsal Root Ganglion Neurons

Local acidosis has been found in various pain-generating conditions such as inflammation and tissue injury. Cannabinoids exert a powerful inhibitory control over pain initiation via peripheral cognate receptors. However, the peripheral molecular targets responsible for the antinociceptive effects of cannabinoids are still poorly understood. Here, we have found that WIN55,212-2, a cannabinoid receptor agonist, inhibits the activity of native acid-sensing ion channels (ASICs) in rat dorsal root ganglion (DRG) neurons. WIN55,212-2 dose-dependently inhibited proton-gated currents mediated by ASICs. WIN55,212-2 shifted the proton concentration–response curve downwards, with an decrease of 48.6±3.7% in the maximum current response but with no significant change in the EC₅₀ value. The inhibition of proton-gated current induced by WIN55,212-2 was almost completely blocked by the selective CB1 receptor antagonist AM 281, but not by the CB2 receptor antagonist AM630. Pretreatment of forskolin, an AC activator, and the addition of cAMP also reversed the inhibition of WIN55,212-2. Moreover, WIN55,212-2 altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. Finally, WIN55,212-2 attenuated nociceptive responses to injection of acetic acid in rats. These results suggest that WIN55,212-2 inhibits the activity of ASICs via CB1 receptor and cAMP dependent pathway in rat primary sensory neurons. Thus, cannabinoids can exert their analgesic action by interaction with ASICs in the primary afferent neurons, which was novel analgesic mechanism of cannabinoids.     

Genetic Dissection of Behavioural and Autonomic Effects of Δ9-Tetrahydrocannabinol in Mice

Marijuana and its main psychotropic ingredient Δ⁹-tetrahydrocannabinol (THC) exert a plethora of psychoactive effects through the activation of the neuronal cannabinoid receptor type 1 (CB1), which is expressed by different neuronal subpopulations in the central nervous system. The exact neuroanatomical substrates underlying each effect of THC are, however, not known. We tested locomotor, hypothermic, analgesic, and cataleptic effects of THC in conditional knockout mouse lines, which lack the expression of CB1 in different neuronal subpopulations, including principal brain neurons, GABAergic neurons (those that release γ aminobutyric acid), cortical glutamatergic neurons, and neurons expressing the dopamine receptor D1, respectively. Surprisingly, mice lacking CB1 in GABAergic neurons responded to THC similarly as wild-type littermates did, whereas deletion of the receptor in all principal neurons abolished or strongly reduced the behavioural and autonomic responses to the drug. Moreover, locomotor and hypothermic effects of THC depend on cortical glutamatergic neurons, whereas the deletion of CB1 from the majority of striatal neurons and a subpopulation of cortical glutamatergic neurons blocked the cataleptic effect of the drug. These data show that several important pharmacological actions of THC do not depend on functional expression of CB1 on GABAergic interneurons, but on other neuronal populations, and pave the way to a refined interpretation of the pharmacological effects of cannabinoids on neuronal functions.     

Genetic Dissection of Behavioural and Autonomic Effects of Δ9-Tetrahydrocannabinol in Mice

Marijuana and its main psychotropic ingredient Δ⁹-tetrahydrocannabinol (THC) exert a plethora of psychoactive effects through the activation of the neuronal cannabinoid receptor type 1 (CB1), which is expressed by different neuronal subpopulations in the central nervous system. The exact neuroanatomical substrates underlying each effect of THC are, however, not known. We tested locomotor, hypothermic, analgesic, and cataleptic effects of THC in conditional knockout mouse lines, which lack the expression of CB1 in different neuronal subpopulations, including principal brain neurons, GABAergic neurons (those that release γ aminobutyric acid), cortical glutamatergic neurons, and neurons expressing the dopamine receptor D1, respectively. Surprisingly, mice lacking CB1 in GABAergic neurons responded to THC similarly as wild-type littermates did, whereas deletion of the receptor in all principal neurons abolished or strongly reduced the behavioural and autonomic responses to the drug. Moreover, locomotor and hypothermic effects of THC depend on cortical glutamatergic neurons, whereas the deletion of CB1 from the majority of striatal neurons and a subpopulation of cortical glutamatergic neurons blocked the cataleptic effect of the drug. These data show that several important pharmacological actions of THC do not depend on functional expression of CB1 on GABAergic interneurons, but on other neuronal populations, and pave the way to a refined interpretation of the pharmacological effects of cannabinoids on neuronal functions.     

