THESJS Ml ICHIGAN ST TE UNIVERSITY UBRARlESI Ill“illwillminim; 312930171 This is to certify that the dissertation entitled Outward Potassium Currents of Supraoptic Magnocellular Neurosecretory Cells Isolated from the Adult Guinea Pig presented by Michael David Hlubek has been accepted towards fulfillment of the requirements for Ph.D. Neurosciences - Pharmacology degree in If [KM Major professor 11 August 1997 Date MS U is an Affirmative Action/Eq ual Opportunity Institution 0-12771 LIBRARY fl Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MTE DUE DATE DUE DATE DUE use com-.mu OUTWARD POTASSIUM CURRENTS OP SUPRAOPTIC HAGNOCELLULAR NEUROSECRETORY CELLS ISOLATED FROH THE ADULT GUINEA PIG bY Michael David Hlubek A DISSERTATION Submitted to Michigan State University in partial fulfillment of requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 1997 ABSTRACT OUTWARD POTASSIUM CURRENTS OF SUPRAOPTIC HAGNOCELLULAR NEUROSECRETORY CELLS ISOLATED FROM THE ADULT GUINEA PIG By Michael David Klubek (1). Whole-cell voltage-clamp recordings revealed several types of outward K’ current present in somata of magnocellular neurosecretory cells (MNCs) dissociated from the supraoptic nucleus of the adult guinea pig were. These currents were identified on the basis of their voltage dependence, kinetics, pharmacology and Ca” dependence. (2) The predominant K’ current evoked from a holding potential of ~40 mV was slowly-activating, long-lasting, tetraethylammonium (TEA)-sensitive and showed little steady- state inactivation. Also, this current was reduced by extracellular Cd”) These data suggest that in supraoptic MNCs classical Cabeinsensitive, delayed rectifier channels (K9) and CabFsensitive, non-inactivating channels (Kuafl both contribute to the sustained current. (3) A transient, low-threshold K. current which was 4- aminopyridine (4AP)-sensitive and showed significant steady- state inactivation was evoked along with the sustained current from a holding potential of -90 mV. Based on these characteristics, this current corresponds to the A-current (1,) described in other neurons. (4) I. was activated when Caz. influx was blocked or when Cab was absent from the extracellular medium, suggesting that Cab influx is not necessary for activation of the current. (5) In many recordings, a transient, 4-AP-insensitive outward current was evoked from a holding potential of -40 mV. This high4threshold transient 3? current was abolished by extracellular Cd?) Charybdotoxin (ChTX) or tetraethylammonium (TEA) and was absent when extracellular Cab was replaced by Sr”) suggesting that it is a transient Cab-dependent Ki current. (6) Current-clamp recordings revealed that the _temporal activation of several K3 channel types shapes MNC action potential repolarization. Also, it was shown that frequency-dependent spike broadening in dissociated MNCs results from a reduction in Iran during repetitive firing. (7) We conclude that the presence of multiple types of If current may, in part, underlie the complex firing patterns of oxytocinergic and vasopressinergic MNCs. ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Peter Cobbett, for helping to define this project and encouraging me to press forward when the road got rough. I also wish to thank my thesis committee members, Dr. James Galligan, Dr. William Atchison, and Dr. Cheryl Sisk, for their guidance. Special appreciation goes to Dr. Galligan, who provided many of the tools (and Friday evening beverages) necessary to complete my research. I wish to acknowledge my mother, Phyllis Hlubek, for her love, understanding and generosity. Mom, perhaps I will get a job in the near future - I don't want to rush into anything, however! Finally, I would like to thank my wife, Deborah, and canine companions, Molly (my "little girl"), Kelly (my "big buddy"), Mariah (my "big girl"), and Bugsy (my "little buddy") for their unconditional love and support throughout my graduate career. iv LIST OF TABLES . LIST OF FIGURES . ABBREVIATIONS . . INTRODUCTION . . I. Neuronal K TABLE OF CONTENTS Channels and Currents . . . . . . . A. Types of K Channels and Currents . . . 1. 2. 3. 4. 5. B. Delayed Rectifier K Channels (Ky) . A-Type K Channels (K) . . . . . . . Ca ~Dependent K Channels (KCa) . . . Inward Rectifier K Channels (Km) . . Other Types of K Channels . . . . . General K Channel Structure . . . . . . . II. Overview of Neuronal Ca' A. Low Voltage-Activated (LVA) Cab a. High Voltage-Activated (HVA) caz' Channels . .‘. . . . Channels Channels III. General Hypothalamo-Neurohypophysial System (HNS) Features . . . . . . . . . . . . . . . A. Historical Overview of HNS Research . . B. Anatomy of the ENS . . . . . . . . . . . . C. Physiology of the ENS . . . . . . . . . . D. Mechanisms Underlying the Electrical Behavior of Supraoptic MNCs . . . . . . l. Preparations Used to Study Mechanisms 2. 3. OBJECTIVES . . . Which Underlie the Electrical Behavior of Supraoptic MNCs . . . . Extrinsic Mechanisms Underlying the Electrical Behavior of Supraoptic MNCs . . . . . . . . . . . Intrinsic Mechanisms Underlying the Electrical Behavior of Supraoptic MNCS . . . . . . . . . . . . . viii ix 35 39 41 49 TABLE OP CONTENTS (CONT'D) MATERIAIs AND “T3003 0 O I O O O O O O O O O O O O O O 51 I. Preparation of Dissociated Supraoptic MNCs . . 51 II. Immunocytochemistry and Morphological Examination of Dissociated MNCs . . . . . . . 52 III. Voltage-Clamp Recording from Mtle . . . . . . 55 IV. Current—Clamp Recording from MNCs . . . . . . 58 v. Drugs . . . . . . . . . . . . . . . . . . . . . 59 RESULTS........................61 I. Outward Ki Current Components in Dissociated MNCs....................61 A. Profile of Macroscopic Outward Current . . 61 B. Activation and Inactivation Kinetics of the Low-Threshold Transient K' Current . 69 C. Voltage Dependence of Activation and Steady-State Inactivation of In» and %MN) . . . . . . . . . . °.° . . . . . . 75 D. Ca Sensitivity of Outward K Current Components . . . . . . . . . . . . . . 82 E. Pharmacology of Outward K Current Components............... 98 II. Spike Repolarization and Frequency—Dependent Spike Broadening in Dissociated MNCs . . . 106 A. Spontaneous Action Potentials . . . . . 106 B. Current-Evoked Action Potentials . . . . 109 C. Effects of K' Channel Blockers on Action Potential Duration . . . . . . . . . . 118 D. Effects of K' Channel Blockers on Frequency-Dependent Spike Broadening . 125 DISCUSSION..................... 140 I. Components of Outward K' Current in Somata of Supraoptic MNCs . . . . . . . . . . . . . . 140 A. Low—Threshold Transient K Current (Inn) 140 B. Sustained Outward K Current . . . . . . 143 C. High-Threshold (Ca2 -Dependent) Transient Outward K: Current . . . . . . . . . . 144 II. Comparison of K Current Components in Somata and Axon Terminals of MNCs . . . . . . . . 146 III. Involvement of Specific Ki Channel Currents in Action Potential Repolarization and Frequency-Dependent Spike Broadening . . . 153 IV. Other Possible Functions of K Channel Currents 163 vi TABLE 0? CONTENTS (CONT'D) BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 167 vii Tgblg 2 Table 1. Table 2. Table 3. LIST 0? TABLES Biophysical and Pharmacological Properties of Voltage-Dependent K Channels . . . . Biophysical and Pharmacological Properties of Ca -Dependent K Channels . . . . . . Biophysical and Pharmacplogical Properties of Voltage-Dependent Ca Channels . . . . viii 10 ll 18 Figure 1. Figure Figure Figure Figure Figure Figure Figure Figure Figure 10. LIST OF FIGURES Page 2 Anatomy of the hypothalamo—neurohypophysial system (HNS) . . . . . . . . . . . . . . . Photomicrographs of dissociated supraoptic cells . . . . . . . . . . . . . . . . . . Supraoptic MNCs can be identified based on size . . . . . . . . . . . . . . . . . . . Profile of outward current in supraoptic MNCs evoked by depolarizing voltage steps from a holding potential of -90 mV . . . . Profile of outward current in supraoptic MNCs evoked by depolarizing voltage steps from a holding potential of -40 mV . . . . High—threshold transient outward current in supraoptic MNCs evoked by depolarizing voltage steps from a holding potential of -40 mv O O O O 0 O O O O O O O O O O I O O Inactivation of the high-threshold transient current by a depolarizing prepulse . . . . . . . . . . . . . . . . . Separation of low-threshold transient outward current from total outward current . . . . . . . . . . . . . . . . . . Voltage dependence of activation of Iron and I‘(V) O O O O O O O O O O O O O O O O 0 Voltage dependence of steady—state inactivation of Iran and Iran . . . . . . . ix 22 S3 56 62 65 67 7O 73 77 79 LIST OF FIGURES (CONT'D) Figure i Page E Figure 11. Effect of extracellular Cdzi on Iron and Im, amplitudes . . . . . . . . . . . . . 83 Figure 12. Effect of extracellular Cdz‘ on the voltage dependence of activation and steady-state inactivation of Iron . . . . . . . . . . . 86 Figure 13. ' Effect of extracellular Cdzi on the voltage dependence of Ixm activation . . . . . 88 Figure 14. Voltage dependence of activation and steady-state inactivation of In“ recorded in Ca -free extracellular solution . . . . 90 Figure 15. Effect of extracellular Cdzi on the voltage dependence of activation and steady-state inactivation of Iron recorded in Ca ~free extracellular solution . . . . . . . . . . 92 Figure 16. Effects of extracellular Cdzi on the high- threshold transient outward current . . . . 96 Figure 17. Effects of extracellular Srz' on the high- threshold transient outward current . . . . 99 Figure 18. Pharmacological properties of Iran and ........... 102 1,0,, . . . . . . Figure 19. Pharmacological properties of the high- threshold transient outward current . . . 104 Figure 20. Differential sensitivities of the low- and high-threshold transient outward currents to charybdotoxin (ChTX) . . . . . . . . . 107 Figure 21. Spontaneous action potentials in supraoptic mes O O O O O O O O O O O O O O O O O O 110 Figure 22. Voltage response of a supraoptic MNC to a single injection of suprathreshold depolarizing current . . . . . . . . . . 112 LIST OF FIGURES (CONT'D) E' l Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure Figure 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 3S. Eese_i Voltage response of a supraoptic MNC to repetitive injections of suprathreshold depolarizing current . . . . . . . . . . 114 Action potential duration in supraoptic MNCs is frequency-dependent . . . . . . . 116 Effect of Cth on the duration of isolated, current-evoked action potentials . . . . Effect of TEA.on the duration of isolated, current-evoked action potentials . . . . Effect of 4-AP on the duration of isolated, current-evoked action potentials . . . . Cab current is necessary for maximal action potential broadening produced by K channel blockers . . . . . . . . . . . . ChTX does not prevent frequency-dependent spike broadening . . . . . . . . . . . . ChTX increases the extent and rate of frequency-dependent spike broadening TEA does not prevent frequency-dependent spike broadening . . . . . . . . . . . . 4—AP prevents frequency-dependent spike broadening . . . . . . . . . . . . . . TEA, but not 4-AP, increases the extent and rate of frequency-dependent spike broadening . . . . . . . . . . . . . . . Temporal contributions of different ionic currents toward MNC action potential dynamics 0 O O O O O O O O O O O O O Roles of different ionic currents in determining the dynamics of frequency- dependent spike broadening . . . . . . . xi 119 121 123 126 129 131 133 136 138 156 160 ACh o-Aga IVA AHP CAMP 4-AP ATP o—CgTX GVIA ChTX CSA DAB DAP DHPs DTX EGTA EPSPS GABA HAP HEPES HNS 5-HT HVA IbTX IPSPs LVA MNCs NP OT OVLT PIPES PVN mRNA SON TEA TTX VP ABBREVIATIONS acetylcholine o-agatoxin IVA after-hyperpolarization cyclic adenosine monophosphate 4-aminopyridine adenosine triphosphate o-conotoxin GVIA charybdotoxin cross-sectional area 3,3'-diaminobenzidine depolarizing after—potential dihydropyridines dendrotoxin ethyleneglycol-bis-(fi-aminoethyl ether) N,N,N',N'-tetraacetic acid excitatory postsynaptic potentials y-aminobutyric acid hyperpolarizing after—potential 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid hypothalamo-neurohypophysial system S-hydroxytryptamine high voltage-activated iberatoxin inhibitory postsynaptic potentials low voltage-activated magnocellular neurosecretory cells neurophysin oxytocin organum vasculosum lamina terminalis piperazine-N,N'-bis-(2-ethane- sulfonic acid) paraventricular nucleus messenger ribonucleic acid supraoptic nucleus tetraethyammonium tetrodotoxin vasopressin xii INTRODUCTION I . Neuronal K’ Channels and Currants K’ channels comprise a family of integral membrane proteins which control membrane excitability in a variety of cell types by selectively catalyzing K. current flow. In general, the equilibrium potential for K‘ (E‘ = the potential at which there is no net KO current flow) is more negative than the resting membrane potential (Eh = the potential at which there is no net current flow) in neurons. 15 channel currents therefore draw the membrane potential closer to E‘ and thus farther from the threshold for action potential initiation. In this way K' currents are intrinsically inhibitory in that they oppose excitatory events. The importance of K' currents in controlling membrane excitability, along with the tremendous diversity of K. channels relative to that of other ion channels, may indicate that the characteristic and diverse electrical behavior exhibited by different neurons is largely defined by their complement of K' channels. Accordingly, any attempt to describe the intrinsic mechanisms which underlie the electrical behavior of specific neurons must consider the complement of K’ channels present in the neuronal membrane. 2 A. Types of K’ Channels and Currents The ionic selectivities of different K' channels appear to be identical. They all exhibit an ion permeability sequence of Tl°>K'>Rb'>NH", are relatively impermeable to Na. and Cap, and are blocked by Cs' (Hille, 1992). Nevertheless, K. channels may be distinguished based on several different criteria such as voltage dependence of activation and inactivation, kinetics, pharmacology (including Caz. sensitivity), and molecular structure. 1. Delayed Rectifier K’ Channels (xv) Two main criteria must be met in order for a K' channel to be classified as a delayed rectifier (Rudy, 1988) . First, the voltage dependence and kinetics of the macroscopic current must be similar to that described by Hodgkin and Huxley (1952) for the "delayed rectifier" current (Iron) in giant squid axons. The current described by Hodgkin and Huxley exhibited delayed activation and little or no inactivation over the duration of a depolarizing voltage step. Also, the membrane conductance (ease of current flow) changed with voltage, a property called rectification in electric circuit theory. The second criterion which defines Kv channels is one of exclusion. Activation of these channels does not depend on intracellular Cab concentration ( [Caz']i) . The non- dependence on [Caz']i distinguishes Kv channels from Caz.