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Effects of [mu]-opioids on neurons of the medical geniculate nucleus Ota, Takayo 2000

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EFFECTS OF ^-OPIOIDS ON NEURONS OF THE MEDIAL GENICULATE NUCLEUS by Takayo Ota M.D., Kinki University, 1996  A T H E S I S SUBMITTED IN PARTIAL F U L F I L M E N T O F THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUTE STUDIES (Department of Pharmacology and Therapeutics)  W e accepyftys thesis as conforming \oMJ\e required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A September, 2000 © Takayo Ota, 2000  In  presenting  degree freely  at  the  available  copying  of  department publication  this  of  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or  thesis  reference  thesis by  this  for  his  and  scholarly  or  thesis  study.  for  her  purposes  financial  gain  of  | ^  a  y\A/L£\ UP ( 0  The University of British C o l u m b i a Vancouver, Canada  Date  DE-6  (2/88)  L^.  C  shall  that  agree  may  representatives.  permission.  Department  I further  requirements  be  It not  that  the  be  Library  an  advanced  shall  permission for  granted  is  for  by  understood allowed  the  make  extensive  head  that  without  it  of  copying my  my or  written  11  Abstract Immunohistochemical and hybridization studies have revealed the presence of u-, but not K- and 5-opioid receptors in the medial geniculate body.  Using whole-cell  patch clamp techniques, I examined the effects of opioid agonists on gerbil M G B neurons (P9-P16) in vitro.  The opioid effects were  concentration-dependent.  Opioids produced different actions on the input conductance (Gj) when applied in low, compared with high concentrations. increase in K inactivation.  +  The increase in Gi might be due to  conductance whereas the decrease in Gj might be due to l  H  When Gi was increased, the reversal potential was —65 mV; this  implicates opioid actions on K , among other ion channels. In the case of decreased +  Gi, the opioid currents did not reverse from - 1 0 0 mV to - 5 0 mV, implying the involvement of cationic channels, other than K . +  D A M G O , a u-selective opioid  agonist, had a reversal potential that was similar to that observed when morphine increased Gi, implying that opioids activate u-opioid receptors in M G B neurons. Tetrodotoxin altered the concentration-dependent action of morphine.  Here, the  suggestion is morphine's actions involve neurons that presynaptic to the patch clamped neuron.  Morphine application blocked spike-frequency adaptation and  reduced firing rates in response to depolarizing current pulse injection. application may block Ca -mediated K 2+  adaptation.  +  Morphine  channels that inhibited spike-frequency  Such blockade may be expected to increase the spike frequency.  However, the increased conductance due to morphine would shunt the N a current, +  resulting in a lower spike frequency.  The results of this study have revealed that  opioids have both excitatory and inhibitory effects on M G B neurons.  iii  Table of Contents Abstract  ••  Table of Contents  iii  List of Figures  v  Tables  vi  1. Introduction 1.1 1.2 1.3 1.4 1.5  Historical perspective Opioid receptor-binding Opioid receptors Practical rationale for animal studies Qustions asked in these investigations  2. Methods  1 2 4 5 7 10 11  2.1 Preparation of slices  11  2.1.1 Surgical preparation  11  2.1.2 Physiological solutions 2.1.3 Tissue slicing 2.1.4 Incubation of slices  12 12 13  2.2 Electrical Recording 2.2.1 2.2.2 2.2.3 2.2.4 2.2.5  Microelectrodes Electrical hardware Microelectrode advancement Electrical parameters Recording stability  14 14 14 15 16 16  2.3  Pharmacological agents  16  2.4  Data and statistical analyses  17  3. Results 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9  4.  Passive membrane properties Firing modes Effects of morphine on membrane properties Effects of morphine during tetrodotoxin (TTX) blockade Effects of morphine on firing patterns...... Mu-opioids Blockade by naloxone Blockade by B a Effect of low-Na 2+ +  Discussion  4.1 4.2 4.3 4.4  5.  19  Influence of gerbil age on passive membrane properties and action potentials Effects of morphine on passive and active membrane properties Effects of opioids on firing properties Conclusion  References  19 20 23 39 45 54 57 60 70  73  73 74 79 82  83  V  List of Figures Fig. Fig. Fig. Fig. Fig. Fig.  3.1 3.2 3.3 3.4 3.5 3.6  Fig. 3.7A Fig. 3.7B Fig. 3.8A Fig. 3.8B Fig. 3.9 Fig. 3.10 Fig. 3.11 Fig. 3.12 Fig. 3.13 Fig. 3.14A  Fig. 3.14B  Fig. 3.15A Fig. 3.15B Fig. 3.16 Fig. 3.17 Fig. 3.18  Firing modes of gerbil M G B neurons Effects of morphine on input conductance (Gi) Morphine decreased input conductance Effects of morphine on input conductance (Gi) Effects of morphine on input conductance (Gi) Relationship of the changes in input conductance to initial input conductance (Gi) Relationship of morphine-induced increase in input conductance (Gi) to membrane potential (V ) Relationship of morphine-induced decrease in input conductance (Gj) to membrane potential (V ) Relationship of morphine-induced increase in input conductance (Gi) to membrane time constant (x ) Relationship of morphine-induced decrease in input conductance (Gi) to membrane time constant (x ) Current-voltage relationship in showing a morphineinduced increase in slope conductance Current-voltage relationship shows a morphineinduced decrease in slope conductance Effects of TTX Effects of morphine on input conductance (Gi) during tetrodotoxin (TTX) application Effects of morphine on input conductance (Gi) with tetrodotoxin (TTX) in cumulative application Relationship of morphine-induced increase in input conductance (Gi) to thresholds for action potential firing (V ) Relationship of morphine-induced decrease in input conductance (Gi) to thresholds for action potential firing (V ) Relationship of morphine-induced increase in input conductance (Gi) to action potential (AP) amplitude Relationship of morphine-induced decrease in input conductance (Gi) to action potential (AP) amplitude Effects of morphine on firing frequency and latency to the first spike Effects of morphine on frequency adaptation Effects of morphine and naloxone on input conductance (Gi) and membrane potential (V )  22 24 25 26 28 30  r  32  r  33  m  35  m  36 37 38 40 41 43  thr  46  thr  47  r  48 49 52 55 58  vi  Fig. 3.19  Antagonistic action of a delayed application of naloxone to morphine on input conductance (Gi) and membrane potential (V ) Reversed effects of advance application of naloxone to opioids on input conductance (Gi) and membrane potential (V ) Effects of B a to morphine response Effects of B a on cumulative application of morphine Effects of B a on morphine response B a acts in a concentration-rependent manner to alter morphine responses Effects of low-Na to morphine on input conductance (R|) and membrane potential (V ) r  Fig. 3.20  r  Fig. Fig. Fig. Fig.  3.21 A 3.21 B 3.22A 3.22B  Fig. 3.23  2 +  2 +  59  61 65 66 67  2 +  72  +  r  72  Tables Table 3.1 Table 3.2 Table 3.3  Effects of Effects of neurons Effects of  morphine on input conductance (Gi) morphine on membrane potential (V ) in during action potential with tetrodotoxin morphine on spike-half width  29  r  44 51  1  1. Introduction  In this thesis, I will describe my observations of the effects of opioids in the auditory thalamus of the central nervous system (CNS).  These effects, while  fascinating, remain somewhat enigmatic for straightforward interpretation. This is largely because a system dedicated to hearing is not known to mediate pain sensation. Nevertheless, several studies have demonstrated the existence of receptors for opioids in the auditory system and associated nuclei, specifically in the medial geniculate nucleus (MGB) of the thalamus and the central vestibular nucleus (Ding et al. 1996; Mansour et al. 1994; Sulaiman and Dutia 1998). Moreover, several groups have recently attempted to clarify the function of opioids in the auditory system. In the light of my observations, I will discuss near the end of the thesis, interpretations of the proposed functions and make plausible suggestions, in relation to known neurological mechanisms of opioid pharmacology. In the Introduction, I will review the scientific literature, summarizing some important effects of opioids, especially analgesia. From a historical perspective, I will describe what is currently known about opioid receptors and their distribution in the C N S , including the M G B . Then, I will discuss why I chose Mongolian gerbils as an animal model to study neurons of the auditory thalamus.  For the  reader's perusal, I will raise issues that are relevant to these investigations and the development of hypotheses for opioid actions.  2 1.1 Historical  perspective  In humans, perceived pain is an unpleasant sensory and emotional condition, implied by the communication of the painful experience and overt body reactions to painful stimuli (Kandel et al. 2000).  Some of the earliest ways of  dealing with pain involved the ingestion of "opium" (a Greek word for juice), obtained from the poppy plant, Papaver somniferum.  Substances from the  poppy head have received use for pain relief for over a millennium (e.g., Egyptians during the reign of Cleopatra) although some claim that there is little evidence of opium use before the 1 1 century (Kirkup 1988). After isolation as th  an opium alkaloid in 1803, morphine has become and remains the standard treatment for relief from acute postoperative pain as well as for chronic cancer and neuropathic pain. influence  on  other  Actions of morphine at its receptors or the inhibitory  transmitter  systems  produce  analgesia.  Tolerance,  dependence and, the withdrawal symptoms observed on abrupt discontinuation chronically treated with morphine in humans and experimental animals, are outstanding features of its pharmacology. In  the  brain, morphine  acts at  membrane  receptors  on  neurons  characterized by a classical dose-response relationship or saturating function curve. For any drug, a demonstration of competitive antagonism would support the premise of drug-action at membrane receptors. The first report of a morphine antagonist was that of Pohl (1915) who studied N-allylnorcodeine.  This  substance inhibited the effects of morphine and produced a rightward, parallel shift in the dose-response relationship. cogeners  led to the  In the 1940s, investigations of its  discovery of other  morphine  antagonists,  including  3  nalorphine.  Clinical investigation of nalorphine in the early 1950s revealed  antagonism of morphine poisoning as well as the identification of unanticipated analgesic effects of nalorphine at higher doses. Whereas this represented the first instance of agonist/antagonist properties, nalorphine did not receive further clinical use because it caused anxiety and dysphoria. Following its synthesis, Blumberg et al. (1961) demonstrated that naloxone was a potent antagonist of morphine in experimental animals, at least ten times as active as nalorphine. Subsequent studies have shown that naloxone is a competitive antagonist of morphine with no analgesic or toxic properties of its own (Jasinski et al. 1967). Subsequently, naloxone has figured prominently in studies that have attempted to demonstrate the existence of morphine receptors. In the 1960s, it was recognized that there were C N S mechanisms that can strikingly alter the perception of painful stimuli.  A discovery that came from  behavioural experiments on pain perception stimulated the rapid development of concepts about receptors for morphine and for substances endogenous to the brain.  