Cannabinoids affect dendritic cell (DC) potassium channel function and modulate DC T cell stimulatory capacity

Cannabinoids affect diverse biological processes, including functions of the immune system. With respect to the immune system, anti-inflammatory and immunosuppressive effects of cannabinoids have been reported. Cannabinoids stimulate G protein-coupled cannabinoid receptors CB1 and CB2. These receptors are found primarily on neurons. However, they are also found on dendritic cells (DC), which are recognized for their critical role in initiating and maintaining immune responses. Therefore, DC are potential targets for cannabinoids. We report in this study that cannabinoids reduced the DC surface expression of MHC class II molecules as well as their capacity to stimulate T cells. In the nervous system, CB1 receptor signaling modulates K(+) and Ca(2+) channels. Interestingly, cannabinoid-treated DC also showed altered voltage-gated potassium (K(V)) channel function. We speculate that attenuation of K(V) channel function via CB1 receptor signaling in DC may represent one mechanism by which cannabinoids alter DC function.     

Kinetic Analysis of Miniature Synaptic Currents in Rat Substantia Gelatinosa Neurons

Interneurons of the substantia gelatinosa (SG) form a complex synaptic network in the dorsal horn of the spinal cord. The properties of miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively) were studied in spinal cord slices of 3- to 4-week-old rats. The reversal potentials of the currents were close to 0 mV for excitatory and –70 mV for inhibitory events. Under recording conditions close to physiological ones (holding potential –40 mV, temperature 32°C, low intracellular [Cl^–]), the mean rise times of these currents were, respectively, 1.0 and 1.8 msec. The decay of the currents was monoexponential in the majority of occurrences (94 and 91.4%), with a time constant (τ) of 2.7 msec for mEPSCs and 7.2 msec for mIPSCs. A part (8.6%) of mIPSCs had an additional slow component with τ = 30.1 msec. All mEPSCs were blocked by 10 mM CNQX, an antagonist of the AMPA/kainate subtype of glutamate receptors. Monoexponential mIPSCs were blocked by 1 mM strychnine, an antagonist of glycine receptors, while two-component mIPSCs required the additional presence of 10 mM bicuculline, a blocker of GABAA receptors. Only two cells of 23 (~9%) demonstrated pure GABA-ergic mIPSCs (τ = 26.2 msec). It is concluded that, under physiological conditions, AMPA/kainate but not NMDA receptors mediate excitatory synaptic transmission in SG neurons. Synaptic inhibition is mediated predominantly by glycine receptors, with mild fractions of IPSCs provided by GABA-ergic transmission and GABA/glycine co-release.     

Properties of Proton-Gated Ion Channels in Sensory Neurons of Rats

In experiments on the somata of sensory neurons isolated from the spinal and trigeminal ganglia of rats, we characterized three subclasses of proton-gated currents differing from each other in their kinetics of desensitization and characteristics of stationary desensitization (but not in the characteristics of stationary activation). A voltage clamp technique in the whole cell configuration and intracellular perfusion were used. Expression of the channels providing currents of each subclass depended on the soma diameter but not on anatomical localization of the neuron. Proton-gated channels of type I were characterized by mono- or biexponential kinetics of current desensitization with the duration of complete decay within a 1 to 15 sec range; the mean pH₅₀ of the curve of stationary desensitization was 7.21 ± 0.02. Channels of type II possessed mostly monoexponential desensitization kinetics with the duration of decay within a 1 to 3 sec range; their pH₅₀ of the stationary desensitization curve was 7.11 ± 0.02. Channels of type III showed mostly biexponential desensitization kinetics; the complete current decay lasted about 5 sec, while the mean pH₅₀ was about 6.78 ± 0.02. Channels of type I were typical of small neurons (soma diameter 10-20 m), while those of types II and III were found mostly in large cells (35-60 m).     