- dependent K' channels (Meech and Standen, Hermann and 3 Hartung, 1983: 1975; Sah, 1996) which produce macroscopic currents that are otherwise indistinguishable from Ixrvr Although all neurons appear to express at least one type of K‘, channel, the properties of K, channels can vary greatly among neurons - an indication that different K' channels are probably expressed. Most Kv channels, however, exhibit a "high threshold" for activation, opening at membrane potentials more positive than -40 mV, and are sensitive to block by extracellular tetraethylammonium (TEA) in millimolar concentrations (Thompson, 1977; Numann et a1. , 1987; Hille, 1992) . Functionally, current catalyzed by K, channels in neurons contributes to action potential repolarization and thus helps determine action potential duration (Hodgkin and Huxley, 1952; Thompson, 1977: Storm, 1987) . Kv currents can also contribute to the generation of the after-hyperpolarization (AHP) immediately following individual action potentials (Storm, 1987) . 2. A-Type K. Channels at.) A rapidly-activating and inactivating current carried through K, channels was first described in Anisodoris snail neurons (Neher, 1971; Connor and Stevens, 1971) and later in many other neurons (Rogawski, 1985) . Because K, channels inactivate rapidly following activation despite a maintained activating stimulus, the current through these channels is transient. The time-dependent inactivation of an A-current (Inn) typically proceeds with a time constant 1 (the time 4 required for the macroscopic Iran to fall to l/e'th (337%) of its peak value) of 10 to 100 ms. K, channels also exhibit a "low threshold" for activation, opening at potentials subthreshold to that required for action potential initiation, and almost complete steady-state inactivation at membrane potentials more positive than -50 mV. Thus K, channels can only be activated by depolarizing stimuli after a period in which the membrane is "hyperpolarized" to potentials more negative than -50 mV. The voltage-dependence of K, channel inactivation is such that in neurons which exhibit an E, more positive than ~50 mV, K, channels are largely unavailable for activation. Dendrotoxin (DTX), a peptide toxin present in eastern green mamba snake venom, is a very potent blocker of K, channels in central neurons (Halliwell et a1., 1986: Dreyer, 1990) . This compound typically blocks I,(,) with an Icso (the concentration that inhibits the macroscopic In” by 50 percent) of less than 50 nM. The convulsant 4-aminopyridine (ll-AP) blocks K, channels at millimolar concentrations but is less effective at blocking other K. channels (Gustafsson et a1, 1982; Numann et al., 1987). Sensitivity to block by 4-AP is thus commonly used to detect the presence of K, channels. Connor and Stevens (1971) were the first to show that 1“,, could regulate the frequency of repetitive action potential firing in neurons. Although K, channels are largely inactivated (due to steady-state inactivation) near 5 E,, the AHP that follows individual action potentials removes a portion of the inactivation. Hence K, channels are activated as the cell depolarizes and the resultant Iron slows the return of the membrane potential toward the threshold for action potential initiation. Over time, however, 1“,, inactivates and the cell is again allowed to depolarize unimpeded. In this way, activation of 1“,, prolongs the interspike interval and slows the firing frequency in a repetitively firing neuron. Im) can also contribute to the repolarization of the action potential (Tanouye et al., 1981: Storm, 1987) and to the delay before the generation of an action potential following a depolarizing stimulus (Daut, 1973; Byrne, 1980). These latter functions of 1m) are observed in neurons which have a sufficient number of K, channels available for activation near E, . 3 . Caa-Dependent r’ Channels (Kn) Activation of K, and K, channels is voltage-dependent and does not depend on [Caz’]i. Activation of Kc, channels, on the other hand, is strongly dependent on [Cay]i (Meech and Standen, 1975: Hermann and Hartung, 1983; Sah, 1996) . The probability that these channels will activate increases with elevations in [Caz‘]i. Accordingly, the voltage dependence of Kc, channel current (Ixrm) mirrors that of Caz. current. This is because changes in Caz. influx through voltage-dependent Caz‘ channels affects [Cazi],, and thus the 6 activation of Kc, channels. Current-voltage (I-V) relationships for Kc, channel currents (Inca) therefore exhibit a characteristic "n-shape" which reflects voltage- dependent changes in Ca” influx. Kc, channels can also be activated by Caz. released from intracellular storage sites via the action of neurotransmitters coupled to second messenger systems (Nicoll, 1988) . There are at least two types of neuronal Kc, channels (Sah, 1996) , the so-called high- or "bigfl-conductance (BKQ) channels (Marty, 1981: Blatz and Magleby, 1984: Romey and Lazdunski, 1984) and the low- or "small"-conductance (SKQ) channels (Romey and Lazdunski, 1984; Blatz and Magleby, 1986). BK“ channels exhibit single-channel conductances of 100-250 ps and are voltage-dependent even at constant Caz. concentrations. These channels require 1-10 uM Caz. for activation at potentials near E, (-50 to --70 mV) . In contrast, SK“ channels exhibit single-channel conductances of 18-50 ps and little voltage dependence at constant Caz. concentrations. These channels require 100-400 nM Cab for activation, and thus are much more sensitive to Ca” than BK“ channels. Both channel types produce macroscopic currents characterized by delayed activation and little time-dependent inactivation, thus resembling Iron kinetically. 8K, and SK“ channels can also be distinguished based on their pharmacological properties. BK“ channels can be blocked by nanomolar concentrations (IC50<100 nM) of the 7 scorpion toxins charybdotoxin (ChTX) and iberatoxin (IbTX) , and by low millimolar concentrations of TEA (Miller et al., 1985; Galvez et al., 1.990: Sah, 1996). These channels are not sensitive to block by apamin (Romey and Lazdunski, 1984: Pennefather et a1., 1985) , a peptide toxin isolated from bee venom. SK“ channels, on the other hand, are not sensitive to block by Cth, IbTx or TEA, but are blocked by nanomolar concentrations (Icso<5° nM) of apamin (Blatz and Magleby, 1986; Lang and Ritchie, 1990: Park, 1994). Activation of BK“ channels produces a current (1mm) which contributes to action potential repolarization. Block of these channels results in action potentials of increased duration (Romey and Lazdunski, 1984; Pennefather et al., 1985; Storm, 1987) . Activation of SK“ channels produces a current (Isaac), which is thought to underlie the prolonged AHP following individual action potentials, as demonstrated by the action of apamin (Romey and Lazdunski, 1984: Pennefather et a1., 1985; Kirkpatrick and Bourque, 1996) . In addition to the above Cab-dependent channel currents, there have been reports of a Cab-dependent transient K. current which contributes to action potential repolarization in some neurons (Ribera and Spitzer, 1987: Neely and Lingle, 1992) . This current is distinct from 1“,, in that it is 4-AP-insensitive and can be activated from membrane potentials at which 1“,, is fully inactivated. The pharmacological properties of the current are identical to those of BK“ channel currents, leading to speculation that 8 it is carried by a BKCa channel variant which has as part of its molecular structure a component which confers a mechanism for inactivation. 4. Inward Rectifier 3’ Channels (rm) Many neurons express K’ channels which show increased activation during hyperpolarizing rather than depolarizing stimuli (Adams and Halliwell, 1982: Constanti and Galvan, 1983) . The I-V relationship for current carried through these channels reveals a decrease in slope conductance with depolarization, a property known as "anomalous" or "inward" rectification. K"t channel currents (Iain) are characterized by a lack of time-dependent inactivation and a strongly voltage-dependent block by intracellular Mgz' (Vandenberg, 1987) . It is believed that the inward rectification exhibited by these channels results from a voltage-dependent block of K. current flow by cytoplasmic Mgzi ions which plug the K"I channels. There are no known pharmacological agents which selectively block Km channels by acting from the extracellular side of the cell. K"t channels do pass some outward K. current in the range of membrane potentials between E, and E,. Under physiological conditions, the membrane potential of a neuron rarely becomes more negative than E . It is believed that K" channels help stabilize E, near E, by conducting outward current in the voltage range just positive to 3,. Increased depolarization then increases the block ome channels by 9 Mg”, thus allowing the membrane potential to change more freely. The biophysical and pharmacological properties of the major voltage- and Ca2'-dependent K’ channels are summarized in Tables 1 and 2, respectively. 5. Other Types of a’ Channels A number of other neuronal K. channels have been discovered which cannot be placed in any of the major classes described above. The most notable of these are either receptor-coupled or modulated by intracellular metabolites. Two receptor-coupled K. channels have been studied extensively - the muscarinic-inactivated KO channel (K,) and the 5-hydroxytryptamine (5-HT)—inactivated K' channel (K5,,r) . Kfl channels exhibit voltage-dependent, delayed activation at membrane potentials more positive than -65 mV and little inactivation (Brown and Adams, 1980: Brown, 1988) . Activation of these channels is inhibited by muscarinic acetylcholine (ACh) receptor agonists. Functionally, these channels contribute a K' current (Inn) that, when decreased by the neurotransmitter ACh, enhances the responsiveness to depolarizing stimuli. K5,," channels are weakly voltage-dependent and normally activated at E, (Klein et a1., 1982: Shuster et al., 1985) . The neurotransmitter 5-HT inactivates these channels via cyclic adenosine monophosphate (cAMP)-dependent phosphorylation, resulting in a decreased K’ current (Inns-rm) and enhanced membrane excitability. In the absence of 5-HT, K5,," channel 10 Table 1 Biophysical and.Pharmacological Properties of Voltage-Dependent K Channels ‘ Channel type K; K, K“ . Activation >-40 <-50 <-50 ‘ threshold (mV) inactivation slow fast slow kinetics r s ?? r < 100 ms r a ?? Single-channel 5-60 1-20 5-30 ; conductance (p8) J Block by TEA + - - Block by 4-AP t + - J Block by DTx - + - Data modified from.Rudy, 1988: TIPS 1996 Receptor and ion channel nomenclature supplement (seventh ed.). 11 Table 2 Biophysical 3nd Pharmacological Properties of Ca -Dependent K Channels Channel type BK“ Sign,ll I ma”), needed 1-10 an 100-400 nM . for activation (at -50 to -70 mV) Voltage-dependence + - Single-channel 100-250 18-50 conductance (pS) Block by TEA + f Block by ChTX/IbTX + ‘ Block by apamin - Data modified from Rudy, 1988: Sah, 1996: TIPS 1996 Receptor and ion channel nomenclature supplement (seventh ed.). 12 currents help stabilize E, near 13,. Voltage-insensitive K’ channels regulated by changes in intracellular adenosine triphosphate (ATP) have also been described in neurons (Ashford et al., 1989: Politi et al., 1989) . These inwardly-rectifying, ATP-sensitive K' channels (K,,,) tend to be open when the concentration of intracellular ATP is low, but tend to close as the concentration (ICso between 15-100 nM) of this purine nucleotide rises. Although the exact function of K," channels in neurons is not known, such channels would contribute a hyperpolarizing current (I,,,,,,) as cellular energy stores are depleted. B. General K' Channel Structure The protein sequences of cloned K’ channels from invertebrate and vertebrate neurons exhibit a remarkable degree of similarity which has been conserved during evolution (Yokoyama et al., 1989) . From analysis of these sequences (see review by Catterall, 1995) , it is believed that K. channel gene products represent a single subunit that consists of amino- (N) and carboxy-terminal (C) segments which reside in the intracellular compartment, and a core region which contains six membrane-spanning segments (51-86) surrounding a pore-forming loop consisting of two short segments (SSl-SSZ) that dip into the membrane. Evidence suggests that the 881-582 segment constitutes the entire or a major part of the channel pore. Mutations in 13 this segment have been shown to modify channel conductance properties (Hartman et al., 1991), ion selectivity and susceptibility to pharmacological block.(MacKinnon and Miller, 1989: MacKinnon and Yellon, 1990). Four separate subunits are believed to come together to form a single, functional homomultimeric or heteromultimeric K' channel. The S4 segment in each subunit contains a number of positively-charged amino acid residues that lie within the transmembrane electrical field. It is believed that these residues function as a sensor for voltage-dependent gating of K. channels. Mutations which neutralize the amino acid charges shift the voltage dependence of channel gating in a predictable manner (Logethetis et al., 1991: Papazian et al., 1991), indicating that the S4 segment represents a - major part of the voltage-sensing apparatus. One theory proposes that the positively-charged amino acid residues move through the membrane in response to changes in transmembrane potential, causing conformational changes of the channel protein which open or close the channel pore (Catterall, 1988). The N-terminal segment is believed to confer a mechanism for inactivation. Mutations of this segment (Zagotta et al., 1990) or removal of the segment by proteolytic enzymes applied to the intracellular side of the channel (Hoshi et al., 1990) disrupts inactivation. Furthermore, application of synthetic peptides containing amino acid sequences found in this segment to the l4 intracellular side of the channel can convert a normally non-inactivating channel current to one that inactivates (Zagotta et al., 1990). Observations such as these have led to the belief that a "ball and chain" mechanism leads to inactivation of K' channels, as has been proposed for Na' channels (Armstrong, 1981). According to this model, an inactivation particle (or "ball”), tethered (by an amino acid "chain") to the channel but free to diffuse in the cytoplasm, moves into the intracellular mouth of the pore and blocks it, resulting in inactivation of the ionic current. Finally, the N- and C-terminal segments each contain many potential sites for phosphorylation by protein kinases (Catterall, 1993), which allow for the regulation of channel behavior under different physiological conditions. II. Overview of Neuronal Cab Channels Like K. channels, voltage-dependent Caz' channels appear to be ubiquitous components of excitable cells, including neurons (See reviews by Carbone and Swandulla, 1989: Scott et al., 1991: Tsien et al., 1995). Cab channels have been studied extensively over the past decade, partly as a result of recognition that they contribute to the regulation of a diverse array of Cab-dependent cellular functions. These functions, such as the regulation of membrane excitability, the regulation of enzymes and ion channels (e.g. , Kc, channels), and the triggering of exocytosis are carefully regulated, in part by Ca” channels mediating Cab influx. 