In 1969, Mayer completed a PhD thesis with startling observations that  short-duration mesencephalon  electrical  stimulation  of  resulted in a profound  the  periaqueductal  gray  of  the  long-lasting analgesia in the  rat.  Subsequent studies from that laboratory revealed that naloxone also completely antagonized the analgesic effects of the electrical stimulation (Mayer et al. 1971). This ushered in a search for endogenous brain "opioids" or substances mimicking the effects of substances derived from the opium poppy.  This  culminated in the discovery of endogenous opioids (e.g., Ieu -enkephalin) in an 5  unlikely tissue source, which were mouse vas deferens and guinea pig myenteric  4  plexus (Hughes et al. 1975) and stimulated the opiate-binding investigations of the early 1970s.  1.2 Opioid receptor-binding In the early 1970s, the independent laboratories of Avram Goldstein, Eric Simon, Solomon Snyder and Lars Terenius demonstrated using tritiated opioid agonists, and later antagonists, opioid receptor-binding sites in the CNS of rodents (Goldstein et al. 1973; Pert and Snyder 1973; Simon et al. 1973; Terenius  1973).  Throughout  the  neuraxis,  these  studies  delineated  stereospecific opioid receptor-binding mainly localized to areas primarily involved in the perception (e.g., limbic system in higher mammals) and reactive components (e.g., substantia gelatinosa of both trigeminal brainstem complex and spinal cord) of nociception. The thalamus and periaqueductal gray area have pathways that are crucial for the transmission of nociceptive information to the cerebral cortex. The lateral thalamus controls somatotopic pain sensation, whereas the medial thalamus regulates pain influenced by emotions (Snyder 1977). In view of the limbic system functions associated with emotion and attention, the localization of opioid receptors to limbic areas implies that endogenous opioids mediate or modulate somatosensory inputs, including pain and emotional behavior. On the other hand, there are numerous reports that opioid antagonists like naloxone do not affect pain threshold measured under various conditions in humans (e.g., Gronroos and Pertovaara 1994; Olausson et al. 1986).  In summary, the  distribution of opioid receptor-binding is regional within the brain, to a certain  5  extent, depending on a functional involvement in the transmission, perception and emotional aspects of nociceptive processing. The actions of opioids at the receptor-sites on neurons can account for therapeutic and addictive effects of opioids (Kanjhan 1995).  1.3 Opioid receptors Opioid receptors are classified into three major groups, u, 5 and K receptors, based on differences in pharmacological actions (Pasternak 1993). In the older literature, there are descriptions of a receptors as a fourth type of opioid receptor (Martin et al. 1976).  SKF-10047, phencyclidine and cyclazocine are  agonists that compete with each other at this receptor (Gilbert and Martin 1976a; Martin et al. 1976; Quirion et al. 1981).  Since the three  drugs  have  hallucinogenic properties, there is a hypothesis that drug actions at the a receptor lead to dysphoric effects.  Several clinically used opioids, including  pentazocine, a partial agonist, and nalorphine which has almost disappeared from clinical use, stimulate the cr receptor (Gilbert and Martin 1976b; Martin et al. 1976). These and other opioids with agonist actions at the a receptor, indeed, can produce clinically significant dysphoria (Gilbert and Martin 1976b). Naloxone does not antagonize some of the effects of a agonists (Kemp et al. 1988). Hence, recent literature ignores a receptors in the opioid receptor classification (Henderson et al. 1999).  Studies using anti-sense technology and rat models  have established the functional significance of these cloned receptors (Chen et al. 1993; Yasuda et al. 1993). Investigators have cloned members of each class  6  of opioid receptor from human cDNA and have obtained the respective amino acid sequences (Knapp et al. 1994; Wang et al. 1994; Zhu et al. 1995). The three opioid receptor classes have subtypes, respectively identified Ui and u-2, 5i and 5 , and K 2  1 (  K and K3 receptors (Pasternak 1993). 2  Investigators  have used agonists and/or antagonists to selectively label these subtypes.  receptor  Recently, an opioid receptor-like receptor (OP ) has been cloned, 4  joining the opioid receptor family (Bunzow et al. 1994). The effector mechanism for opioid  receptors involves coupling via pertussis-sensitive GTP-binding  proteins to inhibition of adenylyl cyclase activity (Childers 1991), activation of K +  currents and depression of Ca -currents (Duggan and North 1983). 2+  All opioid subtypes have a role in reducing the intensity of pain (Pasternak 1993; Ho et al. 1997).  Systemically administered morphine and many other  opioids are more selective for |ii receptors than u receptors. The ^-effects are 2  attributable to supraspinal interactions (Bodnar et al. 1988) whereas the n 2  effects result from spinal interactions (Pick et al. 1992). Activation of \i receptors 2  produces analgesia and other  prominent  effects  such as depression of  respiration and gastrointestinal motility, pupillary constriction, euphoria, sedation and physical dependence (Fowler and Fraser 1994). While morphine action at \a  2  receptors there are suggestions that morphine actions at \i  2  produce  dysphoria  and  psychomimetic  effects,  u, -receptor 2  receptors may activation  is  associated with dependence (Reisine and Pasternak 1995). Psychomimetic actions are not uncommon with pentazocine, a partial agonist at fi receptor, and are attributable to agonist interactions at KI and K receptors.  2  The analgesia produced by pentazocine is mostly attributable to  7  spinal interactions at KI receptors (Reisine and Pasternak 1995). The absence of psychomimetic actions of pentazocine might reflect that a main site for this drug is spinal.  Nalorphine may produce analgesia by activating K3 receptors  (Paul e t a l . 1991). Recent studies have implicated opioid actions at 5 receptors in hormone regulation, feeding behaviour and peripheral analgesia. These receptors exist in supraspinal, hypothalamic-pituitary  regions spinal, and peripherally in joints  (Nagasaka et al. 1996). Whereas sufentanil is the only known clinical opioid with agonist actions at 8 receptors (Freye et al. 1992), the peripheral analgesia recently demonstrated for morphine (reviewed by Herz 1996) may be partly attributable to its actions at 8 receptors.  1.4 Practical rationale for animal studies The distribution of opioid receptors in the C N S of gerbils, the animals used in the present investigations, is unknown, but is probably similar to the rat C N S . Using the extensively studied rat brain as a substitute, the M G B contains u. opioid receptors, but not K- and 8-receptors or O P 4 receptors according m R N A and immuno-histochemical studies (Ding et al. 1996; Mansour et al. 1994; Neal et al. 1999). The role of opioid receptors is gradually being elucidated in the auditory system. A s with neurons in other C N S areas, leu-enkephalin, an endogenous opioid peptide, may influence the development of the central auditory system (Kungel and Friauf 1995). Opioids have a role in increasing thresholds in several auditory tests in experimental animals. In chinchilla, intravenous administration  8 of (-)pentazocine, which has no affinity for a receptors, increased the baseline values for the threshold of a compound action potential  (Sahley and Nodar  1994). In comparisons of nociceptin receptor-knockout mice to wild-type mice, investigators have shown that thresholds for the auditory brainstem responses (ABR) became more positive in homozygote mice, without changing nociception or locomotion (Nishi et al. 1997). Opioids in the auditory system might have a role in perceiving pain stimulation.  Lesions of the MGB block the hypoalgesia  produced by white noise stress (Bellgowan and Helmstetter 1996).  Systemic  injection of naltrexone, a potent, non-selective opioid antagonist, has the same effect as lesioning the MGB (Helmstetter and Bellgowan 1994). Generally speaking, the opioid system in thalamic nuclei, may regulate neuronal activity and also have a pathophysiological role, such as in generalized absence  epilepsy.  Recent studies have shown that the activation of opioid  receptors in the thalamus may cause epilepsy in WAG/Rij rats, a genetic model for absence seizures (Przewtocka et al. 1998). The intrathalamic administration of  D-Ala -N-methyl-Phe -Gly -ol-enkephalin 2  agonist, greatly  4  5  (DAMGO),  increases the number of spike-wave  a jx-opioid  receptor  discharges in the  electroencephalogram (EEG). Given a thalamic origin for the epileptogenesis of absence seizures and the abilities of thalamocortical neurons to profoundly influence cortical activity (Steriade et al. 1997), such effects of DAMGO imply a pathophysiological role for opioids in the thalamus. Among the thalamic nuclei, more specific sites have been studied with respect to the involvement of opioid receptors in the epilepsies. Intrathalamic injection of met-enkephlalin into the mediodorsal nucleus (Frenk et al. 1978), but  9  not a relatively ^.-opioid receptor selective agonist, dermorphine, (Greco et al. 1994),  induces  epileptic  discharges  in  rats.  However,  met-enkephalin  microinjection into the centromedian nucleus of baboons does not elicit seizure activity behaviourally or in the E E G (Meldrum et al. 1981). The use of different strains of rats or other species might account for the differing effects of u receptor activation. Systemic and intracerebroventricular injections of numerous opioid agonists induce different effects of opioids in various animal species (Meldrum et al. 1981). Furthermore, met-enkephalin is a mixed agoinst at u and 6 opioid receptors and both u and 5 opioid receptors exist in the mediodorsal nucleus (Mansour et al, 1995). Hence, the different observations might be due to diverse functions of u and 5 opioid receptors. If the activation of opioid receptors in the thalamus has excitatory effects on neurons, the number of receptors would be small if they maintain inhibitory neuronal activities.  Conversely, autoradiographic studies have demonstrated  that opioid receptor binding in several brain sites of seizure-sensitive gerbils during pre-seizure states was larger than the opioid receptor binding of seizureresistant gerbils (Lee et al. 1986).  M G B was one of the sites that had  significantly higher opioid binding activities. The epileptic events diminished the opioid receptor binding in seizure-sensitive gerbils. Therefore, the activation of endogenous opioid receptors may have an anti-convulsant effect. Frey and Voits (1991) suggested that opioids have a pathophysiological role in absence seizures. They deduced this from the observations that systemic administration of opioids increase spike-wave discharge and endogenous opioids suppress tonic-clonic seizures. However, their hypothesis does not explain the  10  observations that injection of opioids into certain area of the thalamus did not induce seizures. Most plausible explanations may invoke the thalamic activation of opioids having dual effects on seizures.  1.5 Questions asked in these investigations Typically, opioid actions on neurons are inhibitory, however opioids also might have excitatory effects.  