Dual Neuroprotective and Neurotoxic Effects of Cannabinoid Drugs in Vitro

Either protective or toxic effects of cannabinoids on cell survival have been reported extensively in the literature; however, the factors that determine the direction of the effect are still obscured. In this study we have used the neuroblastoma cell line N18TG2 that expresses CB1 cannabinoid receptors to investigate several factors that may determine the consequences of exposure to cannabinoid agonists. Cells that were grown under optimal, stressful, or differentiating conditions were exposed to cannabinoid agonists and then assayed for cell viability by measuring MTT, LDH, and caspase-3 activity. Various cannabinoid agonists (CP 55,940, ∆9-THC, HU-210, and WIN 55,212-2) failed to affect cell viability when the cells were grown under optimal conditions. On the other hand, the same agonists significantly reduced cell viability when the cells were grown under stressful conditions (glucose- and serum-free medium), while enhancing the viability of cells grown in differentiation medium (0.5% serum and 1.5% DMSO). The toxic/protective profile was not dependent on the type or the concentration of the cannabinoid agonist that was applied. The cannabinoid agonist CP 55,940 similarly affected the non-neuronal HEK-293 cells that were grown under stressful conditions only when they expressed CB1 receptors. Our results shed light on the conflicting reports regarding the protective or toxic effects of cannabinoids in vitro and indicate that cannabinoids may activate different intracellular signaling mechanisms, depending on the state of the cell, thus leading to different physiological consequences.     

Perampanel Inhibition of AMPA Receptor Currents in Cultured Hippocampal Neurons

Perampanel is an aryl substituted 2-pyridone AMPA receptor antagonist that was recently approved as a treatment for epilepsy. The drug potently inhibits AMPA receptor responses but the mode of block has not been characterized. Here the action of perampanel on AMPA receptors was investigated by whole-cell voltage-clamp recording in cultured rat hippocampal neurons. Perampanel caused a slow (τ∼1 s at 3 µM), concentration-dependent inhibition of AMPA receptor currents evoked by AMPA and kainate. The rates of block and unblock of AMPA receptor currents were 1.5×10⁵ M⁻¹ s⁻¹ and 0.58 s⁻¹, respectively. Perampanel did not affect NMDA receptor currents. The extent of block of non-desensitizing kainate-evoked currents (IC₅₀, 0.56 µM) was similar at all kainate concentrations (3–100 µM), demonstrating a noncompetitive blocking action. Parampanel did not alter the trajectory of AMPA evoked currents indicating that it does not influence AMPA receptor desensitization. Perampanel is a selective negative allosteric AMPA receptor antagonist of high-affinity and slow blocking kinetics.     

Distinct cannabinoid sensitive receptors regulate hippocampal excitation and inhibition

One of the well-known effects of cannabinoids is the impairment of cognitive processes, including short-term memory formation, by altering hippocampal and neocortical functions reflected in network activity. Acting on presynaptically located G protein-coupled receptors in the hippocampus, cannabinoids modulate the release of neurotransmitter molecules. CB1 cannabinoid receptors, so far the only cloned cannabinoid receptor type in the CNS, are selectively expressed on the axon terminals of a subset of GABAergic inhibitory interneurons containing the neuropeptide cholecystokinin. Activation of CB1 receptors reduces GABA release from presynaptic terminals, thereby increasing the excitability of principal cells. Novel, non-CB1 cannabinoid sensitive receptors are present on the hippocampal excitatory axon terminals, which suppress glutamate release. These cannabinoid receptors have distinct pharmacological features compared to CB1, i.e. WIN 55212-2 is an order of magnitude less potent in reducing glutamatergic transmission than in inhibiting GABAergic postsynaptic currents, and the novel receptor binds vanilloid receptor ligands. Thus, at least two different cannabinoid sensitive presynaptic receptors regulate network activity in the hippocampus, CB1 via the GABAergic interneurons, and a new receptor via a direct action on pyramidal cell axon terminals.     

A patch-clamp study of GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons.