15 Voltage-dependent Caz. channels can be broadly classified on the basis of their voltage range of activation. Those channels which are activated at membrane potentials more negative than ~50 mV are often referred to as "low- threshold" or "low voltage-activated“ (LVA) , while those activated at membrane potentials more positive than -20 mV are often referred to as "high-threshold" or "high voltage- activated" (HVA) . a. Low Voltage-Activated (an) ca” Channels To date, only one type (the "T-type") of LVA Caz. channel has been characterized (Nowycky et a1. , 1985: Fox et al., 1987a, 1987b) . T-type Caz. channels exhibit a low threshold for activation (<-50 mV) and rapid inactivation kinetics (r<50 ms). Also, these channels carry tiny and transient (hence the term T-type channel) unitary Baa. ' currents (slope conductance z 8 p8) and are resistant to organic compounds known to block Caz. channels. T-type Caz. channels are more sensitive to block by Niz' than to Cdz’, whereas the reverse is usually true for HVA Caz. channels. 8. High Voltage-Activated (HVA) Ca" Channels As stated previously, HVA Caz. channels are activated at membrane potentials more positive than -20 mV. All channels in this class tend to be more sensitive to block by Cdz’ than to Niz’. Subtypes of HVA Caz. channels can be distinguished based on differences in their inactivation 16 rates, single-channel conductances, and pharmacology. "L-type" Cab channels (Nowycky et al., 1985: Fox et al., 1987a, 1987b) carry large unitary Bab currents (slope conductance z 25 ps) and produce macroscopic currents which are long-lasting (hence the term L-type channel). The 1,4- dihydropyridines (DHPs) represent a class of synthetic organic compounds used to detect the presence of L-type Cab’ channels. At low concentrations, DHP antagonists block L- type Cab channels but are less effective at blocking other Cab'channels. The peptide toxin o-conotoxin GVIA (o-CgTX GVIA), isolated from the venom of the cone snail Conus geographus, blocks a component of DHP-resistant, HVA Cab channel current present in neurons (Aosaki and Kasai, 1987: Plummer et al., 1989: Randall and Tsien, 1995). This o-CgTX GVIA- sensitive current component is contributed by "N-type" Cab channels ~ so called because they were initially described as being neither T nor L and found only in neurons (Nowycky et al., 1985). N-type Cab channels exhibit slope conductances of intermediate value (12-18 p5) and inactivation kinetics (1:50-500 ms) which vary considerably in different.neurons (see review by Scott et al., 1991). First described in cerebellar Purkinje neurons (hence the term "P-type" channel), P-type channels are largely resistant to DHPs and u-Cng GVIA (Llinas, et al., 1989, 1992a). These channels have conductances between 10 to 20 pS and produce macroscopic currents which exhibit little l7 inactivation. Low concentrations (ICsozz nM) of the peptide toxin 0~agatoxin IVA (o-Aga IVA), isolated from the venom of the funnel web spider Agelenopsis aperta, selectively block P-type channels (Mintz et al., 1992a). o-Aga IVA can be used to reveal a second component of HVA current resistant to DHPs and o-CgTX GVIA (Wheeler et al., 19944 Randall and Tsien, 1995). This component which exhibits slow inactivation (335% inactivation over 100 ms) is blocked by high concentrations of o-Aga IVA (Icwzloo nM) The channels responsible for the current have been designated "Q-type" to distinguish them from P-type channels. A residual or resistant component of Ca” current is not sensitive to DHPs, o-Cng GVIA, or o-Aga IVA (Mintz et al., 1992a, 1992b). Consequently, the channels responsible for the current have been designated "R-type". As with the T-type channels, no selective blocker of R-type channels has been identified. Table 3 summarizes the biophysical and pharmacological properties of the types of voltage-dependent Cab channels found in neurons. III. General hypothalamo-Neurohypophysial Bystea (BN8) Features A. Historical Overview of BN8 Research Two nonapeptide hormones, oxytocin (OT) and vasopressin (VP), are secreted in the posterior pituitary gland from axon terminals of magnocellular neurosecretory cells (MNCs) which have their cell bodies located in the supraoptic 18 ...mo gu=o>omv aconoammsa ensueaocoeo: docsoco so« one nounooom come mane “name .emene can nuances lemma ..«q|uu uuoom none cadences «use + as: an goose . .aoo an goose I :2 8536: :2 case: . + + a t . «>H nm«-a an scene a n r s + u «H>o xenon: an scene. i r u + u . name an sooen _ Ammv oocouosucooM «as «an «we wanna mma on Huccnnouoemceml .aaue. In «.6 no>o «nae .awnec Innate .ne cemVCV .ne omvtc «chances cachetoa oucuomos 30am 30am oucwomoe umou scauo>wuooCH «>2 «>2 «>2 «>m «>m «>4 noeun>euo«a m o a q z a menu «oceano_ maocseno .co usomcoaoauomeuao> uo nowuuoeoum Hc0¢moHoocsucsm one a~0ah>£h04m n flflflfla l9 nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. These neurohormones play important roles in regulating body-fluid homeostasis and reproductive function. The first description of the PVN as a distinct hypothalamic nucleus was provided by Malone (1910) who gave the nucleus the name "nucleus paraventricularis". A year later, the SON was described by Cajal (1911) who called it "nucleus tangentalis". The name "nucleus supraopticus" was not applied until twenty years later (Loo, 1931). An anatomical and functional relationship between the cells of the two hypothalamic nuclei and the posterior pituitary gland was not established until many years after the nuclei were described. Sir Henry Dale (1909) is credited with the earliest description of a posterior pituitary hormone when he reported that extracts from the posterior pituitary gland induced uterine contractions. The discovery that an antidiuretic hormone is also present in the posterior pituitary gland is credited to von den Velden (1913) and Farini (1913). These two clinicians, working simultaneously and independently, found that extracts from the posterior pituitary gland reduced the diuresis in patients with diabetes insipidus. This observation, along with their earlier observations that many patients with diabetes insipidus exhibited signs of pathological pituitary injury, suggested that the posterior gland secreted a hormone which produced an antidiuretic effect. 20 More than a decade later Verney (1926) provided evidence corroborating the findings of von den Velden and Farini. verney noted that a kidney included in a Starling "heart-lung” circuit exhibited an exaggerated flow of dilute urine. If extracts from the posterior pituitary gland were injected into the circuit, urine flow decreased and urine concentration increased. verney concluded that the pituitary gland contributed a factor to the general circulation which was involved in urine regulation at the level of the kidney. Evidence of an anatomical and functional relationship between the two hypothalamic nuclei and the posterior pituitary gland was first provided by Broers in 1933. He showed that selective destruction of the SON or pituitary stalk produced polyuria. He also noted that either lesion resulted in atrophy of both the SON and PVN. The anatomical studies of the Sharrers in 1939 confirmed the findings of Broers, and led to their concept of neurosecretion (Sharrer and Sharrer, 1940) in which they postulated that hypothalamic neurons can act in an endocrine fashion to induce physiological effects in distal target organs. This concept was later validated by Bargmann and Sharrer (1951) who were able to take advantage of improved staining techniques to establish that posterior pituitary hormones were indeed produced in the hypothalamus. A few years later, the purification, characterization and synthesis of OT (du Vigneaud et al., 1954a) and of VP (du Vigneaud et 21 al., 1954b) would define these substances as the principal hormones secreted from the posterior pituitary gland (du Vigneaud, 1956). An enormous growth.in HNS research has since followed this epoch of HNS research, contributing to our considerable and growing knowledge of HNS anatomy and physiology. B. Anatomy of the ENS The cell bedies of MNCs (ls-30 pm in diameter) reside primarily in two pairs of distinct bilateral hypothalamic nuclei - the PVN, which lie adjacent to either side of the dorsal aspect of the third ventricle, and the SON, which cap the lateral aspects of the optic chiasm (Figures 1A and 18). The PVN and SON receive afferent synaptic inputs from diverse sources within the central nervous system (see review by Hatton, 1990). MNCs of the PVN and SON represent sites of integration for afferent signals concerning the status of electrolyte and water balance, and reproductive function. These cells thus represent a final common pathway for output of information following integration of afferent signals. Unmyelinated axonal fibers from MNCs of the PVN and SON form the paraventriculo-hypophysial and supraoptico-hypophysial tracts, respectively. These fiber tracts run through the internal layer of the median eminence, and subsequently form the infundibulum (pituitary stalk). Axonal projections continue through the infundibulum to the posterior pituitary gland (also referred 22 Figure 1. Anatomy of the hypothalamo-neurohypophysial system (ENS). A, shown here (in a drawing of a coronal section of brain) are the relationships of the paraventricular nuclei (PVN) and supraoptic nuclei (SON) to the third ventricle (3V) and optic chiasm (OX). B, magnocellular neurosecretory cells (MNCs) of the PVN and SON project axons through the infundibulum (Inf) to the posterior pituitary (PP) which together with the anatomically-distinct anterior pituitary (AP) comprises the mammalian pituitary gland. 23 Figure 1 PVN MNCs Rostral 6—9 Caudal SON MNC: 0X AP 24 to as the neurohypohysis or neural lobe of the pituitary gland) where they terminate near the basal lamina ~ a thin matrix which lines the perivascular space surrounding fenestrated capillaries of the posterior pituitary. The posterior pituitary consists almost entirely of axon terminals, resident glial cells and a dense network of fenestrated capillaries. Upon entering the posterior pituitary, neurosecretory axons become characterized by numerous focal swellings known as "Herring bodies", which are approximately 20 pm in diameter (Cross et al., 1975). The Herring bodies contain a variety of organelles including lysosomes and mitochondria. The Herring bodies also contain numerous membrane-bound, electron-dense neurosecretory granules with diameters of 150-200 nm (Palay, 1955), each containing packaged neuropeptide. Each neurosecretory axon ultimately gives rise to an estimated 2000 terminals with diameters of 1~12 um (Nordmann, 1977). This high degree of axonal branching provides an anatomical means for signal amplification at the level of the neurohypophysis. The axon terminals contain numerous neurosecretory granules and other subcellular organelles including lysosomes, mitochondria and small, electron-lucent microvesicles with diameters of 40-60 m (Smith, 1970). At least three anatomical characteristics distinguish axon terminals from Herring bodies (Cross at al., 1975). First, Herring bodies are, on the average, larger in diameter than axon terminals. Second, Herring bodies do not 25 contain the electron-lucent microvesicles which are contained within and perhaps define axon terminals. Third, the axon terminals are found immediately adjacent to the perivascular spaces surrounding fenestrated capillaries of the hypophysial vasculature, whereas Herring bodies are further removed from the capillaries. Intimate with the axons and terminals in the posterior pituitary are specialized astrocytic glial cells, known as "pituicytes". These cells make up approximately 25-30% of the posterior pituitary volume (Nordmann, 1977), and it has been suggested that they play an active role in modulating neurosecretion. Studies have shown that pituicytes can interact dynamically with other posterior pituitary elements (Tweedle and Hatton, 1980: 1982: 1987). Under basal conditions, these cells interpose their processes between neuronal elements of the posterior pituitary and between the axon terminals and basal lamina membrane lining the perivascular space. During conditions of increased hormone demand, the pituicytes can alter their morphology, allowing increased contact between neuronal elements and between axon terminals and basal lamina. Observations such as these have led to speculation that pituicytes participate in neurosecretion by modulating inter-neuronal communication and the degree of access that axon terminals have to the hypophysial vasculature. Other ways pituicytes may affect neurohormone release are by modulating the extracellular ionic milieu surrounding axons and terminals (Wu and Barish, 26 1994), and by releasing neuroactive substances which can act on neuronal elements to modulate neurosecretion (Tweedle and Hatton, 1982). The posterior pituitary is perfused by a dense network of fenestrated capillaries originating from the inferior hypophysial artery. Neurosecretory endings within the posterior pituitary terminate onto the basal lamina, separated from the fenestrated capillaries by perivascular space. This anatomical arrangement renders the neuronal elements and glial cells of the posterior pituitary outside the blood-brain barrier. Neurohormones secreted from axon terminals into the extracellular space adjacent to the fenestrated capillaries can freely enter the general circulation and be transported to peripheral target tissues. Also, blood-borne hormones and substances of peripheral origin can freely diffuse across the vasculature and act at receptors located on axon terminals or pituicytes. C. Physiology of the ENS As peptide-secreting neurons, MNCs synthesize and package their neurohormones in the cell bodies (sonata) within the hypothalamus where the appropriate protein synthetic machinery exists (see review by Richter and Ivell, 1985). Expression of hormone genes in the cell nucleus generates messenger ribonucleic acid (mRNA) transcripts, which are subsequently translated at the rough endoplasmic reticulum in the cell body to produce a large preprohormone. 