Electrophysiological studies have shown their  inhibitory effects on various cells, e.g., locus coeruleus neurons (North and Williams 1985) and the centrolateral thalamic neurons (Bruton and Charpak 1998). Thus far, three groups have studied the effects of opioids on the auditory system (Chan-Palay et al. 1982; Lin and Carpenter 1994; Sulaiman and Dutia 1998; Sulaiman et al. 1999). In the vestibular nucleus, Chan-Palay et al. (1982) and Sulaiman and Dutia (1998) have demonstrated the inhibitory effects of opioids, i.e., an increased membrane conductance, hyperpolarization  and  reduction of firing rate. On the other hand, Lin and Carpenter (1994) have shown that morphine increase firing frequency. Using morphine or D A M G O applications, I studied the effects of opioids on neurons of the gerbil M G B and asked the following questions: (1) What effects do opioids have on membrane properties of M G B neurons? (2) If opioids have inhibitory  or excitatory  effects on  membrane currents or channels are involved?  M G B , which  11  2. Methods  2.1 Preparation of slices The animal experiments were conducted according to the  guidelines  approved by the Committee on Animal Care of the University of British Columbia and in compliance with guidelines of the Canadian Council on Animal Care.  2.1.1 Surgical preparation. Meriones  unguiculatus,  For each experiment, I used two Mongolian gerbils, of  either  gender  (9-16  days).  They were  deeply  anesthetized with halothane (MTC Pharmaceuticals, Ontario, Canada). Each gerbil was placed into a wide-mouth, 350 ml bottle containing two cotton balls saturated with - 1 . 8 ml of halothane.  A s an approximate endpoint for deep anesthesia, I  observed the respiratory movements at the level of the gerbil's abdomen; just before respiration ceased completely, the gerbil was taken out of the bottle.  The animal  was decapitated by with a scalpel. A midline incision was made in the dorsal dermis of the head. The midbrain was severed with a stainless steel spatula at -0.1 cm away from the decapitated site, facilitating reflection of the brain from the cranium vault.  The brain was rapidly removed from a cranial cavity by a stainless steel  spatula. The brain was immersed in ice cold (1-4 °C) artificial cerebrospinal fluid (ACSF) containing sucrose as a replacement for NaCI for 5-7 min in a 20 ml beaker. The glass container was placed on ice. The entire surgical procedure required less than 1.5 min.  12  2.1.2 Physiological  solutions.  Normal A C S F was used for most of the  experiment and a modified A C S F was used for the slice preparation. The "normal A C S F " contained (in mM): 124 NaOH, 26 N a H C 0 MgCI , and 1.25 K H P 0 . 2  2  4  3 )  10 dextrose, 2.5 KCI, 2 C a C I  2l  2  For the preparation of slices, a "modified A C S F "  contained a low [Na ], substituted with approximately equimolar sucrose (125 mM). +  The low [Na ] A C S F was used to enhance the viability of the slices (Aghajanian and +  Rasmussen 1989).  For use, the solutions were saturated with 9 5 % 0 / 5 % C 0 . 2  2  The pH of a saturated solution was 7.4, measured by a pH meter (pH Meter 215, Corning, N Y or 30O pH meter, Altex-Beckman, USA).  2.1.3 Tissue slicing. On the morning of an experiment, a 250 ml beaker of modified A S C F , previously saturated with 9 5 % 0 / 5 % C 0 , was kept in a freezer for 90 min 2  2  until an ice-slurry formed. After removal from the cranial vault, the brain was cut into a block that measured 0.8 cm x 0.8 cm, containing the medial geniculate nuclei of both hemispheres, and placed on a filter paper soaked with modified A C S F , bubbled with 9 5 % 0 / 5 % C 0 , in a Petri dish. I cut the brain in two along the midline with a 2  2  double-edge razor blade (Personna, VA, USA). The blades were cleaned with methanol-soaked tissue paper to remove the manufacturer's oil. The blades were snapped in half at the middle such that one edge could be used for cutting the brain and the other for making slices. The first cut was parallel to, and - 0 . 5 cm away from the decapitation plane. Then, the brain was trimmed to a block by making two cuts, each parallel to, at -0.4 cm from the midline, in the lateral aspects of the cerebral hemispheres. After this trimming, the block was returned to the Petri dish for a few  13  minutes until the plastic stage was prepared with a-cryanoacrylate glue (Acuu-flo 8 or Instantbond 8, Lepage, Ontario, Canada). The plastic stage was set in the bath of a Vibratome slicer (Campden Instruments, London, England), filled with the ice-slurry of modified A C S F . A small amount of the glue was spread on the stage with the tip of the glue container. The block was removed from the Petri dish with a stainless steel spoon-shaped spatula. Excess A C S F around the tissue-block on the spatula was removed as much as possible with the capillary action of filtered paper. The block then was placed onto the stage, inserted into the Vibratome, and modified A C S F was poured into the bath, covering the block by ~1 mm. Coronal sections of the brain were cut at 300 um. The cutting speed of the Vibratome was adjusted such that white matter was cut more slowly than the grey matter of the brain.  2.1.4 Incubation of slices. After cutting, the slices were gently lifted and placed into a 250 ml beaker containing normal A C S F .  The beaker had a fine plastic mesh  screen and the slices descended onto this screen which was - 0 . 5 cm below the level of normal A C S F . They were allowed to warm up gradually to 30-33 °C. Slices were incubated for 1 hour in normal A C S F (pH 7.4), saturated with 9 5 % 0 / 5 % C 0 2  2  and maintained with gentle bubbling so as to avoid agitation. After the initial heating, they were allowed to return to room temperature (2325 °C). I used these slices for up to 3 hours after the incubation.  14  2.2 Electrical recording Slices containing the medial geniculate nuclei, were identified visually by a dissection microscope. The medial geniculate nuclei were identified as protruded sections from the brain.  The slices were transferred using a glass tube to the  recording chamber of the microscope. There, the slices were stabilized on a nylon mesh with a platinum wire ring. The slices in the recording chamber, (volume ~1 ml) made from Plexiglass, were superfused by gravity with aerated normal A C S F at a flow rate of ~1.5 ml/min continuously bubbled with 9 5 % 0 / 5 % C 0 . 2  2.2.1 Microelectrodes.  2  The electrodes were made from borosolicate glass tubing  with filaments (WP, Instruments, Sarasota, Florida, USA) and drawn with a puller (Narishige Instruments, Japan). Adjustment of the puller settings allowed me to obtain electrode resistances of 5 to 9 MQ. The electrode solution contained (in mM) 140 K O H , 5 KCI, 10 ethylene-glycol-£»/s-(p-aminoethyl ether)- /V,/V,/V',/V-tetraacetic acid (EGTA), 4 NaCI, 3 MgCI , 10 A/-[2-hydroxyethyl]  piperazine-A/'-[2-ethane-  2  sulfonic acid] ( H E P E S ; free acid), 1 C a C I , 2.8-3 N a A T P or Mg-ATP, and 0.3 2  Na-GTP.  2  Just before the experiment, 2.8 to 3 mM N a - A T P or 3 Mg-ATP, and 2  0.3mM Na-GTP were added to this solution. The electrode solution was adjusted to pH 7.4 with 50 % gluconic acid. A 30 gauge synthetic needle connected to a 1 ml syringe was used to fill an electrode with the solution.  2.2.2 Electrical hardware.  Current-clamp whole-cell patch clamp recordings from  M G B neurons were performed with an Axoclamp 2B amplifier (Axon Instruments,  15  Foster City, C A ) . The recordings were made at room temperature (23-25 °C). Signals were lowpass filtered at 5 kHz, digitized at 10 kHz with a 12 bit data acquisition board (TL1, Scientific Solutions) in a Pentium computer, using pClamp 6 software (Axon Instruments, Foster City, California). Data acquisition, storage and analysis were done by pClamp 6.0.4 software running on a Pentium computer.  2.2.3 Microelectrode  advancement.  I visually observed the cells in the slice with  differential interference contrast microscopy (Zeiss Axioscope FS) with white or infrared illumination. The images were displayed on a video screen (Sony, Japan). The cells were selected visually for microelectrode recording. Before approaching a cell with an electrode, I visually confirmed that there was no contamination at the electrode tip, applying a gentle positive pressure (-100 millibars) to the syringe. This pressure was maintained and controlled manually with a 10 ml syringe, connected to the electrode and holder assembly (Axon Instruments) by plastic tubing.  Approaching the cell with an electrode was accomplished in micrometer  advancements under manual and visually aided control using a micromanipulator (Narishige Instruments, Japan). On mechanical communication of the electrode tip with the cell, the postive pressure was reduced to zero.  A gigaohm seal was  established between the electrode and the cell by suction with the 10 ml syringe. Additional suction was applied with the lips to break through the cell membrane, achieving the whole-cell configuration (Edwards et al. 1989).  16  2.2.4 Electrical  parameters.  I distinguished neurons from glial cells by  evoking action potentials with depolarizing current pulse injections.  The resting  membrane potentials were usually — 6 0 mV in most neurons, after correction for tip potential. The conductance of the each neuron was calculated using Ohm's Law (G = A l / AV) from the steady-state voltage response to hyperpolarizing current pulses (1000 ms duration) of less than 10 mV amplitude injected into a neuron.  The  conductance also was obtained from the main linear portion of the slope in the current-voltage relationships derived from a composite of such responses.  2.2.5 Recording stability.  Generally, the recording conditions (resting membrane  potential, input resistance) were stable, commonly for hours. This was verified by visual observations of the pipette-neuron "patch" condition. When it was apparent that the communication between the electrode and the neuron was compromised, e.g., by debris, plugging of the tip, distortion of the neuron due to the electrode), the recording was abandoned.  All voltage values were later corrected for a liquid  junction potential of -11 mV (Tennigkeit et al. 1997).  2.3 Pharmacological agents Stock solutions of the drugs used in these experiments were prepared using distilled water or normal A C S F and frozen until needed.  Morphine hydrochloride  was obtained from F.E.Cornell & C o . Ltd. (Montreal, Canada).  Naloxone was  purchased from Sigma Chemical (Canada) and Endo Laboratories (New York, USA).  [D-Ala ,N-Me-Phe ,Gly-ol ]-enkephalin (DAMGO), 2  4  5  N-methyl-D-glucamine  17  (NMDG), B a C I and tetrodotoxin (TTX) were purchased from Sigma Chemical 2  (Canada). experiment.  Stock solutions were freshly diluted with normal A C S F before each The drug solutions were kept in inverted 60 ml plastic syringes and  bubbled with 9 5 % 0 / 5 % C 0 . The five syringes were connected to the recording 2  2  chamber with polyethylene tubing for purposes of applying A C S F or drugs by gravity flow. The outflow of each syringe was controlled manually with a valve to change the type of application. The volumes within the reservoirs were adjusted such that a change from solution to another occurred with minimal flow disruption due to differences in hydrostatic pressure. When I only changed syringes without applying morphine, I call it "sham applications" and showed "0 \iM" in the present thesis. Drug(s) were applied for a duration  (3 min) that would allow a reasonable  assumption that steady-state concentrations had been achieved in the bath and tissue. Both control and drug applications were at a rate of 1.5 ml/min.  It took at  least 1.5 min for the 'leading edge' of a solution to reach the recording chamber.  