GABA- and strychnine-sensitive glycine-activated currents in post-natal tissue-cultured hippocampal neurons were studied by using patch-clamp techniques. Current-voltage relations for both agonists in symmetrical Cl- solutions showed outward rectification. Strychnine-sensitive glycine-activated currents have not been studied in detail before in post-natal tissue-cultured hippocampal neurons. Partial desensitization of whole-cell currents was observed in symmetrical Cl- solutions during bath application of GABA- and glycine. In F-/Cl- solutions, both agonists gave a PF/Pcl value of about 0.06. The reversal potentials in mixtures of Cl- and SO4(2-) solutions were close to the equilibrium potentials of Cl- in the presence of both GABA and glycine. Single channels in inside-out excised patches with 2 mumol l-1 GABA and 5 mumol l-1 glycine in the pipette showed outward current rectification. The dose-response curves for GABA and glycine gave Kd values of 52 and 61 mumol l-1, respectively, and Hill coefficients close to 2. GABA and glycine binding were competitively blocked by their respective antagonists (bicuculline and strychnine). The similarities between GABA- and glycine-activated currents and the response in a combination of saturating concentrations of both GABA and glycine implied that the two agonists activated comparable numbers of anatomically distinct channels with very similar permeation properties.     

Blockade of desensitization augments quisqualate excitotoxicity in hippocampal neurons

Glutamate neurotoxicity is thought to play a role in the pathogenesis of several neurodegenerative diseases. While prolonged activation of either NMDA or non-NMDA receptors causes neuronal damage, NMDA receptors appear to mediate most of the glutamate toxicity. The reasons why NMDA toxicity predominates are uncertain but may relate to more effective neuroprotective mechanisms acting at non-NMDA receptors. To determine whether desensitization is one such mechanism, we studied the effects of the lectin wheat germ agglutinin (WGA) on quisqualate currents and toxicity in cultured postnatal rat hippocampal neurons. After WGA treatment, quisqualate currents exhibit little desensitization and a 4- to 8-fold increase in steady-state amplitude. WGA also markedly augments the degree of acute, quisqualate-induced neuronal degeneration. These results suggest that non-NMDA desensitization serves a neuroprotective function in hippocampal neurons.     

Cannabinoids activate p38 mitogen-activated protein kinases through CB1 receptors in hippocampus

Cannabinoid receptors (CB1-R) are the target of a novel class of neuromodulators, the endocannabinoids. Yet, their signalling mechanisms in adult brain are poorly understood. We report that, in rat and mouse hippocampal slices, anandamide and 2-arachidonoylglycerol, synthetic cannabinoids, and delta(9)-tetrahydrocannabinol activated p38 mitogen-activated protein kinases (MAPK), but not c-Jun N-terminal kinase (JNK). In contrast, lysophosphatidic acid (LPA), a lipid messenger acting on different receptors, increased both p38-MAPK and JNK phosphorylation. The effects of cannabinoids on p38-MAPK were mediated through activation of CB1-R because they were blocked in the presence of SR 141716 A and absent in CB1-R knockout mice, two conditions that did not alter the effects of LPA. The activation of p38-MAPK by cannabinoids was insensitive to inhibitors of SRC: These results provide new insights into the cellular mechanisms by which cannabinoids exert their effects in hippocampus.     

Cannabinoid signalling

After their discovery, the two known cannabinoid receptors, CB(1) and CB(2), have been the focus of research into the cellular signalling mechanisms of cannabinoids. The initial assessment, mainly derived from expression studies, was that cannabinoids, via G(i/o) proteins, negatively modulate cyclic AMP levels, and activate inward rectifying K(+) channels. Recent findings have complicated this assessment on different levels: (1) cannabinoids include a wide range of compounds with varying profiles of affinity and efficacy at the known CB receptors, and these profiles do not necessarily match their biological activity; (2) CB receptors appear to be intrinsically active and possibly coupled to more than one type of G protein; (3) CB receptor signalling mechanisms are diverse and dependent on the system studied; (4) cannabinoids have other targets than CB receptors. The aim of this mini review is to discuss the current literature regarding CB receptor signalling pathways. These include regulation of adenylyl cyclase, MAP kinase, intracellular Ca(2+), and ion channels. In addition, actions of cannabinoids that are not mediated by CB(1) or CB(2) receptors are discussed.     