27 The signal peptide sequence is removed while the preprohormone is still attached to the ribosome, to yield a prohormone containing the hormone peptide sequence. The prohormone and enzymes necessary for further processing are then packaged into neurosecretory granules within the golgi apparatus, and the granules are shipped via fast axonal transport to the posterior pituitary where they await an appropriate signal for release from the axon terminals. Evidence suggests that processing of prohormone into hormone destined for the posterior pituitary occurs within the neurosecretory granule during axonal transport (Gainer et al., 1977a, 1977b). A consequence of hormone processing during transport is that the time required to synthesize hormone and have it available for release in the neural lobe can be quite short. In fact, one study has suggested that this entire sequence of events can occur in less than 2 hours (Jones and Pickering, 1970). Neurosecretory granules which are not released following arrival at the axon terminals, are shipped back to the Herring bodies where they can be stored and later recalled during periods of increased hormone demand (Heap et al., 1975). In addition to hormone, a hormone-specific peptide known as neurophysin (NP) is also generated by enzymatic cleavage of the VP- or OT-prohormone molecule within the neurosecretory granule during axonal transport (Gainer et al., 1977). The VP-associated neurophysin and OT-associated neurophysin have homologous structures which differ 28 primarily in their N-terminal portions (Pickering and Jones, 1978). Neurophysins bind with low affinity to their associated hormones and are thus believed to serve as "carrier proteins" during the transport of neurohormones down MNC axons. Neurosecretion refers to the release of hormone from axon terminals into the general circulation. A variety of physiological stimuli have been shown to evoke VP and/or 0T release (see review by Hatton, 1990). VP release occurs in response to changes in plasma osmolarity and/or volume. OT release occurs in response to mechanical distension of the vagina and suckling, and by osmotic stimuli._ Release of VP and OT is dependent on the electrical activity of MNCs. Afferent synaptic inputs release chemical neurotransmitters which produce excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) in hypothalamic magnocellular neurons. These inputs are summated at the cell sonata, resulting in the generation and propagation of a Nai~ (Dreifuss et a1. , 1971) and Cali-dependent (Bourque and Renaud, 1985) action potential once a threshold depolarization is achieved. Neurosecretion follows the transmission of an action potential from the cell body in the hypothalamus, down the axon to the nerve terminal where depolarization initiates a series of events resulting in exocytosis of hormone- containing neurosecretory granules. Terminal depolarization results in the opening of voltage-sensitive calcium 29 channels, allowing extracellular calcium to enter the nerve endings where it triggers exocytosis (Douglas and Poisner, 1964a: Douglas and Poisner, 1964b: Nordmann and Dreifuss, 1972). The current and longstanding model of Cab-dependent exocytosis is derived largely from studies of release of non-peptide neurotransmitters (see review by Sfidhof and Jahn, 1991). In this model, intraterminal Ca” interacts with Cab-binding proteins which promote fusion of neurotransmitter-containing synaptic vesicles with the presynaptic membrane, resulting in release of transmitter into the synaptic cleft. Empty synaptic vesicles are then recycled by endocytosis and replenished with neurotransmitter at the level of the nerve terminal. Release of non-peptide neurotransmitters is generally localized to presynaptic membrane specializations called active zones. These distinct presynaptic areas are characterized by clusters of synaptic vesicles along the intracellular side of the membrane, an intricate cytoskeletal network which is involved in vesicle trafficking, and a high density of intramembranous particles. The existence of’microdomains of high [Cab]i (100 uM or more) in the area of active zones (Llinas, et al., 1992b) has led to speculation that the intramembranous particles are Cab channels. Because of this arrangement, release of non-peptides is rapid and tightly-coupled to pathways for Cap influx. The identity of the cab-binding 3O protein(s) involved in triggering exocytosis is not yet known, although synaptotagmin, a protein localized specifically to synaptic vesicle membranes and shown to interact with several different proteins localized.to the presynaptic membrane, remains a strong candidate (Matthew et al., 1981: Bennett et al., 1992: Brose et al., 1992: Leveque et al., 1992). Several lines of evidence suggest that peptide release from neurons is mediated by biochemical mechanisms somewhat different from those mediating non-peptide release. First, release of neuropeptides is not confined to a discernible active zone, but can instead occur along all regions of the nerve terminal (Zhu et al., 1986). Second, higher-frequency stimulation is generally required for efficient release of neuropeptides (Dutton and Dyball, 1979: Morris et al., 1995). This is likely because neuropeptide release is not confined to active zones where microdomains of high [Cab]i occur following stimulation. Global elevations in [Cab]i necessary to trigger efficient release of neuropeptides would be more likely to occur during the massive Cab entry that occurs only during high-frequency firing of action potentials. Third, vesicles retrieved following exocytosis of neuropeptides must be shipped back to the cell some for recycling, since the terminal lacks the appropriate protein synthetic machinery necessary to produce new neuropeptide. This may indiCate that the proteins responsible for recycling of vesicular membrane in peptidergic neurons may 31 differ from those in non-peptidergic neurons. Considerable evidence suggests that exocytotic release of hormone from MNCs is not restricted to axon terminals in the posterior pituitary. Electron microscopy work has demonstrated that MNC dendrites in both the SON and PVN contain numerous neurosecretory granules containing packaged neuropeptide (Pow and Merris, 1989). Moreover, it was estimated that these dendrites contain 70-80% of all the VP and OT in the hypothalamus. These initial observations led to speculation that secretory material can also be released from MNC dendrites within the confines of the hypothalamic nuclei. This possibility was supported by electron microscopy which provided images of neurosecretory granules being exocytosed from MNC dendrites (Pow and Morris, 1989). Although the purpose of dendritic release of secretory material from MNCs is not clearly understood, one possibility is that hormone released within the hypothalamic nuclei by MNC dendrites acts locally as a modulator of MNC electrical activity and thus of hormone release from these cells. Support for this hypothesis has come from a number of observations. 0T (but not VP) administered via the third ventricle to anaesthetized rats suckling their litters selectively facilitates burst firing of action potentials in OT neurons and increases the frequency of milk ejections (Freund- Mercier and Richard, 1984). Furthermore, OT release within the SON increases during suckling, as demonstrated in vivo 32 using microdialysis techniques (MOos et al., 1989). These results suggest that a facilitatory influence of dendritically-released OT on OT release from the posterior pituitary is a critical component of the milk ejection reflex. The exact mechanism by which OT facilitates its own release is not known. To date, the presence of OT receptors in the SON or PVN is not clearly established, although a moderate expression of OT receptor mRNA in both nuclei has been demonstrated (Yoshimura et al., 1993). Nevertheless, there is evidence indicating that OT can act directly on MNCs in an autocrine and/or paracrine manner. Recordings from MNCs in slices of hypothalamic tissue have revealed that application of OT selectively increases the firing rate of putative OT neurons, even when synaptic transmission is blocked (Inenaga and Yamashita, 1986). Also, application of OT to isolated supraoptic MNCs produces a sharp increase in [Cay]i which can be blocked by a specific OT receptor antagonist (Lambert et al., 1994). This is consistent with an action mediated through OT receptors which, in other tissues, are guanine nucleotide-binding protein (G Protein)- coupled receptors which act via phosphotidylinositol hydrolysis and mobilization of Cab from intracellular stores (Morel et al., 1992). Although no association between OT effects on MNC [Cab]i and on electrical activity has been established, these experimental observations support the possibility that OT selectively facilitates its 33 own release via a direct action on OT neurons. Like OT, VP released from.MNC dendrites in the SON and PVN may act locally on MNCs to modulate their electrical activity and the amount of hormone released from these cells. VP is released in the SON and selectively facilitates its own local release, as demonstrated in vivo using microdialysis techniques (Wotjak et al., 1994). Also, application of VP'selectively increases the firing rate of putative VP neurons in slices of hypothalamic tissue via activation of V1-type VP receptors located on 141le (Inenaga and Yamashita, 1986). To date, two subtypes of'v, receptors (Va and V“) have been characterized (Jard et al., 1987). Both are G protein-coupled receptors which act via phosphotidylinositol hydrolysis and mobilization of Cab' from intracellular stores (Morel et al., 1992). In situ hybridization studies have revealed that only‘Vn mRNA transcripts are found in the hypothalamus (Ostrowski et al., 1994). In isolated supraoptic MNCs, VP has been shown to increase [Cab]i and these effects were blocked by a selective‘Vh receptor antagonist (Dayanithi et al., 1996). These results suggest that supraoptic MNCs express functional V“ receptors which contribute to a selective autoregulation of VP neurons by dendritically-released VP. Extracellular recordings of electrical activity in vivo have provided considerable information regarding MNC firing patterns under physiological conditions (reviewed by Poulain and Wakerly, 1982). Under basal conditions, both VP and OT 34 rat MNCs are characterized electrophysiologically by slow (<3 Hz), irregular firing patterns. However, in response to physiological stimuli that increase hormone secretion, each type of MNC develops specific firing patterns in which action potentials are fired in bursts. VP neurons exhibit "phasic" patterns of action potential discharge, consisting of bursts of higher-frequency (7-12 Hz) discharge separated by periods of relative inactivity or silence. OT neurons, on the other hand, exhibit firing patterns consisting of bursts of high-frequency (up to 50 Hz) discharge occurring approximately 10 seconds prior to the rise in intramammary pressure which precedes milk ejection in lactating female animals. The OT bursts may be superimposed on a background of "slow irregular" (<3 Hz) or "fast continuous" (3-15 Hz) firing activity. These electrical characteristics are critical to the neurosecretory process. It has been shown that the pattern of electrical activity exhibited by MNCs is an important determinant of intraterminal free calcium concentration (Cazalis et a1. 1985: Jackson et al., 1991: Stuenkel, 1994) and thus the amount of hormone released from the posterior pituitary (Dutton and Dyball, 1979: Bicknell and Leng, 1981: Bicknell et al., 1982: Cazalis et al., 1985). 35 0. Mechanisms Underlying the Electrical Behavior of Supraoptic HNCs The two most important factors controlling MNC electrical behavior and hormone release are extrinsic synaptic inputs and intrinsic membrane properties. Extrinsic control mechanisms are represented by afferent synaptic inputs which regulate the excitability of MNCs. Intrinsic control mechanisms, on the other hand, are represented by the ionic conductances which underlie individual action potentials and other more complex phenomena such as burst firing. My research efforts are directed at the intrinsic electrical properties of supraoptic MNCs. Accordingly, the following review will focus largely on that which is known about these cells. For information regarding extrinsic and intrinsic mechanisms which underlie the electrical behavior of paraventricular MNCs, the reader should consult other sources (see reviews by: Hatton, 1990: Renaud and Bourque, 1991). 1. Preparations Used to Study Mechanisms lhich Underlie the Electrical Behavior of Supraoptic MNCs Several in vitro preparations are typically used to study extrinsic and intrinsic mechanisms underlying the electrical behavior of supraoptic MNCs. These preparations include: HNS explants, brain slices, cultured MNCs, and acutely dissociated MNCs. Each preparation offers certain advantages and disadvantages for studying MNC function. 36 HNS explants are used to study the properties of physically intact supraoptic MNCs (Bourque and Renaud, 1983). Explants are prepared without the use of enzyme treatment and permit the use of high-resolution intracellular recording techniques on MNCs in situ with their structure and most of their synaptic contacts preserved. An advantage of the explant preparation is that the preservation of the in vivo structure and synaptic contacts permits the study of extrinsic modulation of MNC activity. Also, the neurohypophysis can be retained with the explant, allowing correlative studies on MNC electrical behavior and hormone secretion (e.g., Renaud, 1987). A major problem associated with the use of explants, however, is that the activity of glial cells, presynaptic terminals and other endogenous tissue elements can potentially modulate or mask specific intrinsic neuronal events under study. Explants also do not permit the use of conventional patch-clamp techniques to study specific ionic conductances. This is largely because cells in explants do not exhibit the "clean“ membrane surfaces necessary to obtain the kind of high-resistance seal between the patch-clamp pipet and the cell membrane required fer patch-clamp recording. Voltage- clamp recordings of cells in explants can be made using intracellular electrodes (Bourque, 1988). However, because MNCs in explants are intact, dendritic processes may contribute to spatial distortions of membrane potential ("space-clam " errors) when attempting to clamp the membrane 37 voltage. Thus, although it may represent a more physiologically relevant system, each of the potential problems inherent with explant preparations must be considered when interpreting data obtained from cells in explants. Intracellular recording techniques can also be applied to MNCs in situ in slices of hypothalamic tissue containing SON (Hatton et al., 1978). Additionally, slice preparations offer an advantage over explants in that they permit the use of patch-clamp techniques to record specific ionic conductances from MNCs within the slices (Nagatomo et al., 1995). This is because a relatively simple, mechanical procedure can be applied to "clean" the extracellular membrane surfaces of exposed neurons in thin slices such that a high-resistance seal between a patch electrode and a cell membrane can be easily obtained (Edwards et al., 1989). As with explants, many of the synaptic contacts are preserved within slice preparations, allowing the study of excitatory and inhibitory postsynaptic currents in MNCs. Problems associated with the use of slice preparations are similar to some of those discussed for explants. Because much of the in vivo structure is preserved within the slice, the activity of glial cells, presynaptic terminals and other endogenous tissue elements can potentially modulate or mask specific intrinsic neuronal events under study. Also, intact dendritic processes may contribute to space-clamp problems in voltage-clamp experiments. 