2.4 Data and statistical analyses The data are presented as means ± S . E . M . and represent observations made in different, visually identified neurons of slices that had been exposed to drugs. In most cases, once an experimental protocol involving morphine application was complete, the slice was discarded and replaced with a fresh slice before further experimentation. Representative graphs were constructed using Corel Draw version 8.0 software (Corel, Ottawa, Canada) or Prism version 2.0 software (Graphpad, San Diego, U.S.A.).  The data were analysed by Clampfit of p C L A M P 6.0.4. or Prism  18  version 2.0 software. Different conditions of experiments were assessed with a Student's unpaired t-test (two-tailed p value). A Bonferroni post test was used to compare means of different concentration applications. I used one sample t-test to analyse in the same condition to evaluate the difference from estimated population means. The data were considered significant if there were less than a 0.05 chance that they would occur randomly (P<0.05).  E C o of concentration-response curve 5  was estimated by sigmoidal concentration-response curve fits by Prism version 2.0 software.  The latter spike half-width was measured the spike-half width after the  peak of action potentials.  19  3. Results I analyzed the results from neurons with resting potentials that were -50 mV or less (corrected for junction potentials). The action potentials evoked with current pulse injection generally had amplitudes of -60  mV (cf. Fig. 3.1).  If the input  resistance (Ri) was extremely high, I considered the possibility of electrode tip plugging by debris. With the aid of DIC-microscopy, I often visually confirmed this situation and usually terminated the experiment. In any case, I chose neurons with a Ri of <1000 M Q for analysis. Control data were selected according to the following: "Group 1" where applications of morphine started >8 min after establishing a whole-cell configuration; and, "Group 2" where morphine applications were made ~3 min after formation of the electrode-neuron seal.  3.1 Passive membrane  properties  The average resting membrane potential (V ) was -59.2 r  Group 1 and -58.5  ± 1.5 mV (n = 11) in Group 2.  + 1.1 mV (n = 21) in  The input conductance (Gi) or  1/Rj, (see Methods) ranged from 1.01 to 6.02 nS (Ri = 989 to 166 MQ). The average Gi in Group 1 was 2.4 ± 0.2 nS (Ri = 501 ± 46 MQ; n = 21) and in Group 2, 1.9 ± 0.2 nS (Ri = 601 ± 59 M Q ; n = 11).  The mean membrane time constant (T ) was  estimated from single exponential fits.  m  For this analysis, I used only data from  neurons that were well-fitted by a single exponential curve. The average x  m  Group 1, 56 ± 4.3 ms (n = 17) and in Group 2, 75 ± 9.1 ms (n = 10).  was in  Using an  unpaired t-test, means of the V and the Gi between Group 1 and Group 2 were not r  20  significantly different (p = 0.6756 for the V ; p = 0.1909 for the G|), but not means of r  the x (P = 0.0432). m  3.2 Firing modes Strictly speaking, it often was difficult to evoke only a single action potential on current injection. Therefore, I have defined "just-threshold" for action potential firing (Vhr), as the voltage value resulting from a minimal amount of injected current t  required to evoke one or more action potentials for about 5 0 % of consecutive recordings in a neuron. The Vmr was in Group 1, -50.1 ± 1.5 mV (n = 21) and in Group 2, -40.8 ± 1.6 mV (n = 11). When I observed >1 spike, even by the minimum current for the justthreshold criterion, I used the first spike for the analysis of spike amplitude and halfwidth. The mean spike amplitude was in Group 1, -61.6 ± 2.5 mV (n = 21) and in Group 2, 59.7 ± 2.3 mV (n = 11). The mean spike half-width was in Group 1, 2.52 ± 0.15 ms (n = 21) and in Group 2, 2.95 ± 0.23 ms (n = 11). By an unpaired t-test, means of the V h between Group 1 and Group 2 were significantly different (p = t  r  0.0005). On the other hand, means of spike amplitude and spike half-width between Group 1 and Group 2 were not significantly different (p = 0.6233 for spike amplitude; p = 0.1190 for spike half-width). With  current  pulse  injection,  the  neurons  exhibited  tonic  firing  on  depolarization from the resting potential or from threshold (Fig. 3.1.A). When the neurons were hyperpolarized by DC-current to <-80 mV, depolarizing current-pulse injection led a single spike or spike bursts that included a low threshold C a  2 +  spike  21 (LTS; Fig. 3.1.B). I used neurons from relatively young M G B gerbils, however, I did not observe the spike doublets on depolarizing current injection as in previous studies of young rat neurons in the ventral partition of the M G B (Tennigkeit et al. 1998).  I sometimes could not detect an L T S on the depolarizing responses, or  rebound L T S following hyperpolarizing current pulse injection, in neurons near the resting potential.  The lack of an L T S feature can imply that the neurons are  interneurons (Pape and McCormick 1995; Pape et al. 1994). Or, since immature rat neurons do not apparently have an LTS (Tennigkeit et al. 1998), the absence of an L T S may reflect a development stage related to the age of gerbil. Thus far, there are no studies in the literature, examining the ratio of interneurons to thalamocortical neurons in the gerbil M G B . Winer et al. (1988) reported that in Sprague-Dawley rats, immunoreactive neurons, expressing glutamic acid decarboxylase (GAD) or y-aminobutyric acid totaled only ~1 % (Winer and Larue 1988).  Gerbil brain has a distribution of GAD-positive neurons that is  analogous to that of the rat, where the activity of G A D in the whole brain or thalamus is higher than in gerbils (Chalmers et al. 1970). In view of the above and absence of more information, I considered neurons with no L T S as thalamocortical neurons, and included them in my analysis. I will first deal with the effects of morphine in sub-threshold membrane properties and then, return later to the effects of morphine in action potentials.  22  200ms  Fig. 3.1. Firing modes of gerbil MGB neurons. The cell produced a tonic firing mode, held at -71 mV (A) and a burst firing mode, held at -91 mV (B). Arrow indicates a low threshold C a spike (LTS). For the rest of the figures in the present experiments, on the scale, the upper line indicates voltage and the lower line indicates current. 2 +  23  3.3 Effects of morphine on membrane  properties  Application of morphine produced both increases (Fig. 3.2) and decreases (Fig. 3.3) in input conductance (Gi). Figure 3.4 and Table 3.1 show data from Group 1 and Figure 3.5 shows data from Group 2. I only used two different concentrations for Group 2.  The effects were irreversible (Fig. 3.2), despite washing for periods  longer than 30 min. I did not observe a reversal of the induced conductance change in either direction, despite long washout periods (e.g., 1 to 2 hrs).  A s shown in  Figure 3.2 and extensively below, applications of naloxone, a non-specific opioid antagonist, also did not seem to accelerate the recovery from morphine application, i.e., reverse morphine's effects, even when naloxone was applied after a washout period of longer than 20 min. The change in the Gj did not apparently depend on the initial value of Gi, at all morphine concentrations (Fig. 3.6). Compared to Figure 3.4, eight out of eleven neurons in Figure 3.5 exhibited a remarkable increase in Gi on morphine application.  The extent of the increased Gi had an impact on the  difference in the concentration-Gi response curves between Groups 1 and 2 (Figs. 3.4.A and 3.5). The largest response was an 80.33 ± 45.41% increase, normalized to the initial Gj in the 1 0 concentration  - 8  M morphine group.  The increase in Gi at the same  from Group 1 was 7.24 ± 5.33 %.  In Group 1, the  higher  concentrations of morphine produced greater increases in Gi in a larger number of neurons. Figure 3.4.B shows the Gi responses, calculated in the same way as above, to injected depolarizing current pulses. The concentration-dependent Gi responses to morphine were quite similar to those obtained with hyperpolarizing pulses. On the  0.1 jaM Morphine -65 mV  wash (24 min)  Fig. 3.2. Effects of morphine on input conductance (Gj). A n application of morphine (0.1 pM) for 3 min increased the input conductance by 126 %. The application of morphine started ~3 min after establishing a whole-cell configuration. The increased Gj produced by morphine was not reversible by washing for > 30 min and or by subsequent application of naloxone (10 p.M for 3 min) and observation for an additional 5 min.  25  Control  Fig. 3.3. Morphine decreased input conductance. Application of morphine for 3 min (0.1 u.M) decreased the initial input conductance by 36%. The responses were obtained during a cumulative application of morphine (0.1 nM to 0.1 uJvl). The currentvoltage relationship was shown in Fig. 3.10.  26  A. Hyperpol. 40-1 — a>  2 g  Si] o sO 0 s  c O o  20-  3  2  4  X  X  X  2  X  0o r  X  T  -20-40-|  3*  2<  B. Depol. 40-1  O  o  o 13 sP  3  2  20-1  X  2  I  0-  O  X  -20-  X -40-  -1  0  1 Q  1  -10  1 Q  -9  1 1 Q  -8  1  1  -I Q-7 10'  1  6  10'  Morphine Concentration (Molar)  5  27  Fig. 3.4. Effects of morphine on input conductance (Gj). (previous page) For each neuron, the change in Gj was normalized to the initial input Gj. Application of morphine began > 8 min after establishing a whole-cell configuration. Morphine concentration is 0 |iM stands for sham applications. Number of neurons at each concentration is indicated above and below the bars. A: Responses to injected hyperpolarizing current pulses (1000 ms duration). Using a Bonferroni post test, we analyzed the population mean difference in Gi relative to the Gj change in asterisk-marked neurons. In the increased Gj, there was no statistically significant difference between the 10" M group or the 10" M group or sham applications. In the decreased Gj group, the changes in Gj in the asterisk-marked neurons are not statistically different from neurons at the 10" M group or the 10" M group or sham applications. B: Responses to injected depolarizing current pulses (1000 ms duration). The Gj was calculated as we described in the Methods. The steady-state voltage response was obtained without the presence of action potentials. W e injected depolarizing current pulses at -10-20 pA into the neuron. Data from three out of 38 neurons include neurons with A V responses that were 10 to 11.5 mV in amplitude. The concentration-response curve of the increased Gi was similar to that of Fig. 3.3.A, but not of the decreased Gj. The number of neurons in the decreased Gi increased at the higher concentration of morphine. 7  8  8  10  r  27  100-  o TJ *r T3 O C sP O  •100  J  10"'  10"  Morphine concentration (Molar)  Fig. 3 . 5 . Effects of morphine on input conductance For each neuron, the change in Gj was normalized to Gj. The application of morphine began ~3 min after whole-cell configuration. Note the different percentage 3.4.  (Gj). the initial input establishing a scale from Fig.  29  Table 3.1. Effects of morphine on input conductance  (Gj)  Increased Gi Morphine Gj Concentration (Molar) (% of control) 0 10" 10' 10" 10" 10" 10"  10  9  8  7  6  5  9.11 11.11 13.75 7.24 11.21 29.32 26.72  ±6.00 ±6.51 ± 11.05 ±5.33 ±7.56 ±13.57 ± 13.04  P value  n  0.23 0.34 0.058 0.40 0.28 0.12 0.088  4+ 2 2 2 3 4 6  eased Gi Morphine Concentration (Molar) 0 10" 10" 10" 10" 10" 10'  10  9  8  7  6  5  P value  n  (% of control) 9.8 10.40 18.55 16.17 25.74 17.16 0.84  ±0.71 ±3.80 ±6.19 ±5.89 ±10.01 ±16.86 ± 0.41  0.0002 0.11 0.019 0.11 0.24 0.49 0.29  5 3 4 3 2 2 2  f  t Since we used five syringes, the data were obtained from three different neurons by sham applications.  30  Increased G i  Decreased G j Initial Gj (nS) o.o-  1.5-,  W  C5  <  0 . 1 u M (3)  1.0-1  -0.5H  0.5-\  -1.0-  1 n M (4)  0.0-  -i-1.5  3  0 1.5-.  <g C5  1.0-1  0.0  0 . 