ATP MODULATORY ACTIONS ON HIPPOCAMPAL SYNAPTIC TRANSMISSION
YUANJING YANG
A thesis submitted to the Department of Anatomy and Cell Biology in conforrnity with the
requirements for the degree of Master of Science
Queen's University
Kingston, Ontario, Canada
May 30,200 1
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ATP might play a role in the establishment of Long-term potentiation (LTP) in the hippocampus, which is one of the synaptic modifications proposed to underlie the memory process. In this study, we set out to investigate the modulatory effects and mechanisms of action of ATP on synaptic transmission in these synapses. Our observations indicate that there are at least three different effects of ATP on the hippocmpal synaptic transmission, which rnight be rnediated by different purine receptors (purinoceptors). The first effect was observed at a nanomolar ATP concentration and it consisted of a transitory synaptic transmission enhancement. This ATP action rnight be mediated by a P2Y purinoceptor on which 8-cyclopentyltheophyline (CPT) appears to work as an antagonist. The second action was observed at a micromolar concentration and was characterised by inhibition of the synaptic transmission. Such an effect is mediated by a presynaptic P2Y receptor, which is CPT sensitive and appears to be incorporated in the presynaptic terminais right after the induction of LTP. The third effect was observed with a millimolar concentration of ATP and was characterised by inhibition of synaptic transmission. This inhibition appeared to have two different phases, a short-lasting and a long-lasting. The later resembles long-term depression. These observations indicate that ATP actions on hippocampal synapses are complex and that a diverse population of purinoceptors mediate its effects. 11, ACKNOWLEDGMENTS
First I wish to thank Dr. Carlos Barrages-LBpez for this project and his continua1 advice and supervision throughout the research. 1 have appreciated his very patient, invaluable support, and guidance. Sincere thanks for the two years, Carlos.
1 also would like to thank Drs. Stephen C. Pang and R- David Andrew. Thanks for the encouragement, selfless help, salvation, and guidance. Your contribution to my graduate studies was immeasurabIe.
A special thank you goes to my [ab-mates Rosa Espinosa-Luna, Rustum
Karanjia and Xuzhi Li for the question asking-answering support and valued companionship.
Thank-you to the administrative staff and other members of the Department of
Anatomy and Ce11 Biology, and especialIy, Ms. Anita Lister, 1 will always remember your cornfort.
VI11. RESULTS ...... 23-38
A . Field potentids in the straturn pyramidale and rnolec~rlarlayer ...... 23
B . LTP in the stratrrm pyramidale ...... 25
C . ATP modulation of synaptic transmission ...... , .... 28
1 . Effects of low concentrations of ATP and adenosine ...... 28
2 . Effects of intermediate concentrations of ATP on synaptic
. * transmission ...... 31
3 . Effects of large concentrations of ATP on synaptic
transmission ...... 36
IX . DISCUSSION ...... 39-45
The synaptic potentiation induced by a nanornolar concentration of ATP
appears to be rnediated by ATP itself ...... 39
The synaptic potentiation induced by a nanomolar concentration of ATP
appears to be mediated by P2Y-CPT sensive receptors ...... 40
The synaptic potentiation induced by a nanomolar concentration of ATP is
different than LTP ...... 1
P2Y-CPT receptors are rapidly incorporated in the putative new synapses
formed during the LTP ...... 42
Activation of P2X receptors might mediate long-term depression
(LTD)...... 43
X . CONCLUSIONS ...... 46
XI. REFERENCES ...... 47-60
iv XII. APPENDIX A (CURRICULUM VITAE) ...... *.---...-.-.-. .-.- 61-62 IV, LIST OF ABBEWVIATIONS
Long-Term Potentiation (LTP), Long-Term Depression (LTD), Field Excitatory
Postsynaptic Potentials (fEPSPs), 8-Cyclopentyltheophyline (CPT),
Pyridoxalphosphate-6-Azophenyl-2',4'-Disulfonic Acid (PPADS), Adenosine
Triphosphate (ATP), Adenosine Diphosphate (ADP), Adenosine Monophosphate (AMP),
Artificial Cerebrospinal Fluid (aCSF), Excitatory Postsynaptic Potentials (EPSPs), N-
Methyl-D-Aspartate (NMDA), Alpha-amino-3-Hydroxy-5-Methyl-4-IsoxazoLepropionic
Acid (AMPA), Persona1 Computer (PC), High Frequency Tetanic Stimulation (Tetanus), and Cornu Arnmonis (CA, Latin for Ammon's hom). V. LIST OF FIGURES