L-type channel inhibition by CB1 cannabinoid receptors is mediated by PTX-sensitive G proteins and cAMP/PKA in GT1-7 hypothalamic neurons

Using immortalized hypothalamic GT1-7 neurons, which express the CB1 cannabinoid receptor (CB1R) and three Ca²⁺ channel types (T, R and L), we found that the CB1R agonist WIN 55,212-2 inhibited the voltage-gated Ca²⁺ currents by about 35%. The inhibition by WIN 55,212-2 (10 μM) was reversible and prevented by nifedipine (3 μM), suggesting a selective action on L-type Ca²⁺ channels (LTCCs). WIN 55,212-2 action exhibited all the features of voltage-independent Ca²⁺ channel modulation: (1) no changes of the activation kinetics, (2) equal depressive action at all potentials and (3) no facilitation following strong prepulses. At variance with WIN 55,212-2, the CB1R inverse agonist AM-251 (10 μM) caused 20% increase of Ca²⁺ currents. The inhibition of LTCCs by WIN 55,212-2 was prevented by overnight PTX-incubation and by intracellular perfusion with GDP-β-S. The latter caused also a 20% Ca²⁺ current up-regulation. WIN 55,212-2 action was also prevented by application of the PKA-blocker H89 or by loading the neurons with 8-CPT-cAMP. Our results suggest that LTCCs in GT1-7 neurons are partially inhibited at rest due to a constitutive CB1R activity removed by AM-251 and GDP-β-S. Activation of CB1R via PTX-sensitive G proteins and cAMP/PKA pathway selectively depresses LTCCs that critically control the synchronized spontaneous firing and pulsatile release of gonadotropin-releasing hormone in GT1-7 neurons.     

Inhibitory Postsynaptic Currents Induced by Activation of Interneurons in Cultured Rat Hippocampal Neurons

Using the patch-clamp technique in the whole-cell configuration, we studied the characteristics of a series of action potentials (APs) induced by a 500-msec-long current pulse applied to a pre-synaptic unit, as well as the kinetic characteristics of post-synaptic currents (PSCs) evoked by the APs in a post-synaptic unit, in synaptically connected pairs of cultured hippocampal neurons. Presynaptic inhibitory units were identified as GABA-ergic interneurons; they were divided into two groups according to the size of the soma and the number of processes. The kinetic characteristics of PSCs, which were induced in the post-synaptic neuron by a series of the APs generated in the pre-synaptic cell, demonstrated a certain dependence on the morphological characteristics of these cells. In interneurons with large-sized somata, the kinetics of the currents were more fast, and the reversal potential was close to the equilibrium Cl potential. In interneurons with small-sized somata, currents were slower, and the reversal potential was shifted. We conclude that under conditions of culturing, a pre-synaptic cell not only directly provokes the development of PSC in a post-synaptic neuron and determines the amplitude of this current but also significantly influences the kinetics of this current. Neirofiziologiya/Neurophysiology, Vol. 37, No. 2, pp. 116–123, March–April, 2005.     

Expression of tachykinin receptors in Xenopus oocytes injected with poly(A)+RNA from cat dorsal root ganglion

The expression of the types of tachykinin receptors in the dorsal root ganglion (DRG) neurons by means ofXenopus oocyte expressing system was studied. Poly(A)⁺ RNAs were extracted from cat cervical and lumbar DRG. Two days after injection of Poly (A)⁺ RNAs, the oocytes were recorded with the two-electrode voltage clamp technique. In the oocytes injected with DRG poly(A)⁺ RNA, [Sar⁹, Met(O₂)¹¹]-substance P(Sar -SP, 1 μmol/L), neurokinin A (NKA, 1 μmol/L) or [β-Ala⁸]-neurokinin A₍₄₋₁₀₎ (Ala-NKA, 1 μmol/L) produced an inward current comprising a rapid spike and a long sustained oscillatory component for several minutes. Sar-SP induced response was blocked by NK-1 antagonist L-668, 169 (1 μmol/L), but not by NK-2 antagonist L-659, 877(1μmol/L). In contrast, Ala-NKA and NKA responses were only blocked by L-659, 877. The oocytes injected with DH Poly(A)⁺RNA also responded to Sar-SP and NKA with similar inward currents, which were selectively blocked by L-668, 169 and L-659, 877, respectively. These tachykinins-induced responses had a potent desensitization. The present data indicate expression of NK-1 and NK-2 receptors in DRG neurons, suggesting that there may be tachykinin autoreceptors on the nociceptive primary afferent terminals. Project supported by the National Natural Science Foundation of China (Grant No. 39370249).