0 1 U.M ( 3 )  0.5H  ,-1.5-J 0.0-  1.5  1.0  -i 4  -0.5H  1 u M (4)  0.0-  W  J  2  1 0 u M (6)  -0.5-^  0.1uJv1(2) -1.0H  0.5H  -1.5-  0.0  Initial Gj (nS)  Fig. 3.6. Relationship of the changes in input conductance (Gj) to initial input conductance (Gj). The application of morphine in 5 different concentrations changed Gj to hyperpolarizing current pulses. The changes tended to be independent of the initial Gj at all concentrations. W e chose data at the concentration where there were significant effects on conductance (see Fig. 3.4.A). The number of neurons is indicated in parentheses.  31  other hand, in the decreased Gj category, the two sets of responses showed dissimilar trends. At concentrations of morphine less than 10" M, a decreased Gi 6  was  observed in fewer  neurons tested with depolarizing  pulses than  with  hyperpolarizing pulses. An extensive comparison would require a greater number of experiments at low morphine concentrations, particularly with depolarizing test pulses.  At the higher concentrations of morphine, different from the response to  hyperpolarizing current pulses, most neurons decreased the Gj with depolarizing current pulses. In Figure 3.4.A, I compared the asterisk-marked increases in Gj and increases in Gi evoked by 10" and 10" M concentrations of morphine and sham applications. I 8  7  also compared the asterisk-marked decreases in Gi and the decreases in Gi evoked by 10"  and 10" M concentrations of morphine and sham applications. Whereas  10  8  one might assume sigmoidal concentration-response relationships in Fig. 3.4.A for the mean changes in Gi in neurons tested with hyperpolarizing pulses, a Bonferroni post test showed that the population mean difference was not statistically significant with regard to asterisk-marked bars. Using sigmoidal concentration-response curve fits (see Methods), an estimate of the  EC50 in the  (hyperpolarizing test pulses) was 3.18 x 10" M. 7  increased Gi category  An estimate of the EC50 in the  decreased Gi category (hyperpolarizing pulses), consisting of responses to 10" and 10" M, was 8.91 x 10" M . . 7  8  Morphine application typically hyperpolarized the membrane potential (V ; Figs. 3.7A and 3.7B). r  In neurons where morphine increased Gi, the V  r  hyperpolarized by 1-5 mV. The frequency of hyperpolarizations increased at  10  M  32  AV  40H  r-5.0 r  1  2 2  20-  -2.5  3 2  I  i  Ii  1  o  Q. 0)  •5 "  1  20H  ^  T  -40H 10  10"  9  10"  8  10"  10"  7  6  N  h-2.5 ro  o o. >_ o  2 10"  It ra  2  0  ^  CC  -o.o  o o  ra  10  S  •-5.0 Q.  Morphine concentration (Molar)  Fig. 3.7A. Relationship of morphine-induced increase in input conductance (Gj) to membrane potential (V ). The V was recorded at 3 min after starting application of morphine. The V hyperpolarized at higher concentrations of morphine. One cell at 10" M and 10" M, and two cells by sham applications did not change the V . r  r  r  5  6  r  33  a> u c  r  40 H  c o AV  CO  4-1  o  re  r  •2.5  2  20-  X  3 T3  «  c o o H-  re  o a  n  C  o o  5.0  a>  T3  O.O  o\£  o-  re  VI TI  U  -20-1  1 -40H  _  10"  10  10'  9  re  o a i_  LI-  2 2  o  N  h-2.5  3  1  10"  8  <u  2  10"  1CT  7  6  10"  5  Q.  >-5.0  Morphine concentration (Molar)  Fig. 3.7B. Relationship of morphine-induced decrease in input conductance (Gj) to membrane potential (V ). The V was recorded as for Fig. 3.7A. Application of morphine hyperpolarized V in most neurons. One cell by sham applications, and two cells at 10" M and 10" M, morphine did not change the V . r  r  r  9  10  r  34  higher concentrations of morphine. morphine  hyperpolarized V  r  In the case of decreased Gj (Fig. 3.7B),  in most neurons.  In summary,  I observed a  hyperpolarization in response to morphine application, despite the direction of the induced Gj change. The morphine-induced change in membrane time constant (x ) was almost m  correlated to the change in Gi, when Gj was increased (Figs. 3.8A), but not when Gj was decreased (Fig. 3.8B). A s described above, I estimated the x from the fit with m  a single-exponential curve. I excluded neurons that did not apparently fit the curve, i.e., when the fit was not exact, x  m  increase in x  was very large. The bias might cause the large  observed with 10" M morphine (Fig. 3.8.A). Alternatively, morphine 5  m  could affect the input capacitance especially at the higher concentration.  When  morphine decreased Gi, there was no association between the change in Gi and x . m  The reversal potential for morphine was determined from the intersection of control and morphine curves in the current-voltage relationship (Fig. 3.9). The mean reversal potential in the increased Gi, was -61.3 ± 6.4 mV (n = 3) in Group 1 and 68.2 + 2.3 mV (n= 9) in Group 2. The calculated K -equilibrium potential is -85 mV, +  which implies that morphine activates several ion channels as well as K channels. +  In neurons with a morphine-decrease in Gi, the current-voltage  relationship  demonstrates that the decrease of the slope conductance caused by morphine was clear when the V was > -80 mV (Fig. 3.10). r  35  Morphine Concentration (Molar) Fig. 3.8A. Relationship of morphine-induced increase in input conductance (Gi) to membrane time constant (x ). The T was estimated using a single exponential fit. When the trace did not apparently fit a single exponential curve, we omitted the cells from this figure. m  M  36  Fig. 3.8B. Relationship of morphine-induced decrease in input conductance (Gi) to membrane time constant (x ). The x was analyzed and chosen in the same way as in Fig. 3.8A. The x decreased in most neurons and was not correlated to the change in Gj. m  m  m  37  -100  J  Fig. 3.9. Current-voltage relationship for morphine-induced increase in slope conductance. Data were obtained from the steady-state voltage changes in control and during application of 10 u,M morphine for >3 min. A s shown from the intersection of the control and morphine curves for this neuron, the reversal potential for morphine was -74 mV.  38  Fig. 3.10. Current-voltage relationship for morphine-induced decrease in slope conductance. Data from neuron of Fig. 3.3. The responses were obtained during a cumulative application of morphine (0.1 nM to 0.1 pJvl). 1nM of morphine decreased the initial input conductance by 35 %, calculated from the exact values. Application of morphine decreased the slope conductance at potentials depolarized to V = -80 mV in 1 nM (A) and to V = -85 mV in 0.1 u.M (B). r  r  39  3.4 Effects of morphine during tetrodotoxin (TTX)  blockade  The administration of T T X (300-600 nM) blocked action potentials in 7 1 % of neurons (12 out of 17 neurons; Fig 3.11.A) and reduced the voltage responses to depolarizing current pulses.  Application of T T X diminished the responses to  hyperpolarizing current pulses in 5 9 % of neurons (10 out of 17 neurons).  The  average increase of Gi was 28.7 ± 9.0 %, normalized to the initial input Gj (n = 10). Application of T T X caused an apparent increase in the slope conductance, calculated from steady-state voltage responses to subthreshold current pulses (Fig. 3.11 .B). The remaining 41 % of neurons exhibited a decreased Gi, averaging 16.6 ± 3.6 % to the control response, when tested with hyperpolarizing current pulses. Application of T T X did not greatly affect V . The mean change in the V was ~1 mV, r  r  i.e., near the limits of detection (1.3 ± 0.5 mV, n = 17). When  applying  TTX  (300-600  nM)  to  block  action  potential-induced  transmitter release, the application of morphine continued to increase or decrease the Gi (Fig. 3.12). The effects of morphine on G, remained irreversible. Compared to the typical responses illustrated in Fig. 3.3, morphine had different effects on Gi during concomitant T T X application. In the absence of T T X and with hyperpolarizing current pulses, morphine at 10" M and 10" M, increased or decreased Gi (Fig. 6  3.3.A).  7  However, during T T X application, morphine at the same concentrations,  induced a decrease in G,.  The effects of morphine on Gi were smaller when  depolarizing current pulses were used. Note that in the neuron of Fig. 3.13.A, the responses measured with depolarizing current pulses do not seem to depend on the morphine concentration.  y  40  Fig. 3.11. Effects of TTX. A: Application of T T X (300nM) for longer than 5 min, abolished tonic firing. The neuron was held at -71 mV. Under both conditions, the same current was injected. The current-voltage relationship is show in B. B: 5 min application of T T X (300 nM) reduced the slope conductance. The input conductance increased by 75 %. The subsequent application of morphine did not change the slope conductance.  41  A . Hyperpol. 60-.  O  O  40 H  c eg  o "S  20J  o c 6  s  3  N o TTX  X  4  i  o  X  6  -20-1  X  6  B. Depol. 60 -i — © 40-  o u £ c  o t> v9s  0  O o  N o TTX  20  4  3  4  J l  0•20H T  T  1  0 10" 10" 10 10 10 10" Morphine Concetration (Molar) 10  9  8  7  6  5  42  Fig. 3.12. Effects of morphine on input conductance (Gi) during tetorodotoxin (TTX) application, (previous page) For each neuron, the change in Gj was normalized to the initial input Gj. Application of morphine began more than 8 min after establishing a wholecell configuration. T T X (300-600 nM) was applied until action potentials were no longer evoked in response to injecting depolarizing current pulses at - 1 0 0 pA morphine was then applied cumulatively. Gi in T T X was considered as a control value. A: Responses to injected hyperpolarizing current pulses. No TTX was applied during Gi measurements at the zero morphine concentration. Different from Fig. 3.3.A, in the 10" and 10~ group, the change in Gi decreased in all neurons. At 10" M, the large mean response was due to the large increase in Gj by one neuron. B: Responses to injected depolarizing current pulses. The responses were not dependent on the morphine concentration. 6  10  7  43  Morphine Concentration (Molar)  Fig. 3.13. Effects of morphine on input conductance (Gi) with tetorodotoxin (TTX) in cumulative application. Two examples from Fig. 3.12 for the effects of cumulative application of morphine on the Gi determined from responses to injections of depolarizing and hyperpolarizing current pulses. For each neuron, all Gi were normalized by fractions, regarding the largest Gj as 1.0 and the smallest Gi as 0. C stands for the control value and T T X stands for the Gi after action potentials were completely abolished by TTX application (300-600 nM). In both neurons, the responses to hyperpolarizing current pulses yileded similar curves. In Fig. 3.11. A., the responses by depolarizing current pulses were independent of the morphine concentration.  44  Table 3.2. Effects of morphine on membrane potential (V ) in neurons during action potential blockade with tetrodotoxin. r  Increased Gi Morphine Concentration (Molar) 10" 10' 10"  10  9  5  AV (mV)  n  0.0 0.0 -1.67 ± 1 . 2 0  2 2 3  r  Decreased Gi Morphine Concentration (Molar) 10' 10" 10" 10" 10"  10  9  7  6  5  AV (mV) r  -2.00 -1.00 -1.83 -1.92 -1.00  ±1.00 ±1.00 ±0.60 ±0.64 ±0.00  n  2 3 6 6 2  45 During application of TTX, morphine hyperpolarized V in all neurons (n = 6; r  Table 3.2). The change in the V (AV) was smaller than the AV observed in the r  r  r  absence of TTX. Compared to changes measured at 0 u,M morphine, one may assume that the changes in the V are independent of the morphine concentration. r  In summary, the responses evoked by morphine application were modified by TTX.  The effects of morphine did not necessarily result from actions only on the  postsynaptic membrane.  