Figure 1. Schematic representation of a coronal section of the rat hippocampus and
dentate gyms showing the main excitatory connections ...... 4
Figure 2. A rise in intraceIlular calcium concentration plays a central role in the induction
of both long-term potentiation (LTP) and depression (LTD) in the CAL
hippocampal area ...... 9
Figure 3. Equipment used to record and to analyze the field potentials from rat
hippocampal slices ...... 1 8
Figure 4. Equipment used to superfuse and visualize hippocampal slices...... 19
Figure 5. Typical field potential recorded in the stratum pyramidale of CA1 hippocampal
area -...... ,..,,...... ,...... 2 1
Figure 6. Field excitatory postsynaptic potentials (EPSPs) recorded in the molecular
layer (A) and stratum pyramidale (B) of the CA1 have opposite
polarities ...... 2 4
Figure 7. Typical field potentials recorded in the stratum pyramidale of CA1
hippocampal is stimulus dependent ...... 26
Figure 8. Application of a high frequency tetanic stimulation (Tetanus) increased both
the population spike amplitude (A) and the slope of initial wave (Pl) of the
field potentials (B) ...... 2 7
Figure 9. A low ATP concentration (300 nM) transitorily increases the population spike
amplitude in the CA1 area ...... 29 .. Figure 10. 8-Cyclopentyltheophyline (CPT) blocks the effects of low ATP concentrations
(300 nM) but it increases the amplitude of the population spike by
itseIf ...... 3 O
Figure Il. Low adenosine concentration (300 nM) fails to modify the evoked population
spike...... 3 O
Figure 12. Adenosine also fails to change the population spike after inhibition of GABA,
receptors with picrotoxin ...... 3 2
Figure 13. A micromolar concentration (100 PM) of ATP reversibly blocks synaptic . . transmission ...... ,...... 3 3
Figure 14. Electrically potentiated field potentials were also inhibited by a micromolar
concentration (100 PM) of ATP but did not prevent the subsequent appearance
of Long-term potentiation (LTP) ...... 3 4
Figure 15. ATP-induced inhibition of electrically potentiated field potentials occurs very
rapidly (< 140 seconds)...... 35
Figure 16. ATP also inhibits the field potentials after total saturation of the long-term
potentiation (LTP)...... 3 7
Figure 17. A short application of a high ATP concentration (30 rnM) induces a Long-term
synaptic depression which is partially blocked by the P2X receptor antagonist,
PPADS ...... 3 8 VI. INTRODUCTION
"...die mecfianisms of rnentaC aczivity are ceriain (y accom.anied moCecuhr rnoïfzztions in tzerve
ceTl andpreceded6y conydéx changes in the relationsliip 6e~weenneiirons. $0 understand mental
activity it is necessary to understarzdtfiese molecular rnoïfzificarions..." mmdny caja~(igii).
A. Overview
Rm6n y Cajal (19 11) was the first to demonstrate that neurons are the basic anatornical and functional units of the neural tissue. He was also the first to suggest that the contacts between acon tenninals and the dendrites or the soma of another neuron are the sites at which the functional information crossed from one neuron to the next. These specialized junctions through which neuronal elements interact were later called synapses by
Shemngton (see Hamrnond, 1996a; Kandel & Siegelbaum, 2000). Since then, a lot has been learned about synaptic transmission, which despite its cornplexity can be functionally classified in two groups: electrical and chemical. In electrical synapses the pre- and post- synaptic membranes are joined by interceilular channels, named gap junctions. At chemical synapses, these membranes are separated by a synaptic cleft. Electrical changes in the presynaptic membrane leads to the influx of calcium ions and the subsequent release of a chemical transmitter (neurotransmitter). This substance diffuses across the synaptic cleft and binds to specialized proteins (transmitter receptors) in the postsynaptic membrane. Activation of these receptors opens or closes ion channels in the postsynaptic membrane generating a postsynaptic potential. Postsynaptic potentials can be excitatory, if L they increase the probability of action potential generation in the postsynaptic membrane, or inhibitory, if they decrease this probabiiity. The importance of the synapse is that it is not only a relay site but also a integrative sise, and it is indeed the target for many other endogenous (neuromodulators) and exogenaous substances.