38 To circumvent some of the problems associated with explant and slice preparations, some investigators have used MNCs cultured from the area of the SON (Cobbett and Mason, 1987: Cobbett et al., 1989). These neurons have membrane surfaces suitable for patch-clamp recording and are generally devoid of synaptic contacts. However, because the neurons in these cultures are harvested from prenatal or early postnatal animals, the ionic channels in these cells may differ in terms of total complement or behavior from that of MNCs of adult animals. V Recently, methods have been developed to dissociate MNCs of the adult SON using a combination of enzymatic and mechanical procedures (Cobbett and Weiss, 1990: Oliet and Bourque, 1992). These neurons are free of synaptic contacts and glial investments, and exhibit clean membranes that are suitable for patch-clamp recording. Also, only the most proximal dendritic structure is preserved, thus making space-clamp problems less of a concern when conducting voltage-clamp experiments. Available data suggests that the electrical characteristics of these neurons are largely unimpaired by the dissociation procedure. Nevertheless, when interpreting data from acutely dissociated neurons, one must be aware of the possibility that proteolytic digestion by enzymes used in the dissociation procedure can potentially alter the behavior of membrane proteins such as ion channels. 39 z. Extrinsic Mechanisms Underlying the Electrical Behavior of Supraoptic MHCs VP and OT release from supraoptic MNCs can be modulated at different levels within the HNS. Within the hypothalamus, afferent synaptic inputs to the SON release chemical neurotransmitters which can act at the dendrites and some of MNCs to modulate their electrical behavior and thus the amount of hormone released. Neurotransmitters released within the SON may also act at receptors located on terminals of other afferent inputs to the SON and on resident glial cells, thus regulating their participation in the control of hormone release (see review by Hatton, 1990). Although the primary level of control for hormone release from supraoptic MNCs is within the SON, there is considerable evidence that hormone release can also be modulated by neurotransmitters or circulating hormones at the level of the neurohypophysis. These substances may also act at receptors located on axon terminals or pituicytes within the neurohypophysis to modulate hormone release from MNCs. For a comprehensive review of neurotransmitters and hormones implicated in the modulation of OT and VP release at the level of the neurohypophysis, the reader is referred to Falke (1991). The remainder of this section will address specifically the extrinsic control mechanisms which underlie stimulus-secretion coupling at the level of the SON. Supraoptic MNCs receive diverse synaptic inputs from select brainstem and forebrain structures. The MNCs thus 40 represent sites of integration for afferent inputs which help control VP and OT release. Evidence suggests that glutamate and y-aminobutyric acid (GABA) represent the dominant neurotransmitters released in the SON to regulate MNC activity; The organum vasculosum lamina terminalis (OVLT) appears to provide glutamatergic input to the SON (Renaud et al., 1993). Electrophysiology studies have shown that glutamate is an excitatory neurotransmitter mediating fast EPSPs in supraoptic MNCs (Van Den Pol et al., 1990: Oliet and Bourque, 1992). The prominent source of GABAergic input to the SON has not yet been defined. However, electrophysiology studies have shown that GABA mediates fast IPSPs in supraoptic MNCs (Randle et al., 1986: Randle and Renaud, 1987: Oliet and Bourque, 1992). Many other substances are also thought to provide regulatory influences on the activity of supraoptic MNCs at the level of the SON. Studies have demonstrated that a variety of neurotransmitters are packaged within terminals located presynaptic to MNCs of the SON. The caudal ventrolateral and dorsomedial medulla have been shown to project norepinephrine-containing inputs (from the A1 and A2 cell groups respectively) to the SON (Sawchenko and Swanson, 1981, 1982). Neuropeptide Y appears to be co-localized in norepinephrine-containing inputs from the caudal ventrolateral medulla (Harfstrand et al., 1987). Inputs from dorsomedial medulla neurons also contain the peptide neurotransmitter inhibin (Sawchenko et al., 1988). The SON 41 receives innervation from 5-HT-containing fibers originating from the B7, 88 and B9 cell groups in the midbrain (Sawchenko et al., 1983) and from angiotensin II-containing fibers originating from the subfornical organ (Jhamandas et al., 1989). Although their exact sources have not yet been defined, other neurotransmitters which may act at the level of the SON to control MNC activity include acetylcholine (Mason et al., 1983), histamine (Panula, 1986), dopamine (Buijs et al., 1984), enkephalin (Martin and Voigt, 1981), cholycystokinin (Vanderhaegen et al., 1981), substance P (Shults et al., 1984) and atrial natriuretic peptide (Standaert et al., 1987). Thus, in addition to glutamate and GABA, a variety of other neurotransmitters are believed to influence the activity of supraoptic MNCs. Available data on these neurotransmitters indicate that they do not mediate fast synaptic potentials as has been described for glutamate and GABA. It therefore seems likely that these substances mediate slow synaptic events and thus function as neuromodulators of MNC activity. For a review of the specific effects of these neurotransmitters on the activity of supraoptic MNCs, the reader is referred to Renaud and Bourque (1991). 3. Intrinsic Mechanisms Underlying the Electrical Behavior of Supraoptic MNCs Intracellular recordings of membrane potential in in vitro preparations (particularly brain slices and HNS 42 explants) have provided a general picture concerning the electrical properties of supraoptic MNCs. For example, somatically-recorded MNC action potentials are known to consist of TTX-sensitive, Nai~dependent (Andrew and Dudek, 1984) and Cab-dependent components (Bourque and Renaud, 1985). The Cab component is represented by a distinct 'shoulder' on the repolarization phase of individual action potentials which contributes to the quite long (up to 5 ms) duration of MNC action potentials. Removal of K? from the intracellular and extracellular compartments dramatically prolongs (up to 100 fold) the duration of somatically- generated MNC action potentials (Bourque et al., 1985), suggesting that outward Ki currents underlie action potential repolarization as in other neurons (see review by Rudy, 1988). Individual MNC action potentials are immediately followed by a hyperpolarizing after-potential (HAP) that Bourque et a1. (1985) has suggested may result from the activation of a transient Cab-dependent K’ current (Ito) . HAPs can be temporally summated following repetitive spikes and may play a role in controlling the firing pattern of MNCs by setting an upper limit on the maximal frequency of firing which can be achieved during burst activity. HAPs may also facilitate the removal of channel current inactivation which occurs during the time course of the action potential. Following the post spike HAP, a slow depolarizing 43 after-potential (DAP) is observed (Andrew and Dudek, 1983). Temporal summation of DAPs following repetitive spikes is involved in the generation of a small (<10 mV) sustained depolarization ("plateau potential") which appears to provide the basis for the intrinsic generation of burst activity. Current-clamp studies (Bourque, 1986) suggest that the DAP results from the spike-induced activation of a voltage-gated Caz. current (In) . Andrew and Dudek (1984) have shown that burst firing of spikes in MNCs is followed by a prominent, frequency- dependent AHP, which is believed to result from the activation of a slow Cab-dependent Ki current (I‘m) . Current-clamp studies (Bourque and Brown, 1987: Kirkpatrick and Bourque, 1996) have suggested that law is contributed by apamin-sensitive, Cay-dependent K’ channels. Functionally, the AHP acts to regulate intraburst firing frequency and perhaps also firing patterns since it can help negate the late DAP. Burst firing of spikes in MNCs also results in frequency-dependent spike broadening (Andrew and Dudek, 1985: Bourque and Renaud, 1985) which is represented by a progressive increase in spike duration with each successive action potential at the onset of a burst. A similar phenomenon is also observed in MNC axon terminals (Gainer et al., 1986: Jackson et al., 1991) and it may contribute to facilitation of hormone release by enhancing Cab' accumulation within the terminal (Jackson et al., 1991: Stuenkel, 1994). The exact events which underlie 44 frequency-dependent spike broadening in MNCs are not clearly understood, although a reduced voltage-activated K. current and/or an increased voltage-activated Cab current following repetitive stimulation may be involved (O'Regan and Cobbett, 1993). Clearly, data from intracellular recordings of membrane potential in slice and explant preparations have provided valuable insights into the intrinsic electrophysiology of supraoptic MNCs. A necessary prerequisite to understanding the mechanisms which underlie membrane potential phenomena, however, is a thorough characterization of the specific ionic conductances present in the MNC membrane. These specific ionic events are not easily revealed using intracellular recording techniques. The application of patch-clamp techniques on neurons in slice preparations and on neurons isolated from the SON has facilitated the study of specific ionic conductances which underlie membrane potential phenomena. Using whole-cell voltage-clamp techniques on MNCs acutely isolated from adult rat SON, Fisher and Bourque (1995a, 1995b) were able to evoke inward macroscopic Cab' current that exhibited both inactivating and non- inactivating components during prolonged membrane depolarizations from a holding potential (V,) of ~80 mV. Contributions to the inward macroscopic Cab current from at least four (possibly five) types of voltage-activated Cab' channel currents were distinguished based on their 45 thresholds for activation, rates of inactivation and their sensitivities to a series of calcium channel blockers. A portion of the inactivating component exhibited a low activation threshold (>~60 mV) and rapid inactivation kinetics (r = 42 ms at -10 mV). This component was more sensitive to block by the divalent cation Nib than to Cd”’ and was insensitive to 0.5 nM o-Cng GVIA, a peptide toxin reported to produce a preferential block of inactivating N- type Cab channels in other neurons (Aosaki and Kasai, 1987: Plummer et al., 1989: Randall and Tsien, 1995). Based on these results, the low-threshold, rapidly-inactivating Cab' current component in MNC somata was identified as being carried by T-type Cab channels which have been described in other neurons (Fox et al., 1987). A high-threshold, inactivating component of Cab’ channel current was also reported. This component activated at membrane potentials more positive than -30 mV and exhibited slow inactivation kinetics (r = 1790 ms at ~10 mV). Application of 0.5 uM 0~Cng GVIA blocked a significant portion of this current component. Together, these results were taken to indicate the presence of an N- type Ca” channel current component. The non-inactivating component of the macroscopic Cab' channel current exhibited a low-activation threshold, activating at membrane potentials more positive than ~60 mV. A portion of this current component was sensitive to 10 pH nifedipine, a DHP compound which produces a preferential 46 block of L-type Ca” channels in other neurons (Fox et al., 1987: Hille, 1992). The activation threshold (>-60 mV) of the nifedipine-sensitive current in MNC somata was low, however, compared to values reported for L-type currents in other cell types (Hille, 1992), suggesting that MNCs express a novel, low-threshold, L-type Ca” channel. Another portion of the non-inactivating current component was not sensitive to block by nifedipine, but was blocked by o-Aga IVA, a peptide toxin reported to block non-inactivating P— type Cab channels (Mintz et al., 1992) at low concentrations (Icmzz nM) and inactivating Q-type Cab channels (Wheeler et al., 1994) at higher concentrations (Icmzloo nM). Since this portion of current was non- inactivating and was blocked by low concentrations of o-Aga IVA (Icmz3 nM), it was identified as being carried by P- type Cab channels. Fisher and Bourque (1995a, 1995b) concluded that supraoptic MNC somata of the adult rat express T-, N-, L-, and P-type Cab channels. They also noted the presence of a Ca” current component with an intermediate activation threshold (>~50 mV) and rate of inactivation (r = 187 ms at ~10 mV). This current component was unaffected by the organic Cab channel blockers used in their experiments, and thus did not correspond to any identified ca” channel type. The components of somatic K' current in adult supraoptic MNCs have not been thoroughly characterized. To date, voltage-clamp recordings of voltage-activated outward 47 K. current have been reported in three published studies (Bourque, 1988: O'Regan and Cobbett, 1993: Nagatomo et al., 1995) . Using intracellular voltage-clamp techniques on supraoptic MNC somata in explants of adult rat hypothalamus, Bourque (1988) recorded a transient (inactivating) outward K+ current (Iroc) evoked by depolarizations from a holding potential of ~100 mV. This current was significantly reduced by 4~AP (1 mM) or DTX (4 nM) , compounds which have both been shown to produce a preferential block of inactivating A-type KO channel currents in other neurons (see review by Rudy, 1988) . Also, it was reported in the study that Imc was dependent on the presence of extracellular Caz’, implying that Caz. influx into MNCs strongly modulates the gating of TOC channels. This” conclusion was in part based on the observation that I,“ was reduced by up to 90% by addition of the Caz' channel blocker Cdzi (50-400 pH) to the extracellular medium. Also, I,“ was nearly abolished by removal of Caz. from the extracellular medium. The author concluded that MNCs of the adult rat SON display a transient "A-like“ K' current which, although similar pharmacologically to A-type K. currents reported in other neurons, is novel in that it is dependent on Caz. influx (see Tables 1 and 2). O'Regan and Cobbett (1993) reported that whole-cell voltage-clamp recordings from Mlle dissociated from the SON of the adult rat revealed inactivating and non-inactivating components of somatic outward Ki current. However, although 48 the non-inactivating component was observed in all dissociated MNCs studied, the inactivating component was observed in only about one-half