3.5 Effects of morphine on firing patterns In  most  neurons,  morphine  application  decreased the  just-threshold  responses evoked by current pulses (1000 ms, duration). The changes in  V hr t  were  associated with a decreased Gj (Fig. 3.14B), not an increased Gj (Fig. 3.14A). At a morphine concentration of 10" M, however, the action potential threshold increased 5  by an average of - 2 . 5 mV. In neurons where morphine decreased Gi, the magnitude of reduced threshold was roughly proportional to the magnitude of to the Gi decrease, except at the higher concentrations of morphine (Fig. 3.14B). Morphine application typically decreased the amplitude of the action potential (AP; Figs. 3.15A and 3.15B).  In cases where morphine increased Gi, dose-  dependent change in A P was similar to the dose-dependent change in Gj. In cases where morphine decreased Gi, the magnitudes of the A P change were not correlated to the magnitudes of change in Gi ignoring the largest change at 10" M 6  (Fig. 3.15B).  Morphine Concentration (Molar)  Fig. 3.14A. Relationship of morphine-induced increase in input conductance (Gj) to thresholds for action potential firing (V hr)The Vthr was "just-threshold", defined at the amplitude of injected current that evoked the action potentials, approximately 50% of the time during successive current pulses (1000 ms, duration). The Vhr decreased at most concentrations of morphine. However, application of 10" M morphine produced biphasic changes in Vhr which significantly increased and then decreased. t  t  5  t  47  Fig. 3.14B. Relationship of morphine-induced decrease in input conductance (Gj) to threshold for action potentials ( V ) . The Vthr decreased in most neurons. In most cases, the amount of decrease in Vhr is correlated to the concomitant decrease in Gj. thr  t  48  Fig. 3.15A. Relationship of morphine-induced increase in input conductance (Gj) to action potential (AP) amplitude. Morphine application decreased the A P amplitude in most neurons. The changes in the A P (AAP) were greater at higher concentrations and corresponded to changes in Gj.  49  -30  40H o c ro  |G| IAP amplitude  20 E  10  "ST T3  3 0  1 0  -io  1 0  -9  1 0  -s  -jo"7  10"6  10"5  "-30  Morphine Concentration (Molar)  Fig. 3.15B. Relationship of morphine-induced decrease in input conductance (Gj) to action potential (AP) amplitude. Morphine application decreased the A P in most neurons. Unlike changes in the A P found with the increased Gj, there is no association between the change in Gj and in A P .  5 0  Morphine application had little effect on spike half-width (Table 3.3) which reflects the roles of Na -currents and K -currents in the respective ascending and +  +  descending phases of the action potential. either as a whole, or as a latter half width.  I separately analyzed spike duration Application of morphine at 10" M 6  prolonged mean spike half-width, according to these criteria. However, the changes were not significant. Hence, I could not elucidate morphine's effects on the different ion activity in action potentials. Morphine application changed the firing frequency and latency to the first spike evoked by current pulses (1000 ms, duration).  Figure 3.16 illustrates three distinct  examples of the changes. In pattern A, morphine application decreased the firing frequency or prolonged the latency to the first spike. In pattern B, morphine had no effect.  In pattern C , morphine administration increased the firing frequency and  shortened the latency to the first spike. The changes were not closely correlated with the change in the Gj. Even so, in the morphine-increased Gi category, >85 % of neurons showed pattern A or B (n = 24) and in the morphine-decreased Gj category, - 7 0 % of neurons showed pattern B or C (n = 17). In order to study morphine's effects on the spike afterhyperpolarization (AHP) through participation of Ca -dependent K currents, it was necessary to select data 2+  +  from neurons exhibiting the same firing frequency, i.e., for a comparison of control and morphine data.  Unfortunately, I could not find enough data to satisfy this  criterion. The slow A H P is responsible for the alteration adaptation (Foehring et al. 1989).  of firing properties  in  Most neurons (70%) showed spike-frequency  51  Table 3.3. Effects of morphine on spike-half width Whole half width Morphine Concentration (Molar) 0 10" 10" 10" 1010" 10"  10  9  8  7  6  5  control (ms) 2.49 2.67 2.60 2.67 2.60 2.67 2.54  ±0.18 ±0.10 ±0.11 ±0.14 ± 0.086 ± 0.35 ± 0.26  morphine (ms)  n  ±0.18 ±0.15 ± 0.20 ± 0.40 ±0.11 ± 0.22 ± 0.33  8  morphine (ms)  n  2.56 2.82 2.67 2.59 2.76 3.03 2.73  f  5 6 5 5 6 8  .after half width Morphine Concentration (Molar) 0 10" 10" 10" 10 10" 10'  10  9  8  7  6  5  control (ms) 1.77 1.80 1.76 1.80 1.75 1.87 1.84  ±0.14 ± 0.065 ± 0.066 ± 0.065 ± 0.055 ±0.28 ±0.18  1.84 1.85 1.74 1.68 1.82 1.97 1.82  ±0.11 ± 0.079 ±0.13 ±0.25 ± 0.026 ±0.15 ± 0.22  8  f  5 6 5 5 6 8  f At 0 \LM of morphine, the data were obtained from three different neurons.  52  53  Fig. 3.16. Effects of morphine on firing frequency and latency to the first spike, (previous page) Three example patterns for effects of morphine on firing frequency and latency to the first spike to square current pulses (1000 ms, duration). In the increased input conductance, for the firing rate, 46 %, 38 %, 16 % of neurons, and for the first spike latency, 68 %, 29 %, 3 % of neurons showed pattern A, B, C , respectively (n = 24). In the decreased input conductance, for the firing rate, 24 %, 35%, 41 %, and for the latency to the first spike, 24 %, 41 %, 35 % of neurons showed pattern A, B, C , respectively (n = 17).  54  adaptation in M G B (n = 10; Fig. 3.17). I measured the time between peaks of action potentials and used them for an inter-spike interval (ISI) analysis. Figure 3.17 shows a plot of spike-frequency that is the reciprocal of the ISI against the event of interval number. When the spike-frequency was >25 Hz, fast and slow phases were evident (Fig. 3.17.A).  By comparing the responses to the same current pulses, morphine  application reduced the fast and slow phases, as well as the spike-frequency (Fig. 3.17.A and Fig. 3.17.B). The reduction of the spike-frequency adaptation was more obvious in a comparison of data at the same spike frequencies (Fig. 3.17.B).  3.6 Mu-opioids My-opioid receptors are abundant in the M G B . The effects of a selective \iopioid receptor agonist, D A M G O , were similar to that of morphine.  I applied  D A M G O at concentrations of 1 nM to 0.1 u,M in both Groups 1 and 2 (n = 12; data not  shown).  Application  hyperpolarizing the V  r  of  DAMGO  increased  and  to values between 0 and >5 mV.  decreased  the  Gi,  Other investigators  demonstrated the reversible effects of D A M G O in thalamic neurons (Bruton and Charpak, 1998).  However, I did not observe reversal of the effects after 30 s  applications of D A M G O , despite observation periods of 30 min.  The reversal  potential for D A M G O was - 6 9 . 5 ± 11.5 mV (n = 2), which was similar in value to that for morphine in Group 2. receptors in M G B neurons.  Therefore, I conclude that morphine activated u,-opioid  Interval number  56  Fig. 3.17. Effects of morphine on spike-frequency adaptation, (previous page) Spike frequency (1/inter-spike interval) vs. the interval number. Each trace is a response to injected pulse currents (1000 ms, duration) which is shown in right-side of the trace. 70 % of neurons showed spike-frequency adaptation, which had a fast phase and a slower phase. When the frequency was over 25 Hz, the fast phase was observed. Morphine application reduced the frequency and blocked both fast and slower phases. In B, the second trace of control in response to 64 pA and the first trace with 10 jiM of morphine in response to 94 pA are the identical firing rate. The reduction in spike-frequency adaptation is clear in fast phase.  57  3.7 Blockade by naloxone Naloxone blocked the effects of morphine in a time-dependent manner. A s described above, a washing with A S C F did not reverse the effects of 0.01 to 0.1 jxM morphine or 1 nM to 0.1 u,M D A M G O . When the wash was longer than 20 min, a subsequent application of 1-10 jiM of naloxone (3-10 min) did not reverse the change in the Gi (Fig. 3.18; n = 4). Conversely, a naloxone application that started within 20 min after terminating opioid application, counteracted both the opioid induced increase and decrease of Gi (Fig. 3.19; n = 4). By itself and prior to co-application with morphine, naloxone evoked a small decrease in Gi. When co-applied with morphine, naloxone opposed the effects of morphine. In Fig. 3.19, for example, the Gi was increased by morphine and then decreased by a delayed application of naloxone.  This reversal, likely  antagonism, was observed in the absence and presence of TTX. Application of naloxone as a pretreatment blocked the effects of morphine or D A M G O (n = 11). For this procedure, I first applied naloxone (10 u.M) for 5-20 min which, alone, did not greatly alter V (-0.4 ± 0.5 mV; n = 10), but when applied for ~5 r  min, either increased Gj by 18.1 ± 6.3 % (n = 8) or decreased Gj by 25.4 ± 5.5 % (n =3).  I applied either morphine (0.1 u.M) or D A M G O (0.1 u.M) while continuing the  naloxone application (10 u.M) for an additional 3 min. Then, the naloxone application terminated was and opioid application was continued. Despite either the naloxoneinduced increase or decrease in Gi, naloxone reversed the effects of the opioids on V and Gi (Fig. 3.20). Note the differing durations of naloxone pretreatment in Figure r  3.20.  Also, the blockade of opioid action by naloxone was dependent on the  58  Fig. 3.18. Effects of morphine and naloxone on input conductance (Gi) and membrane potential (V ). The graph shows the effects of morphine and naloxone of the same neuron as in Fig. 3.2. An application of morphine (10 u.M) for 3 min significantly increased the Gj and decreased the V . These effects were not reversible, despite termination of the morphine application for 24 min. A subsequent application of naloxone (10 u,M) for 3 min did not reverse these effects of morphine. r  r  59  Fig. 3.19. Antagonistic action of a delayed application of naloxone to morphine on input conductance (Gj) and membrane potential (V ). 0.1 u,M of morphine application (3 min) increased the input Gj by 15.5 % and hyperpolarized the V temporarily and hyperpolarized by 2 mV before starting naloxone application. Brief hyperpolarization might be due to changing solution from control to morphine. Delayed application of naloxone (10 uJM, 3 min) antagonized the effects of morphine on the Gj. The Gi with naloxone was smaller than the Gi in control by 17.3 %. r  r  60  duration of naloxone application. The prior application of naloxone did not reverse the effects of a later application of an opioid applied for 5-10 min (Fig. 3.20.A). When naloxone pretreatment only slightly affected V and Gi (see above) and its r  application time was >15 min, a combined application of naloxone and opioid did not result in changes in Gi and V . r  However, application of opioid counteracted the  effects of naloxone pretreatment when the antagonist produced either an increase or decrease in Gi (Fig. 3.20.B).  3.8 Blockade by Ba  2+  My previous experiments demonstrated that K -currents are involved in +  activation by morphine.  If only K -conductances were increased by morphine +  application, V would hyperpolarize due to an increased Gj. Previous investigators r  have reported that B a  2 +  blocked K -currents activated by morphine and other \i+  opioids in thalamic and locus coeruleus neurons (North and Williams 1985; Bruton and Charpak 1998).  To determine the participation of such currents in the  responses to morphine, I used B a , as well as T T X to block transmitter-release. 