Alterations in the efficiency of synaptic transmission (synaptic plasticity) are accepted by many researchers to be the neurrophysiological basis of memory and learning.
Hippocampal synapses that use glutamate as an excitatory neurotransmitter have been part of the neuronal network implicated in mermory and learning. The apparent role of these neuronal networks in Alzheimer's Disease has prompted considerable research into the mechanisms of synaptic modulation and symaptic plasticity in the hippocampus (see Beggs et al., 1999; Kandel et al., 2000; Kandel, 2~000).
Growing experimental evidence indicates that adenosine S'triphosphate (ATP) and its derivatives (ADP, AMP, and adenosine) dghtplay an key role as functionaIIy important neuromodulators in the hippocampus (see: de Mendonca & Ribeiro, 1997; houe et al.,
1996). The specific effects, the type of punnergic receptors, and the mechanism of action for each of these purines are not completely understood. This is, at least in part, due to the fact that ATP is metabolized in the brain, which generates the other cornponents to the presence of a complex receptor population to these purines in the hippocampus as well as the lack of more specific punnoceptor antagonists (Cunha er al., 1998; Cunha et al., 1996;
Mendoza-Femandez et al., 2000). Therefore, the focus in this study was to characterize the effects and mechanism of action of ATP ona well characterized glutarninergic synapse of the hippocampus with the purpose of understanding the functional role and pharmacological
2 effects of purines in this structure.
B . Anatomical and physiological properties of the hippocampus
The hippocampus is a specialized cortical structure of the cerebrurn located in the
lower-media1 part of the tempord lobe (see Beggs et al., 1999; Hamrnond, 1996b; Brown
& Zador, 1990). It is sornetirnes called archicortex (or allocortex) because it is considered more primitive (evolutionarily) than the rest of the cerebral cortex (neocortex). A distinctive feature between the neocortex and the hippocampus is that the first has six distinctive histological Iayers whereas the latter has only three. The hippocampus is a relatively elongated (rostrocaudally) structure that Iays adjacent to the olfactory cortex. It is an arched and recurved sheet of cerebral cortex. This structure is composed of two intimately interrelated sernilunar-like structures, Ammon's horn and the dentate gyrm (Figure 1). The first is divided in three continuous regions CA1, CA2, and CA3; CA stands for Cornu
Arnmonis (Latin for Ammon's horn). The CA1 and CA3 areas comprise the hippocampus proper. The CA2 area is very srnaII and indistinct in some species and it is often ignored.
The stratzm pyramidale, a layer of approximately 150 pm in thickness, contains the principal neurons called pyramidal cells. The dentate gyrus is smaller than the Cornu
Arnmonis and its principal neurons are the granula cells. Both areas contain a vast number of cholinergic and GABAergic interneurons.
Figure 1 shows a coronal section of the rat hippocampus and dentate gyrus displaying their most important excitatory connections (Hammond, 1996b; Brown &
Zador, 1990). Axons arising frorn the pyramidal neurons of CA3 generate two types of 3 Figure 1. Schematic representation of a coronal section of the rat hippocampus and dentate gyrus showing the main excitatory connections. Two major divisions are seen in the hippocarnpus, CA1 and CA3. The hippocampus is constituted in three layers: 1) the straturn oriens, 2) the straturn pyramidale, and 3) the molecular layer (with two divisions the straturn radiatum and straturn lantnosum moleculare). The shafurn pyramidale is a continuous neuronal layer between CA1 and CA3 areas. It contains the main ce11 type, the pyramidal neuron, which has two dendritic trees that emerge Eom opposite poles of the soma. The basal dendrite arises from the sarne pole as the axon, and the apical dendrite is located in the rnolecular layer. Excitatory inputs firom CA3 to CAL are extensive and a single CAL pyramidal neuron is innervated by about 5000 different CA3 neurons through the Schaffer collaterals. The connections of these cells are also called en passant (on the way) synapses because a single CA3 axon makes contact with the dendrites (mainly with dendritic spines) of many other CA1 pyramidal cells. According to their shape, dendritic spines can be thin, mushroom-like, branched, or stubby. The neck of the spine restricts diffbsion between the head of the spine and the rest of the dendrite. Thus, each spine may fùnction as an independent biochernical structure. This cornpartmentalization is believed to be important for selectively changing the strength of synaptic connections, e.g. during long-term potentiation. This diagram was adapted b y Barajas-L6pez £kom Harnmond (1996). branches. One type leaves the hippocampus and projects to other structures (e-g. hypothalamus) via the fornix. The other branches form axonic bundles and are known as the Schaffer Collateral Pathways. These recurrent colaterals end in axonic terrninals which form synaptic contacts with the apical dendrites of CA1 pyramidal cells. Such synapses are excitatory and use glutamate as their neurotransrnitter.