of the cells. No explanation was offered in the study as to why some MNCs express only non-inactivating K’ current, nor was there any attempt to characterize the kinetics or pharmacology of the two components of K. current. Whole-cell voltage-clamp recordings from supraoptic MNCs in slices containing hypothalamus have also revealed inactivating and non-inactivating components of somatic outward Ki current (Nagatomo et a1. , 1995) . The inactivating component of current evoked during membrane depolarizations from a holding potential of ~80 mV exhibited a low threshold for activation (~60 mV) and rapid inactivation kinetics which were best fit by a single exponential (r = 9.5 ms at +40 mV). Also, this current was nearly abolished by 5 mM 4-AP. These results are typical of A-type K' channel currents (Ian) reported in other neurons (see review by Rudy, 1988). The authors also reported that I; in supraoptic MNCs was reduced by 10 pH angiotensin II. No attempt was made to characterize the non-inactivating component of outward current. OBJECTIVES Secretion of OT and VP is directly controlled by the electrical activity of the hormone-containing neurons. Accordingly, one of the fundamental processes underlying neurosecretion is the relationship between electrical activity and hormone release in neurosecretory neurons. My research efforts were directed at the specific ionic events underlying the electrical activity in supraoptic MNCs. Specifically, my aim was to characterize the components of somatic outward K' current in MNCs and examine their involvement in the intrinsic regulation of MNC electrical behavior. The components of somatic K' current in adult supraoptic MNCs have not been characterized thoroughly. The preparation I used consisted of MNCs acutely dissociated from the adult guinea pig SON using a combination of enzymatic and mechanical procedures (Cobbett and Weiss, 1990: Oliet and Bourque, 1992). Recordings of somatic KO current were made using the tight-seal, whole-cell recording technique described by Hamill et a1. (1981). In addition, I used a variation of this technique to record membrane potential and examine the involvement of identified K' current components in membrane potential phenomena. This 49 50 work will add to our understanding of the intrinsic mechanisms which underlie the electrical behavior of supraoptic MNCs. NATERIALS AND METHODS I. Preparation of Dissociated Supraoptic MNCs Dissociated supraoptic MNCs were prepared according to a modification of the method described by Cobbett and weiss (1990). Adult guinea-pigs (male 250-300 g, obtained from the Michigan Department of Health, Lansing, MI, USA) were decapitated. The brain was rapidly removed and a tissue block containing hypothalamus was prepared. Using a Vibratome, coronal slices 800 um thick were cut from the tissue block while submersed in cold incubation medium containing (mM): NaCl 120: KCl 5: CaCl2 1: MgCl2 1: d- glucose 25: and PIPES 20 (pH 7.3). From the slices, SON- containing tissue explants were dissected out and placed in 10 ml oxygenated incubation medium (32‘C) supplemented with protease (3 mg/ml, Type I, Sigma) for 60 min. Explants were then rinsed three times with and maintained at room temperature in protease-free incubation medium until required. To dissociate MNCs, an explant was removed from the incubation medium and placed in 1 ml dissociation medium ‘which.contained (mM): NaCl 130: KCl 5: CaClz.2: MgCl2 1: d- glucose 10: and HEPES 20 (pH 7.3). The tissue was then triturated using a sequence of fire polished pasteur pipettes of decreasing bore size (0.5-0.2 mm inside 51 52 diameter). The resulting cell suspension was then plated onto 35 mm polystyrene culture dishes for immediate use in experiments. All experiments were conducted at room temperature (22-25'C). II. Immunocytochemistry and Morphological Examination of Dissociated MNCs Plated cells were fixed for 30 min with modified Bouin's fixative (4% w/v paraformaldehyde, 0.2% v/v picric acid, 0.1 M phosphate buffer, pH 7.4). The fixed cells were then subjected to three 5~min rinses in phosphate-buffered saline (PBS: pH 7.4). SON neurons were stained at room temperature according to procedures described for the VECTASTAIN‘ Elite ABC Kit system (Vector Laboratories, Burlingame CA). The primary antibody used for the procedure was directed against rat neurophysins (1:10,000: A8948: Chemicon, Temecula, CA). Detection of immunolabelled neurons was accomplished using 3,3'~diaminobenzidine (DAB) provided in a DAB Substrate Kit for Peroxidase (Vector Laboratories, Burlingame CA). Figure 2 shows several dissociated neurons viewed using phase contrast and normal light optics. Note that only the larger neurons were decorated by neurophysin antibody. To determine if size of isolated guinea pig SON neurons could be used to identify MNCs, the procedure for morphometric analysis of neurons described by Oliet and Bourque (1992) was followed. Briefly, the cross-sectional 53 Figure 2. Photomicrographs of dissociated supraoptic cells. A, phase contrast photomicrograph of a single cell (arrow) obtained after dissociation of the SON of a guinea pig. B, shows the same cell (arrow) as in A viewed under transmitted light following immunostaining for NPs. C, phase contrast photomicrograph of smaller cells (arrows) obtained in the same preparation used to obtain the cell shown in A and B. D, shows the same cells (arrows) as in C viewed under transmitted light following immunostaining for NPs. Note the absence of NP staining in these smaller cells. Bar represents 50 um. 54 Figure 2 55 area (CSA) of isolated neurons was estimated as that of a uniform oval using the following equation: csa =-- 12,12, eqn (1) where Rs and RL are the short and long radius, respectively. Immunostaining and CSA.of the neurons were then examined to determine if there was any correlation between the two parameters. Similar to the findings of Oliet and Bourque (1992), 95% (105 of 111 cells) of the neurons which exhibited a CSA 2 160 and stained positive for neurophysin (Figure 3). III. Voltage-Clamp Recording from MNCs Patch electrodes were double pulled and then fire polished to resistances of 1-5 Mn. For recording K. currents, the dissociation medium supplemented with tetrodotoxin (TTX: 2.5 nM) was used as the extracellular solution in most experiments (see Results and Figure Legends for changes in medium composition in specific experiments). Electrodes were filled with a solution consisting of (mM): KCl 135: MgCl2 1: d-glucose 10: EGTA 0.5: ATP 2: cAMP 0.2: and HEPES 10 (pH 7.3). The electrode solution for recording inward divalent cation current consisted of (mM): CsCl 135: MgClZ 1: d-glucose 10: EGTA 5: ATP 2: cAMP 0.2: and HEPES 10 (pH 7.3). After formation of a tight seal between the electrode and cell membrane, the electrode potential was 56 Figure 3. supraoptic MNCs can be identified based on size. This histogram shows the distribution of 279 isolated neurons based on cross-sectional area (CSA) and either positive (dark bars) or negative (light bars) staining by OT and VP neurophysin antibodies. Note that 95% (105 of 111 cells) of the neurons which exhibited a CSA 2 160 umz stained positive for neurophysin. 57 Figure 3 00¢ $83 E ON :60 13S notable observation regarding the effects of TEA was that after maximum spike duration was achieved during the train, spike duration began to decrease, returning towards the value observed for the first action potential. Unlike ChTX or TEA, extracellular 4~AP (1 mM) did prevent frequency- dependent spike broadening (Figure 32). In fact, in most cases, the first spike in the 30 pulse train exhibited the longest duration and subsequent spikes decreased slightly in duration. Figure 33A shows spike duration plotted against spike number before and after the addition of TEA or 4-AP to the extracellular medium. Control and ChTX data shown previously in Figure 30 are included in the graph for reference. Figure 333 shows the data from figure 33A plotted logarithmically. The 4~AP data were well fit by a straight line with negative slope value (x = -0.76). Because the TEA data appeared to exhibit a polynomial relationship over a 30 pulse train, they could not be described by a straight line. The data points over which progressive broadening occurred, however, were well fit with a straight line with a slope of 5.19, indicating that, as in the presence of ChTX, the rate of spike broadening in the presence of TEA was greater than that observed under control conditions. 136 Figure 32. 4~AP prevents frequency-dependent spike broadening. Successive action potentials elicited by injecting brief (3 ms) pulses of suprathreshold depolarizing current (0.4 nA) at 10 Hz before (A) and after (8) addition of 4-AP (1 mM) to the extracellular medium. In the presence of 4-AP, the first action potential of the 10 Hz train exhibited the longest duration and subsequent action potentials exhibited a progressive decrease in duration. 137 Figure 32 mgrv 138 Figure 33. TEA, but not 4~AP, increases the extent and rate of frequency-dependent spike broadening. A, spike durations plotted against spike number after addition of TEA (O: 5 mM) or 4-AP (I; 1 mM) to the extracellular medium. Control and ChTX data shown previously in figure 29 are included in the graph for reference. Note that the extent of spike broadening is greater in the presence of TEA compared to control and that after maximum spike duration was achieved during a train, spike duration began to decrease. In the presence of 4-AP, the first spike in the 30 pulse train usually exhibited the longest duration and subsequent spikes decreased slightly in duration. 8, when the x-axis is converted to a log scale, it is evident that the rate of Spike broadening is greatest in the presence of TEA. Duration (ms) Duration (ms) -5 d O N N150)” a... ON toe-moo 139 Figure 33 P P F 0 5 1O 15 20 25 30 Event Number t eel - , | "up: u- .IHIIIIII ”1| . ‘ ‘ 33:3 :.:.::::.... P 1 10 30 Event Number (log) DISCUBBION I. Components of outward 1? Current in Somata of supraoptic MNCs Several components of outward K' current recorded from somata of MNCs from adult guinea pig SON were identified on the basis of their voltage dependence, kinetics, pharmacology and Ca” dependence. A. Low-Threshold Transient K’ Current (Ian) A low-threshold transient K’ current was evoked from a holding potential of ~90 mV. The current activated at test potentials more positive than -60 mV and was fully inactivated at membrane (holding) potentials more positive than ~40 mV. Also, the transient current was preferentially blocked by 4-AP. This transient current therefore resembles the A-current (Iron) identified in cultured neonatal rat supraoptic neurons (Cobbett et al., 1989), rat supraoptic (Nagatomo et al., 1995) and paraventricular (Li 8 Ferguson, 1996) magnocellular neurons in hypothalamic slice preparations and other excitable cells (Connor and Stevens, 1971; Neher, 1971; Rogawski, 1985). It was previously reported (O'Regan & Cobbett, 1993) that Iran was evident in only about one half of the 140 141 supraoptic neurons acutely dissociated from the adult rat. In contrast, my experiments show that all supraoptic magnocellular neurons acutely dissociated from adult guinea pig exhibit a prominent.lkuo. It is tempting to speculate that this discrepancy is the result of species differences between rats and guinea pigs. However, this speculation is not supported by a recent study (Li & Ferguson, 1996) of MNCs of the rat paraventricular nucleus in which it was reported that a prominent Law was always present. Another possible reason for differences between the data obtained by O'Regan and Cobbett (1993) and the data of the present study is that methods for preparation of the isolated MNCs were different: the compositions of the enzyme-containing media were not the same and SON neurons of rats (but not guinea pigs) were labelled in vivo with Evans Blue. It is also notable that, even when present, Ina) reported by O'Regan and Cobbett (1993) was significantly smaller in amplitude compared to that reported here. Given those conditions, it is possible that an attenuation of lam) (by Evans Blue labelling, for example) during preparation of cells may have generated a subpopulation of MNCs in which Ixm was not detectable. Additionally, recent studies of identified OT and VP supraoptic neurons of the female rat (Stern & Armstrong, 1995: Stern & Armstrong, 1996) propose that CT and VP neurons may differ in terms of the size and/or voltage dependence of La”. Given the potential for cell dissociation techniques to influence the amplitude of 142 recorded currents, it is possible that O'Regan and Cobbett (1993) recorded from MNCs which normally exhibit a greater I‘m. The possibility that subpopulations of MNCs differ in terms of the size and/or voltage dependence of Imu was not systematically investigated in the present study. Interestingly, the A-current identified here was considerably different than the transient outward current (Imc) identified by Bourque (1988) in whole supraoptic neurons of rat hypothalamic explants in terms of activation threshold, voltage-dependence of steady-state inactivation and sensitivity to divalent cations. He reported a value of -74.5 mV as the activation threshold for Iroc and a value of -82.4 mV as the membrane potential at which half of the channels are inactivated. These values are more negative than the values (~50 mV and -62 mV, respectively) obtained from my experiments. Also, Bourque (1988) reported that I,“ is dependent on the presence of extracellular Caz', implying that Caz. influx into MNCs strongly modulates the gating of TOC channels. This conclusion was, in part, based on the observation that I,“ was reduced by up to 90% by extracellular Cdz’ at concentrations (50-400 nM) which apparently did not produce detectable changes in the voltage dependence of current activation or steady-state inactivation. In contrast, my experiments demonstrate that in acutely isolated supraoptic MNCs extracellular Cdz' at concentrations as low as 125 all not only reduced the amplitude of Iran! but also produced large positive shifts 143 in the voltage dependence of both activation and steady— state inactivation of this current. Conversely, removal of Caz. from the extracellular medium (no Cdz‘ present) produced large negative shifts in the voltage dependence of both Ixm activation and steady-state inactivation. Nevertheless, using either Cab-free or Cdb-containing extracellular solution I was still able to evoke a substantial A-current during depolarizing voltage steps. The fact that Iran is activated in the absence of extracellular Caz. suggests that Caz. influx is not an absolute necessity for activation of the current. The present results were more consistent with those reported by Carignani et al. (1991) which suggest that in cultured rat cerebellar granule cells I“,n behavior is dependent on the inventory of external divalent cations rather than on Caz. influx and, perhaps [Caz']i. B. Sustained Outward A' Current A slowly-activating, sustained outward K’ current was co-activated with I‘m during depolarizing voltage steps to potentials more positive than -30 mV from a holding potential of -90 mV. This current did not inactivate during the test pulse, showed little voltage-dependent steady-state inactivation and was preferentially blocked by TEA. Based on these characteristics, the sustained current probably corresponds in part to the delayed rectifier current (Iron) originally described in squid giant axons and later in almost all other excitable cells (see review by Rudy, 1988) . 