2 +  After completely abolishing action potentials with TTX, I applied B a >12 min prior to morphine application. B a  2 +  (200 u.M) for  2 +  application (with TTX) depolarized the  neurons by 6 mV to 11 mV, reaching a plateau value after >12 min (n = 8). From a V near - 6 0 mV, B a r  2 +  decreased Gj more when measured with hyperpolarizing, than  with depolarizing pulses. B a  2 +  decreased the Gj by - 6 0 % and unmasked a sag in  the voltage response, as previously observed (Fig. 3.21 A; Tennigkeit et al. 1996). At this point, I assumed that B a  2 +  blocked a substantial portion of the K currents and +  time (min)  Fig. 3.20. Reversed effects of advance application of naloxone to opioids on input conductance (Gi) and membrane potential (V ). (previous page) A: 7 min advance application of naloxone could not reverse the effects of morphine. Naloxone application (10 u.M) increased the Gj by 20.7 % and did not change the V . Combined application of naloxone and morphine (0.1 \iM) decreased the Gj and the V . Morphine application alone continued the combined effects. B: 20 min advance application of naloxone could antagonize the effects of D A M G O . Application of naloxone (10 u.M) decreased the input Gj. The V was hyperpolarized briefly when naloxone application was started and recovered to the control level during the application of naloxone. Combined application of naloxone and D A M G O (0.1 |iM) did not change the Gi and the V . Only D A M G O application increased the Gj and hyperpolarized the V . Note different time scales in A and B. r  r  r  r  r  r  63  co-applied morphine.  Cumulative application of morphine from 10" to 10' 9  5  M  produced a greater reduction in Gi with depolarizing, than with hyperpolarizing current pulses (n = 6; Fig. 3.21 B). In Fig. 3.20B, the changes in Gi were greatest with pulses in both directions at 10" M and became smaller with hyperpolarizing 7  current pulses at 10" M. This feature was also observed in an absence of B a 5  2 +  (Fig  3.13). With or without co-application of B a , the greatest changes in Gi occurred at 2 +  morphine concentrations of 10" M in 4 neurons, 10" M in 3 neurons and 10" M in 3 9  7  6  neurons. These effects do not apparently involve IN P and may be a consequence of 3  morphine acting on IH (see Discussion). Furthermore, higher concentrations of morphine increased the amplitude of the L T S in response to hyperpolarizing current pulses (>-100 pA; Fig. 3.21 A). A 10 to 15 min wash with TTX-containing A S C F reversed these effects. Application of B a  2 +  blocked the effects of morphine and unmasked a sag in  the voltage responses to hyperpolarizing pulses. These effects, accompanied by a depolarization of 8 to 11 mV, were apparent if B a after morphine.  2 +  was applied either before, or  In the presence of TTX, application of morphine (0.1 to 10 |xM)  increased Gi (Figs. 3.22A and 3.22B). This change in the Gi was reversed by a subsequent application of B a  2 +  (0.2 to 1 mM).  containing A S C F for 30 min reversed these effects.  A thorough washing with TTX-  TTX  Wash  65  Fig. 3.21 A. Effects of B a o n morphine response, (previous page) After blocking action potentials by TTX, B a (200 u,M) was applied for 12 min. Application of B a increased the input resistance and unmasked a sag in the voltage response. Co-application of 10 u.M of morphine had little effect on the input resistance. Morphine application hightened low threshold C a spike to hyperpolarizing current pulses. These effects were reversible. 2+  2 +  2 +  2 +  66  —o— depol. —•— hyperpol.  Morphine Concentration (Molar)  Fig. 3.21 B. Effects of B a on cumulative application of morphine. After action potentials were abolished by T T X (300-600 nM), B a (200 pM) was applied for 12 min. The Gi was calculated from the voltage response to ~10 pA depolarizing and hyperpolarizing current pulses. From 10" to 10" M of morphine did not change Gj in the presence of Ba . z +  2 +  9  2 +  5  TTX  wash  68  Fig. 3. 22A. Effects of B a on morphine response, (previous page) After blocking action potentials with TTX, 10 |xM morphine (3 min) increased Gj. B a blocked the effects of morphine and decreased Gj more than the control Gj. B a application unmasked a sag in the voltage record. These effects were reversed by washing. 2 +  2 +  2 +  69  0.2 mM  1 mM  Ba 2+  10uM Morphine r 4n  E  10  20 time (min)  Fig. 3.22B. B a acts in a concentration-dependent manner to alter morphine responses. In TTX, 10 u,M morphine application increased G j . Subsequent application of B a (0.2 mM) did not change the V . After B a was increased to 1 mM, B a blocked the effects of morphine and decreased G | below control G j . V depolarized 8 mV. These effects were reversed by washing. 2 +  2+  2+  r  2+  r  70  3.9 Effect of low-Na  +  To elucidate the mechanism of morphine's greater effect on the voltage responses to hyperpolarizing, than depolarizing pulses, I performed experiments where we substituted N M D G for N a , a major ion carried by I H channel. I changed +  the extracellular [Na ] from 150 mM to 26 mM, using N M D G . +  I perfused low N a - A C S F +  containing T T X before and after  morphine  application (n = 2). The low [Na ] A C S F hyperpolarized V by 6 mV and decreased +  r  Gj by 25-35 % (Fig. 3.23) and reduced the changes in V and Gj during morphine r  application. A co-application of T T X and B a  2 +  appear to make clearer, the effects of  low [Na ] on morphine-induced changes in V and Gj. +  r  The effects of morphine on the persistent Na+ current (lNa,p) were investigated on one neuron. W e held this neuron at -71 mV and, at -51 mV where Iwa.p likely would be more than 50% activated. Before starting morphine application, N M D G application for 12 min decreased Gj as revealed by hyperpolarizing current pulses from 1.64 nS to 0.71 nS, just after stopping the N M D G application.  At -71 mV,  N M D G application reduced the morphine-induced change in the Gj (1 uM), where as there was reduction when the neuron was held at -51 mV.  time (min)  72  Fig. 3.23. Effects of low-Na to morphine on input conductance (Gj) and membrane potential (V ). (previous page) In the presence of T T X (300 nM), we changed extracellular N a from 150 mM to 26 mM for 3 min before and after 10" M morphine application. Without morphine application, exchanging extracellular solution hyperpolarized V and decreased Gj in response to both depolarizing and hyperpolarizing current pulses. During morphine application, the change in V and Gj were smaller. +  r  +  8  r  r  73  4. Discussion In these experiments, I have examined the effects of morphine and D A M G O on membrane conductance and spike firing at gerbil M G B neurons. I observed both increases and decreases in membrane conductance.  The increased input  conductance may be due to an increased K conductance, whereas the decreased +  conductance may have resulted from a blockade of the hyperpolarization activated inward current, IH- The application of morphine, a u and 5 opioid agonist, and D A M G O , a u selective opioid agonist, had similar effects.  From their reversal  potentials, I suggest that, their activation of u receptors produced the effects through similar ionic mechanisms.  Naloxone antagonized these effects, consistent with  opioid effects on M G B neurons being mediated via opioid receptors. It now remains to briefly summarize these results and provide feasible explanations for these effects.  4.11nfluence of gerbil age on membrane properties and action potentials Compared to other studies in thalamic nuclei, the V and Rj observed here r  were relatively high and t 1996;  Williams  et  al.  m  was longer than in previous studies (Tennigkeit et al.  1996).  These characteristics  are  repesentative  of  thalamocortical neurons from younger animals (Ramoa and McCormick 1994; Tennigkeit et al. 1998).  In our studies, 20% of the neurons did not display an  evoked L T S . However, Tennigkeit et al. (1998) observed only two such neurons in >P14 animals. In their studies, this may have resulted from the selection of neurons from only the ventral partition of the M G B . With respect to the action potential (AP),  74  V^r, A P amplitude, and A P duration had almost identical values to those from adult neurons (Ramoa and McCormick 1994; Tennigkeit et al. 1998). characteristics of the A P achieve maturity earlier than the other  Hence, the membrane  properties (Ramoa and McCormick 1994). Several studies have shown differential development of membrane ionic channels, pumps or transporters. For example, tetraethlyammonium (TEA) sensitive K  +  channels have a role in determining the duration of action potentials in P26  neurons, but not in P3-5 neurons (Spigelman et al. 1992). V is largely dependent r  on the N a - K pump activity, which develops significantly after P14 (Molnar et al. +  1999).  +  Thus, the disparity in the development of ionic channels and pumps may  account for the different rate of maturity of membrane properties, reversal potentials and firing modes in younger animals versus older animals. In summary, we cannot assume that membrane properties remain homogeneous in neurons sampled experimentally in animals aged P9 to P16 days.  4.2 Effects of morphine on passive and active membrane properties The largest differences in the effects of experiment 1 and experiment 2 were changes in Gj. W e conducted these experiments in two ways: (1) observations in neurons starting >8 minutes after break-through (Group 1) and (2) observations in neurons starting >3 minutes after break-through (Group 2). The large increase in the conductance in Group 2 might have resulted from mechanical damage of the neuronal membrane after its perforation and the equilibration of its contents with internal pipette solution (Ries and Puil 1999). Since morphine application time was  75  the same between Group 1 and Group 2, the involvement of a second messenger system by morphine is an unlikely cause for the large increase in the Gi in Group 2. The effects of morphine and D A M G O were not reversed on washout in the present study. There are no studies on opioid effects in M G B neurons. However, in other neurons (Cherubini et al. 1984) and in rat thalamic neurons (Bruton and Charpak 1998), morphine or D A M G O effects are reversed with washing. Moreover, in my studies, a subsequent application of naloxone did not reverse the effects of morphine and D A M G O when the wash was longer than 20 min. The absence of recovery cannot be attributed to a short control period, e.g., in Group 2, where the control value might have changed before starting morphine application, since the effects of opioid application were also not reversed in Group 1. The absence of reversibility may reflect a second messenger system activation by the opioid. Opioid receptors are members of the G-protein coupled-receptor family (Uhl et al. 1994). Morphine and D A M G O application changed Gi.  Differences in activating  currents at membrane potentials between — 5 0 mV and —70 mV may have induced the different responses to depolarizing and hyperpolarizing current pulses. Below, I will discuss, in more detail, the effects of depolarizing and hyperpolarizing current pulses.  The morphine concentration-response relationship for Gi showed no  statistical significant difference between each concentration (Fig. 3.5). The failure to find a significant effect might be due to the mixed action of the opioids. the  conductance changes into  increased and  concentration of morphine (Figs. 3.4 and 3.5).  