The hippocampus is connected to various cortical areas imrnediately adjacent to it and form the hippocampal system (Beggs et al., 1999; Hammond, 1996b; Brown &
Zador, 1990). These structures include the perirhinat, entorhinal, and parahippocampal cortices. The major input to the hippocarnpus is the entorhinal cortex (Beggs et al., 1999;
Hammond, 1996b; Brown & Zador, 1990). This structure sends information to the hippocampus through a bundle of nerve fibers called the perforant path (Figure 1). These fibers establish synaptic contacts with granular cells of the dentate gyms. Axons of the granular cells (also called mossy fibers) synapse with neurons in CA3.
The hippocampal system is criticai for the establishment of certain types of mernories, in particular the so-called declarative (or explicit) memory (Hammond, 1996b;
Kandel er al., 2000; Kandel, 2000). This memory is required to recognize faces, places, and things, dong with their respective meanings. In agreement with the experimental evidence that supports this theory, human experiments using functional brain imaging (with magnetic resonance imaging or positron emission tomography) show activation of the hippocarnpal system during certain type of rnemory tasks. In addition, lesions of these structures are associated with clinical deficits in learning tasks that specifically required explicit memory. Memory formation, however, requires wide and distributed brain
5 regions, and the hippocarnpus is clearly not the only storage site for memory. Indeed, the hippocampus is not essential to acquire many forms of memory, and even in tasks dependent on it for acquisition, the structure is not required for later retrieval (Hammond,
1996b; Kandel et al., 2000; Kandel, 2000).
The unique histological organization of the hippocampus allows the preparation of coronal hippocampal slices in which the Schaffer collaterals are in the same plane as the
CA 1 pyramidal neurons they innervate. Thus, presynaptic fibers (Schaffer collaterds) cm selectively be stimulated while the corresponding excitatory post-synaptic potentials
(EPSPs) can be recorded in the CA1 region (Figure 1). Extracellular, intracellular, and patch clamp techniques have been used to record these synaptic signals. The histological features of the hippocampus and its importance in rnemory and teaming tumed this structure into one of the favourite models for studying synaptic communication. Indeed, it was in the hippocampus where the best cellular mode1 to explain rnemory, long-term potenriation (LTP), was first described (Stevens & Sullivan, 1998; B liss & Collingridge,
1993; Huang, 1998).
C. LTP as a mode1 of synaptic plasticity
LTP can be defined as a long-lasting increase in synaptic efficacy following the high-frequency stimulation of afferent fibers. LTP has been by far one of the best cellular models to explain memory (Stevens & Sullivan, 1998; Bliss & Collin-gidge, 1993; Huang.
1998). It is, therefore, not surprising that it has attracted a lot of attention fiom researchers interested in the memory process and the associated mechanisms that are responsible for its 6 formation- In several recent reviews, different authors have concluded that not only is LTP
a viable mechanism for the induction and storage of memories, but is also the most
prornising candidate (Stevens & Sullivan, 1998; Bliss & ColIin,oridge, 1993; Huang,
1998). Other authors (Martinez & Derrick, 1996; Nosten-Bertrand et al., 1996) conclude that the link between LTP and memory is in some cases tenuous, and in others even contradictory. Despite these discrepancies, most evidence fdysupports LTP as having a role in learning and memory (see Eichenbaum, 1996). Because of al1 this, the mechanisms underlying LTP have been the topic of many studies since its original description in 1973
(El liss & Gardner-Medwin, 1973).