144 The possibility that more than one channel type contributed to the sustained current was supported by experiments which showed that this current could be reduced by 125 pl! extracellular Cdz', a concentration which blocks Caz. influx but does not affect the voltage dependence of the sustained current. This result was similar to that reported by Cobbett et al. (1989) which showed that the sustained outward current recorded from cultured neurons of rat supraoptic nucleus was reduced by addition of the inorganic Caz’ channel blocker (:02. (1-2 ml!) to Caz.- containing extracellular solution. These observations suggest that in supraoptic MNCs a Cab-sensitive, non- inactivating K' channel (Inca) and a classical Cab- insensitive, delayed rectifier channel both contribute to the sustained current. Li and Ferguson (1996) arrived at a similar conclusion regarding the sustained outward K' current in rat paraventricular MNCs. C. High-Threshold (Cab-Dependent) Transient Outward x’ Current A major finding of my study was a transient outward K' current in acutely isolated MNCs which was altogether different from Raw This current could be activated during depolarizing voltage steps to membrane potentials more positive than -20 mv from a holding potential (-40 mV) at which Iran is fully inactivated. Furthermore, this current was 4~AP-insensitive, TBA- and ChTX-sensitive and appeared 145 to be absolutely dependent on Cab influx. The current was not evident when Cab was absent from the extracellular medium or when Cab influx was blocked. _There have been reports of high-threshold, CaaF’ dependent transient K. currents in other cell types including calf cardiac purkinje fibers (Sigelbaum & Tsien, 1980), rat adrenal chromaffin cells (Neely & Lingle, 1992) and amphibian spinal neurons (Ribera & Spitzer, 1987). However, the Cab-dependent transient KO current described here (recorded under whole-cell conditions) was different from those previously reported. In calf cardiac purkinje fibers (Siegelbaum.& Tsien, 1980) and in rat adrenal chromaffin cells (Neely & Lingle, 1992) the time required to reach peak amplitude of the transient current and the time course of current inactivation appeared to be much longer than the values reported here for MNCs. The time required to reach peak amplitude of the transient current and the time course of current inactivation reported for amphibian spinal neurons (Ribera & Spitzer, 1987) are closer to the values of the high-threshold transient current recorded in this study. However, the transient current in amphibian spinal neurons could be fully activated when extracellular Ca” was replaced with Sr”) The Cadeependent transient K. current reported here was abolished when extracellular Ca” was replaced with Sr”, suggesting an absolute dependence on 2+ Ca influx. Dryer et a1. (1989) reported a TTX-sensitive, 146 Nazactivated transient K. current recorded under whole-cell conditions which closely resembled the Cab-dependent transient K' current reported here in terms of time course of activation and inactivation. It was later determined, however, that their current was likely due to inadequate voltage control to the extent that unclamped Na°-dependent action potentials were present during recording (Dryer, 1991) . Unclamped Na. currents were not a problem during my experiments using isolated MNCs. In my studies, the extracellular solution contained TTX at a concentration (2.5 nM) sufficient to block all Na. current. Therefore the Caz.- dependent transient K' current recorded could not have been the result of unclamped Nan-dependent action potentials. I also considered was the possibility that the Caz.- dependent transient K’ current was the result of an unclamped Cab-dependent action potential. However, no indication of poor voltage‘control was observed during recordings made under conditions for studying Caz’ current. Moreover, the Cab-dependent transient K. current was not observed when extracellular Caz. was replaced with Srz', although Caz. and Srz' currents were of similar magnitude. II. Comparison of A. Current Components in Somata and Axon Terminals of HNCs Although properties of somatic currents have been characterized in a variety of cell types, the properties of voltage-gated ion currents mediating transmitter or hormone 147 release from vertebrate axon terminals have been difficult to characterize. This is largely because electrophysiological studies on these channels are limited by the extremely small size of most individual axon terminals. The axon terminals of VP and OT MNCs, however, are sufficiently large to permit the use of conventional patch-clamp techniques to study their ionic channels and currents. Mbst axon terminals in the rat posterior pituitary have diameters of approximately 2-3 um, but some have diameters of up to 12 um (Nordmann et al., 1987). It is these larger terminals which have been used to conduct patch—clamp experiments aimed at providing a better understanding of the ionic events which underlie neurosecretion. By virtue of their voltage dependence and their ability to be regulated by intracellular second messengers such as Cab} if channels have the potential to serve as important transducers for electrical and chemical signals at the level of the axon terminal. Accordingly, the components of outward K. current present in MNC axon terminals have been examined in several studies. The laboratories which have studied K. current in these terminals, however, have provided conflicting reports regarding the identity of the K' currents present. Whole-cell voltage-clamp experiments on isolated axon terminals ("neurosecretosomes") prepared from rat posterior pituitary have shown that these terminals exhibit both 148 inactivating (Thorn et al. , 1991) and non-inactivating components (Wang et al. , 1992) of outward K. current. The inactivating component of current evoked during membrane depolarizations from a holding potential of -80 mV exhibited a low activation threshold (>-60 mV) and rapid inactivation kinetics (r = 21 ms at +30 mV) . 4~AP blocked the inactivating current in a concentration-dependent manner (Icsoz3 mM), while TEA (100 mM) and ChTX (200 nM), organic compounds reported to block various components of non- inactivating K' current (Hille, 1992) , had no effect on the inactivating current. In contrast to the inactivating K. current (Iroc) in rat MNC somata reported by Bourque (1988) , the inactivating K. current recorded from neurosecretosomes was unaffected by removal of extracellular Caz? or by addition of the Caz. channel blocker Cd” (2 mM) to the Ca”- containing extracellular medium. These results indicate that MNC axon terminals of the rat express a low-threshold, A—type K' channel which is not critically dependent on Caz. influx. The non-inactivating component of outward H. current in rat neurosecretosmes was studied using both single-channel and whole-cell patch-clamp techniques (Wang et a1. , 1992) . This component exhibited a high activation threshold, activating at membrane potentials more positive than -30 mV. Also, the non-inactivating current was determined to be completely dependent on intracellular Caz. concentration and could be abolished by extracellular Cdz‘ (80 nM) . 4~AP (7 149 mM) and dendrotoxin.(100 nM) had no effect on this current, nor did apamin (40-80 nu), a peptide toxin reported to block some.small-conductance CabFactivated channels (Lancaster et al., 1991). The non-inactivating current was reduced by low concentrations of TEA (Icsoz0.5 nM) , but was insensitive to ChTX'(10-100 nu) which.has been.reported to selectively block some large-conductance Cabeactivated channels (Reinhart et al., 1989). Single-channel experiments revealed that MNC axon terminals express a large-conductance (unit conductance = 231 pS in symmetrical 150 mM K') , Caz.- activated K’ channel. Like other reported large-conductance Cap-activated K. channels (termed maxi-K' or BK channels), this channel was blocked by TBA (0.5 mM); but unlike other reported large-conductance Cab—activated K' channels, this channel was insensitive to block by ChTX (100-360 nM). Together, these results suggest that MNC axon terminals in the rat express a novel large-conductance CabFactivated K' channel which contributes to the macroscopic non- inactivating KT current. No evidence of a non-inactivating, Cay-insensitive KV channel was reported in the study. Whole-cell experiments on thin slice preparations of rat posterior pituitary confirmed that the macroscopic K' channel current recorded during sustained depolarization of the axon terminal membrane consisted of multiple components (Bielfeldt et al., 1992). An inactivating component exhibited a low activation threshold (>-60 mV) and rapid inactivation kinetics (r a 22 ms at +50 mV) . While the 150 inactivating current recorded from rat neurosecretosomes (Thorn et al., 1991) was not sensitive to block by TEA, the inactivating current recorded from thin slice preparations of rat posterior pituitary was sensitive to block by relatively low concentrations of TEA (Icsozl mM) . Although it is difficult to account for this discrepancy, one explanation may simply be that K. channels behave differently when studied in slices compared to isolated terminals. The non-inactivating component of K' current recorded from thin slice preparations of neurohypophysial tissue exhibited a low activation threshold (>-30 mV) and little inactivation over the duration (500 ms) of a suprathreshold voltage step. This current component was sensitive to block by dendrotoxin (IC50<20 nM) and was less sensitive to block by TEA (Icso<5 mM) than was the inactivating component. The sensitivity to block by dendrotoxin was atypical, however, since this toxin has been shown to produce a preferential block of inactivating A-type channel currents in most other cell types. The non-inactivating current recorded using single- channel techniques was abolished by removal of extracellular Caz. or by addition of Cdz' (100 M!) to the extracellular medium, suggesting that it was contributed entirely by a non-inactivating, Cab-dependent K' channel. Since the functions of K' channel types might differ between neuronal compartments, it might be particularly informative to compare the properties of the K. currents I 151 recorded from MNC somata to those recorded from the axon terminals of these neurons. It is difficult to make such a comparison, however, because of the conflicting reports described above regarding the identity of K. currents present in axon terminals of the posterior pituitary. Nevertheless, similarities and differences between the complements of K' currents in MNC somata and axon terminals are evident. It should be pointed out first, however, that my experiments were performed using guinea pig supraoptic MNCs. Accordingly, the following discussion regarding compartmental localization of K? channels and currents in MNCs assumes that the inventory of K' channels is the same in guinea pigs and rats. A low-threshold, inactivating K. current is exhibited in MNC somata and in MNC axon terminals. In both neuronal compartments, this current activates between ~60 to —40 mV and exhibits rapid inactivation kinetics. However, whereas the current I recorded from MNC somata appears to be insensitive to extracellular TEA (up to 5 mM), the current recorded from axon terminals may be very insensitive (Thorn et al., 1991) or very sensitive (Bielefeldt et al., 1992) to extracellular TEA. The differences in the reported TEA sensitivities of the inactivating current recorded from axon terminals is not due to species differences, since rats were used in both studies. The differences may, however, be due to differences in the preparation used in the studies. The most striking difference between MNC somata and 152 axon terminals with respect to their complements of K. currents is the apparent lack:of’a.BQ channel current in the latter compartment. Regardless of the preparation used, the non-inactivating current recorded from axon terminals of the posterior pituitary appears to be carried entirely by Kc. channels. In contrast, my experiments suggest that both R9 and K3 channels contribute to the non-inactivating current recorded from somata of MNCs. It has been proposed that the inactivating K. current is largely responsible for action potential repolarization in MNC axon terminals, whereas the non-inactivating, Cab? dependent current acts to uncouple functionally terminal excitability from axonal spikes (Wang et al., 1992: Bielefeldt and Jackson, 1993). Burst firing results in an accumulation of intracellular Ca” in MNC axon terminals (Jackson et al., 1991). This increase in [Cay]i would tend to activate Inn» maximally in axon terminals and produce a long-lasting hyperpolarization. The hyperpolarization may in turn result in a refractory period during which axon terminals are electrically uncoupled from axonal spikes. Support for this hypothesis was provided by Bielefeldt and Jackson (1993) in a study in which they were able to demonstrate that aim:. channel current causes frequency- dependent action potential failures in axon terminals of the posterior pituitary. This may explain, at least partly, why the efficacy of hormone secretion from the posterior pituitary declines when the axon terminals are continuously 153 stimulated at high frequency for >20 5 (Bicknell et a1, 1984; Gainer et al., 1986) The functional roles of somatic . I It current components are discussed below. III. Involvement of Specific 18 Channel Currents in Action Potential Repolarization and Frequency-Dependent Bpike Broadening Both OT and VP supraoptic MNCs exhibit complex electrical behaviors characterized by burst firing of action potentials in certain physiological conditions (see review by Poulain S Wakerley, 1982). Voltage-gated.Naf (Andrew and Dudek, 1984) and Ca” (Bourque and Renaud, 1985) currents both contribute to somatically-generated MNC action potentials. Outward K' currents, are believed ‘to play a prominent role in regulating MNC excitability and in modulating firing patterns (see reviews by Renaud & Bourque, 1991: Legendre & Poulain, 1992). The currentoclamp experiments I performed suggest that the temporal activation of several K. channel types shapes MNC action potential repolarization as in other central neurons (Storm, 1987; Zhang and NcBain, 1995). At least three pharmacologically distinct K' channel currents contributed to action potential repolarization in isolated supraoptic MNCs. The 4-AP- (Iron) and TBA-sensitive (Ixm) currents both contributed substantially to MNC action potential repolarization. However, the two currents contributed differently to the temporal profile of 154 repolarization. 