decreased groups  I separated for  each  In each concentration group (Fig.  3.5), responses were mixed, ranging from 1 % to 89%. The smaller change in Gi  76  may have resulted from a balance between opioid evoked opening and closing of ion channels. It also remains possible that not all of the M G B neurons express opioid receptors to some extent (see Ding et al. 1996). The  large difference in conductance between Group 1 and Group 2  influenced the morphine concentration-response curves (Figs. 3.4.A and 3.5). Group 2, it was difficult to determine an E C .  In  Although Fig.3.4.A shows  5 0  concentration-response curves without TTX that are different from other opioid studies, the responses to hyperpolarizing current pulses (Fig.3.4.A) in cases where DAMGO  increased Gi, yielded similar curves and EC o values in thalamic 5  centrolateral neurons (Bruton and Charpak 1998) and guinea pig nodose ganglionic neurons (Ingram and Williams 1994).  Hence, the control value in Group 2 is  probably inaccurate and more time should be allowed after break-through to the whole-cell configuration, before taking measurements. The changes in Gi coupled with the changes in V , evoked by morphine and r  D A M G O application are consistent with the activation of more than one type of ion channel.  At higher concentrations of morphine, Gj increased and V decreased. r  This might represent increases in K and Cl" conductances. A more positive holding +  current was needed to maintain the same potential after applying morphine and the morphine induced increase in Gj was blocked by B a . These findings are consistent 2 +  with hypothesis that the change in Gj resulted from opioid activation of an outward K  +  current.  At lower morphine concentrations, the increased Gi, coupled with the  increased V , may reflect opioid activation of C a r  hyperpolarization activated inward current,  IH.  2 +  or N a  +  currents or the  Opioids induce increases in  77  intracellular C a  2 +  concentration (Jin et al. 1992; Tang et al. 1996) and also shift the  activation curve of IH along the voltage-axis to more negative potentials (Ingram and Williams 1994). At lower concentrations of morphine, there was a tendency for Gi and V to decrease.  In such cases, morphine application might block K  r  +  ion  channels or the N a - K pumps. Morphine application itself is a known enhancer of +  +  N a - K pump activity (Sykoba et al. 1985). Hence, when opioids decreased Gi and +  +  V , it seems possible that the opioid blocked K channels or maintenance of the [K ] +  +  r  gradient. When opioids decreased Gi and increased V , they may have blocked an r  inward current. However, I observed such actions only on two neurons. In summary, opioid application appears to increase K conductance at higher +  concentrations and to decrease a K conductance, N a - K pump activity or activation +  +  +  of IH, at lower concentrations. Finally, the initial Ri had no correlation to either the increase or decrease in Gi evoked by opioid application. From these data, I suggest that opioid activates K channels as well as other ion channels. +  A change in membrane capacitance ( C ) could account for morphine's action m  on T . The increase in Gj coupled with the change in T , occurred only at lower M  M  concentration of morphine. Thus far, two studies have shown that opioids can alter C  m  (Rusin et al. 1997; Sargent et al. 1988).  However, they demonstrated that K-  opioid receptors, but not |o,- and 5-opioid receptor activation altered C . These might m  reflect methodological and experimental differences.  Sargent et al. (1998) used a  model membrane and Rusin et al. (1997) used rat neurohypophysial endings, which have a high density of K-opioid receptors and a low density of | i - and 8-opioid receptors (Mansour et al. 1994).  Rusin et al. (1997) used three different opioid  78  agonists at 1-3 u.M, a concentration at which I also observed little effect on C . m  Hence, it remains possible that u.-opioids have an effect on C . m  When Gi increased, the reversal potential was between -61 mV and - 6 7 mV, strongly implying that opioids activate K channels along with other ion channels in +  M G B neurons. When the Gi decreased, opioid actions did not reverse in the voltage range from —100 mV to —50mV.  In my experiments, the calculated reversal  potential for K is - 8 5 mV and for CI" is - 5 8 mV. This implies that cationic currents +  are involved in the activation of currents by opioids in M G B neurons. TTX  application  altered  the  morphine  concentration-response  curve,  compared to morphine application alone (Fig. 3.4 and Fig. 3.12). This implies that the action of morphine has an effect at presynaptic sites. In the presence of TTX, morphine application still had effects on the Gi, but did not greatly change V . r  Opioids may activate CI" channels with an equilibrium potential near V , or the sum r  resulting from different changes in Gi to various ions equals 0 mV. Opioids are inihibitory at presynaptic sites (Johnson and North 1993). These inhibitory effects are mediated by diminishing Ca -activated presynaptic transmitter 2+  release. Ca  2 +  Johnson and North (1993) suggested that opioids act on voltage-gated  channels to reduce C a  2 +  influx, resulting in a reduction of transmitter release.  So far, there is no report that opioid acts on CI" channels.  Nevertheless, it is  plausible that morphine application in the presence of T T X activates several ion channel types. In the presence of TTX, I observed different responses to depolarizing and hyperpolarizing current pulses (Fig. 3.13).  The difference is more obvious in the  79  presence of B a . 2 +  From - 7 0 mV to - 5 0 mV, the change in voltage activates and  inactivates several ion channels, which might induce the distinctive actions to depolarizing versus hyperpolarizing current pulses.  With hyperpolarizing current  tests, Gi decreased whereas Gi was unchanged when the neurons were tested with depolarizing current pulses. Such effects are consistent with morphine inhibiting IH. Several investigators have demonstrated the blockade of IH by opioids in the locus coreruleus, nodose ganglion and hippocampus (Alreja and Aghajanian 1993; Ingram and Williams 1994; Svoboda and Lupica 1998).  The different Gi responses to  depolarizing and hyperpolarizing current pulses were concentration-dependent; the responses were largest at 10" to 10" M of morphine concentration. 9  hyperpolarizing shift of l  H  Thus, a  7  might occur primarily at relatively lower concentration of  opioids in M G B neurons.  4.3 Effects of opioids on firing properties Opioid application decreased A P amplitude, depending on the magnitude of the increased Gi and the concentration.  Similarly, a recent study has shown that  extracellular  reduced  application  of  morphine  the  peak  Na -current +  in  a  concentration-dependent manner in rat and human cardiac myocytes (Hung et al. 1998).  In their study, application of morphine did not change the C a  outward current, or inwardly rectifying K current. +  2 +  current,  Therefore, Hung et al. (1998)  concluded that the morphine inhibits a N a current in myocytes. However, in our +  study, morphine application appeared to have effects on K  +  and other currents.  Morphine may have inhibited directly, or shunted the N a - currents. +  80 Opioid application did not have significant effects on spike-half amplitude. An increase in N a conductance comprises the first half part of the A P , and K channels +  +  are involved in the latter half of the A P . The absence of effects imply that opioid application has no blocking actions on spike-generating N a channels or the delayed +  rectifier responsible for repolarization. Spigelman et al. (1992) demonstrated that in mature rat hippocampal neurons, T E A , but not 4-aminopyridine (4-AP), application prolonged spike duration. If we postulate that our neurons contain similar currents as in their experiments, opioid application did not affect TEA-sensitive or 4 - A P insensitive K -currents, such as inward rectifiers or several Ca -activated K +  2+  +  channels. In the case where morphine decreased Gj, there was little change in the A P , except at 10" M of morphine.  Morphine application did not significantly reduce  6  action potential amplitude compared to sham applications.  Hence, the reduction  observed in action potential might be due to cell deterioration.  Since I have data  from only two neurons at 10" M, it is difficult to infer any effect. 6  Morphine may block Ca -activated K channels. Unfortunately, we could not 2+  +  get enough data to analyze the spike afterhyperpolarization (AHP), mediated by a Ca -activated K conductance. Instead, I examined spike-frequency adaptation, in 2+  +  part resulting from activated Ca -mediated K channels. Unlike rat M G B neurons 2+  +  (Tennigkeit et al. 1998), we observed spike-frequency adaptation in gerbil M G B neurons.  In addition, similar to cortical neurons (Foehring et al. 1989)  or  motoneurons (Kernell 1965), the adaptation had an early fast phase and late slower phase and is not observed in other thalamocortical neurons (Ramoa and McCormick  81  1994).  Blockade of spike-frequency adaptation by morphine application indicates  that morphine inhibits C a - or Na -mediated K 2 +  +  +  currents (Foehring et al. 1989).  Ca -mediated K channels have three subtypes (reviewed by Sah 1999). Apamine 2+  +  insensitive Ca -mediated K 2+  +  channels have a role in spike-frequency adaptation  because of their slow activation.  In addition, this channel might regulate the A H P ,  together with small conductance Ca -mediated K channels, which are apamine2+  +  sensitive. Opioid actions have paradoxical effects on the A H P . In guinea pig myenteric plexus cells, morphine prolonged the A H P , following a train of action potentials (Tokimasa et al. 1981; Cherubini et al. 1984). In the rat dorsal root ganglion, several opioid agonists blocked the tail currents following depolarizing current pulses, which are mediated by Ca -activated K channels (Akins and McCleskey 1993). However, 2+  +  in guinea pig or rabbit coeliac ganglion neurons, morphine did not inhibit the slow A H P (Cassell and McLachlan 1987). enkephalin  immunoreactivity.  The coeliac ganglion neurons demonstrate  Cassell  and  McLachlan  (1987)  used  higher  concentrations of morphine, compared to other studies, showing the blocking effects on Ca -activated K channels. Hence, blockade of Ca -activated K channels by 2+  +  2+  +  morphine might be tissue dependent. Opioid  application  induced  contradictory  effects  on  spike-frequency  adaptation and on the excitability of neurons. Blockade of apamine-insensitive C a 2 +  activated K  +  channels reduces spike-frequency adaptation  and increases the  excitability of the neuron. However, we observed opioid application blocked spikefrequency adaptation and reduced the firing frequency when morphine increased Gj.  82  Lidocaine application in the thalamic neurons also induced the same contradictory effects (Schwarz and Puil 1998). A s explained before by Schwarz and Puil (1998), the contradictory effects might result from a shunt of N a current. W e observed the +  blockade of spike-frequency adaptation only at higher concentrations of morphine, when the Gi was increased in most neurons. This observation supports the shunt hypothesis.  4.4 Conclusion I suggest that u.-opioid application has effects on several ion channels. My experiments demonstrate that opioids might reduce the activation of l  H  at lower  concentrations and increase the activation of K currents at higher concentrations. +  A s described in the Introduction,  opioids have roles in both increasing and  decreasing the excitability of thalamocortical neurons. 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