4-AP produced a pronounced broadening during the early phase of spike repolarization. The broadening produced by TEA, on the other hand, was most pronounced during the latter phase of repolarization. Thus Ixm appears to contribute to spike repolarization near the onset of the repolarization phase whereas Law contributes primarily during the latter phase of repolarization. These observations are consistent with my voltage-clamp results which revealed a faster activation of Iumr ChTX also produced spike broadening, suggesting that a Cab-dependent K. channel current (perhaps the high-threshold transient current) also contributes to action potential repolarization in supraoptic MNCs. Like TEA, ChTX produced spike broadening primarily during the latter phase of spike repolarization. The degree of broadening produced by ChTX, however, was not as great as that produced by TEA. This is likely due to smaller size of the ChTX-sensitive current. The fact that ChTX produced spike broadening primarily during the latter phase of spike repolarization is not inconsistent with a role for the fast, high-threshold transient current in spike repolarization. The threshold for activation of this current is higher than that of Ian) and may require the prior activation of a high—threshold Cab channel current during a spike. Inward Cab currents appear to be expressed during the repolarization phase of the MNC action potential, since block of In” or I'm!) produced a Cabrdependent shoulder during this phase. 155 Accordingly, the high-threshold transient current may be expressed predominantly during the latter phase of spike repolarization when [Cab]i is greatest. Figure 34 summarizes the temporal contributions of different ionic currents toward MNC action potential dynamics. Frequency-dependent spike broadening in repetitively- firing neurons can arise via any of several different mechanisms. The mechanism of spike broadening in neurons of the mollusc Archidoris involves a frequency-dependent reduction of a Kw channel current which increases the expression of La as a prominent shoulder on the repolarization phase of the action potential (Aldrich et al., 1979). Frequency-dependent spike broadening can occur by one of two mechanisms in neurons of the mollusc Aplysia californica. In Aplysia bag cell neurons, spike broadening results from a reduction in a slowly-inactivating, TEA- sensitive current carried by Kv channels (Quattrocki et a1. , 1994). However, in R20 neurons from the abdominal ganglion of Aplysia, spike broadening results from a reduction in a rapidly-inactivating, 4~AP-sensitive current carried by K. channels (Ha and Koester, 1995, 1996). Frequency-dependent spike broadening in both Aplysia neuron types was manifested as an increased expression of La as a prominent shoulder on the repolarization phase of the action potential, as in Archidoris neurons (Aldrich et al., 1979). Extracellular recordings in viva (Mason and Leng, 1984) and intracellular recordings in vitro (Andrew and Dudek, 156 Figure 34. Temporal contributions of different ionic currents toward MNC action potential dynamics. (1) Following the onset of a depolarizing stimulus (arrow), the membrane potential (8,.) becomes more positive than the resting membrane potential (8,) . The first current activated is the low-threshold IKm which contributes a hyperpolarizing influence that slows the depolarization. (2) When the membrane becomes sufficiently depolarized, I"a becomes activated and the E" rapidly approaches the equilibrium potential for Na‘ (En) . (3) By the time the action potential reaches its peak, I,“ has begun to inactivate, I“ has begun to activate, and Iran has increased. The strong hyperpolarizing influence of Iron begins to repolarize the E", driving it towards the equilibrium potential for K' (E‘) . The depolarizing influence of I,“ together with an increasing In slows the rate of repolarization. (4) Later in the repolarization phase, Irm and Inc” are sufficiently activated such that, along with Iron! they enhance the repolarizing and drive toward the E‘. (5) Because the E:K is more negative than the ER, the K’ currents become active late in the repolarization phase drive the 8" past the Eu, closer to the E‘. The hyperpolarization immediately following MNC spikes is referred to as the hyperpolarizing after-potential (HAP). 157 Figure 34 (3) (Kw > (I H ) Ne Ca (2) IN. > [Kim (4) (l +l +l K(Ai KN) > | K(Cai) Ca (1 ) 'KUU (5) 1m, +le Hm” 158 1985: Bourque and Renaud, 1985) from supraoptic MNCs have revealed that MNCs exhibit significant action potential broadening following the onset of burst firing. Recordings of action potentials from.isolated neural lobes using optical recording techniques and potentiometric dyes indicate that a similar phenomenon occurs at the level of MNC axon terminals (Gainer et al., 1986). Recent studies using voltage-clamp techniques suggest that a reduction in If current during burst firing may contribute to frequency- dependent spike broadening in MNC somata (O'Regan and Cobbett, 1993) and in MNC axon terminals (Jackson et al., 1991). However, at neither level was the identity of the specific component(s) of K' current responsible for spike broadening in MNCs determined. My experiments suggest that frequency-dependent spike broadening in isolated supraoptic MNCs results from a reduction in Iran during repetitive firing, based on the observation that 4-AP (but not TEA or ChTX) prevents spike broadening. As Inn is reduced during repetitive firing, its contribution to spike repolarization is also reduced and action potentials broaden presumably due to an increased expression of It, as a shoulder on the repolarization phase of the action potential. A similar mechanism has been reported for frequency-dependent spike broadening in R20 neurons from the abdominal ganglion of Aplysia (Ma and Koester, 1995, 1996). Frequency-dependent changes in TEA- (Ifly)) and ChTX- 159 sensitive K' currents (Inca) do not appear to contribute to spike broadening during repetitive firing. Instead, these current components appear to effectively limit the rate and extent of broadening resulting from a frequency-dependent reduction in I‘m. K, and Kc. channel currents contribute to spike repolarization predominantly during the latter phase of repolarization. Accordingly, these currents would be expected to provide a greater contribution to spike repolarization as spike duration increases following the onset of burst firing in MNCs. The increased contribution of these currents to spike repolarization would effectively act to offset the decreased contribution of’Ikuo. Figure 35 summarizes the roles of different ionic currents in determining the dynamics of frequency-dependent spike broadening. The increase in spike duration during repetitive firing may permit Cab channels to stay open longer, resulting in an increased accumulation of intracellular Ca”) Frequency- dependent spike broadening accompanied by an increased accumulation of intracellular Cab has been demonstrated in MNC axon terminals (Jackson et al., 1991). Because of the importance of intracellular Ca” in mediating hormone release, it is possible that the accumulation of intraterminal Cab which accompanies spike broadening underlies the facilitation of hormone release during burst firing in MNCs (Dutton and Dyball, 1979: Bicknell and Leng, 1981). If an analogous accumulation of intracellular Cay 160 Figure 35. Roles of different ionic currents in determining the dynamics of frequency-dependent spike broadening. Following the onset of burst firing, Iron begins to inactivate (due to steady-state inactivation), decreasing the hyperpolarizing drive responsible for spike (action potential) repolarization, and thus permitting an increased expression of Its exhibited as a shoulder on the repolarization phase of the spike. The increased expression of In due to the accumulation of Iran inactivation during burst firing results in a progressive increase in the duration of successive spikes - a phenomenon known as frequency-dependent spike broadening. As spikes increase in duration, Iron and Inc.) are activated more fully. At some point during the burst, the increased repolarizing drive provided by these currents exceeds the depolarizing drive provided by I“, thus preventing further spike broadening. Note that in this model the effect of ICa on spike broadening is two-fold. First, I“ is responsible for the shoulder which underlies the broadened spike. Second, Ita is responsible for the inorease in [Cab]i which leads to an increased activation of In“), thus limiting the rate and extent of spike broadening. 161 Figure 35 Burst Firing 1+ +1 lKiAi lCa Inactivation Activation + + + + Spike Broadening + -- + - (I . IKM lK(C:ai Activation Activation 162 accompanies spike broadening in MNC somata during repetitive firing, it may directly enhance the Cadeependent DAPs in supraoptic MNCs which are recognized to be important in the formation and maintenance of burst firing patterns that promote hormone release from the posterior pituitary (Legendre et al., 1988). Increased Cab influx via voltage-dependent Cab’ channels during repetitive firing may also act indirectly to regulate burst firing. Calbindin-th (calbindin), a potent cytosolic Cab buffer endogenous to supraoptic MNCs, has been shown to play a role in determining intrinsically— generated firing patterns in these cells (Li et al., 1995). The Cab-buffering activity of this protein attenuates transient increases in [Cay]i resulting from Ca? influx through voltage-dependent Caz. channels (Lledo et a1. , 1992: Chard et al., 1993). Li et a1. (1995) showed that in supraoptic MNCs calbindin inhibits the formation of DAPs following individual action potentials and prevents burst firing, presumably by buffering transient increases in [Cab]i. Elevations in [Cab]i resulting from increased Ca?’ influx during broadened action potentials may help overwhelm the Ca” buffering capacity of calbindin and thus unmask the DAPs which promote burst firing. Increased Cab influx during broadened action potentials may also trigger ryanodine receptor-mediated Cafi release from internal stores, which enhances DAPs and promotes burst firing in supraoptic MNCs (Li and Hatton, 1997). 163 Recently, dendrites of supraoptic MNCs have been shown to release hormone by exocytosis into the hypothalamus (Pow and Morris, 1989). It has been hypothesized that hormone released within the SON from MNC dendrites acts locally as a modulator to facilitate MNC electrical activity and thus hormone release from these cells (reviewed in the INTRODUCTION section of this dissertation). Perhaps broadened somatic action potentials facilitate hormone release from MNC dendrites by increasing [Cabji, as has been proposed for hormone release from axon terminals. If this were indeed the case, increased hormone release from MNC dendrites resulting from frequency—dependent spike broadening may represent a positive feedback mechanism which contributes to the facilitation of hormone release from MNC axon terminals observed during high-frequency stimulation (Dutton and Dyball, 1979; Bicknell and Leng, 1981: Bicknell et al., 1982: Cazalis et al., 1985). Iv. Other Possible Functions of E' Channel Currents Other potential functions of the outward K’ currents described in the present study should be considered in light of MNC activity profiles in vivo in rats (see review by Poulain & Wakerley, 1982) and in in vitro preparations of rat hypothalamus (Mason, 1983: Andrew & Dudek, 1984a,b: Bourque & Renaud, 1985: and others) and guinea pig hypothalamus (Erickson et al., 1993). These studies show that firing patterns exhibited by rat MNCs in vitro and 164 guinea pig MNCs in vitro are similar, although there are some minor species differences (such as the range of the duration of bursts of action potentials generated during phasic firing). Much of the following discussion assumes that the electrical behavior of MNCs and the inventory of ion channels in MNCs are essentially the same in both. species. Also, it is clear from.Figure 2 that primarily current conducted by channels present in the somatic membrane of MNCs are reported in my studies. ‘The following discussion considers only the role of these currents, despite the almost certainty that.dendritic channel currents also contribute to the electrical behavior of intact cells. The A-current may modulate the rate of depolarization between two successive action potentials, as is the case in other excitable cells (Hille, 1992). Iran may therefore play an important role in regulating neuronal excitability and determining firing frequency within a burst. The steady-state inactivation characteristics of Iron suggest that the efficacy of a depolarizing stimulus (i.e. the ability to evoke an action potential) will be dependent on the membrane potential immediately prior to and at the time of the stimulus. At ER, Iran is largely inactivated, thus requiring a period of hyperpolarization for the removal of inactivation. Removal of steady-state inactivation by'a hyperpolarization.(such as that provided by an HAP) would allow this transient outward current to counteract a depolarizing stimulus until it is again inactivated. 165 Conversely, if’Ikuo is inactivated or blocked the same stimulus will be unopposed by outward current and thus will generate a greater depolarization (Gustafsson et al., 1982), perhaps sufficient to induce firing. The transient Cab-dependent K. current may also play an important role in removing channel inactivation which occurs during MNC action potentials. Each action potential within a burst is immediately followed by a hyperpolarizing } afterpotential (HAP) which exhibits an amplitude . proportional to the extracellular Ca” concentration and can be abolished by block of Ca” influx (Andrew & Dudek, 1984b: Bourque et al., 1985). Bourque et al. (1985) have suggested that the HAP may result, from the activation of a transient Cab-dependent K' current during an action potential. At least part of the transient Cab-dependent KO current identified here in isolated MNCs is available for activation at relatively depolarized membrane potentials. This current may therefore contribute to the HAP following individual action potentials in supraoptic MNCs. The HAP in turn may facilitate recovery of voltage-dependent ion channels from inactivation. Current-clamp analysis has demonstrated that the slow afterhyperpolarization (AHP) following bursts of action potentials results from the activation of a sustained Cab; dependent K’ conductance (Kirkpatrick & Bourque, 1996) . This conductance has been proposed to modulate firing rate within bursts (Bourque et al., 1985; Kirkpatrick & Bourque, 166 1996). The gradual accumulation of somatic intracellular Ca” during a burst of action potentials, as occurs in MNC nerve terminals (Jackson et al., 1991), may contribute to the gradual activation of this K’ current. 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