D. LTP is found in the central and peripherai nervous systems
The hypothesis that LTP rnight serve as a memory storage tool resulted, at least in part, from its initial description in the hippocarnpus, structure known to be cntical for the formation of certain types of memories (see Beggs et al., 1999; Kandel et al., 2000;
Kandel, 2000). In the hippocampus, LTP is evident at the three major synaptic connections of the structure (Figure 1). Thus, it is induced by stimulation of the perforant path and recorded in the dentate gyrus granule neurons as initially described by Bliss and Lomo
(1973), by stimuIation of the mossy fibers and recorded in the CA3 pyramidal cells
(Yamamoto & Chujo, 1978), and by stimulation of the Schaffer collateral branches of the
CA3 neurons and recorded in the CA1 pyramidal cells (Schwartzkroin & Wester, 1975;
Anderson et al., 1977). LTP was found to occur in other CNS regions, including the piriform (Stripling et al., 1988) and entorhinal (Wilhite et al., 1986) cortices, the septum
7 (Racine et al., 1983), and in the ventral hom of the spinal cord (Pockett & Figurov, 1993).
LTP has also been described in synapses of the peripheral nervous system (Brown &
McAfee, 1982). As expected, LTP is not lirnited to rnamrnais but is also present in other vertebrates such as gold fish (Lewis & Teyler, 1986), bullfrogs (Koyano et al., 1985), and birds (Scott & Bennett, 1993) as well as in invertebrates (Walters & Byrne, 1985). The wide distribution of LTP suggests that this synaptic phenornenon is used in physiological process other than memory and learning.
E. Cellular mechanisms involved in long-term potentiation
LTP is highly dependent on high levels of postsynaptic calcium (Figure 2). The exact rnechanism(s) by which calcium ions induce LTP is a rnatter of debate but elevation of postsynaptic calcium is clearly necessary, and may even be sufficient for the induction of hippocarnpal LTP. Induction of LTP is prevented by a pretetanus injection of calcium chelators into the postsynaptic ce11 (Malenka et al., 1988), and induction occurs when the postsynaptic ce11 is artificially loaded with this ion.
A great deal of evidence (e-g., Kauer et al., 1988; Nicoll et al., 1988; Malenka et al., 1988; Petersen et al., 1998; Malenka & Nicoll, 1997; Kandel & Siegelbaum, 2000;
Hammond, 1996b) indicates that the primary source of calcium influx during the induction of hippocampal LTP occurs through the glutamate-gated channels, N-methyl-D-aspartate
(NMDA) receptors. This receptor is unique in that opening of the channei requires glutamate binding as well as a moderate level of depolarization. At normal resting potentials (- -70 mV), the channel is blocked by magnesium, and glutamate binding is
8 Nerve Terminal
Glutamate release
-** --.
Postsynaptic Membrane
Figure 2. A rise in intracellular calcium concentration plays a central role in the induction of both long-term potentiation WTP) and depression (LTD) in the CA1 hippocampal area. Synaptic release of glutamate can activate metabotrapic (mGlu) or ionotropic receptors in the postsynaptic membrane. Activation of the mGUu receptor cm then activate the IP3/diacylglycerol cascade that produces the release ofcalcium £iom intracellular pools. NMDA are a subtpe of gluarnate ionotropic receptors/channels that are calcium permeable whereas the alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptors are channels that are Iess permeable to calcium but they can activate calcium influx by ceIl depolarization and the subsequent activation of voltage activated calcium channels. A moderate increase in the intracellular calcium concentration can activate protein phosphatases and LTD. A larger increase in this calcium concentration can activate protein kinases and LTP. Modifierd by Barajas- Lope2 C. nom Beggs et aL, (1999). insufficient to open it. However, at depolarized membrane potentials (> -40 mV), magnesium is expeIled from the channel, which can then be opened by glutamate and which displays a high permeability to calcium ions. Thus, the NMDA receptor complex is said to be dually reguiated by two factors: ligand and voltage. These CO-factorscan be recmited through several means. First, a relatively long, high intensity presynaptic burst of activity
(such as a high-frequency train of stimulation) can induce LTP by releasing glutamate ont0 the postsynaptic receptor, while depolarizing the postsynaptic ce11 through stimulation of the non-NMDA type of glutamate receptors; for instance the alpha-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA) receptors. In fact, the synaptic activation of
AMPA receptors is responsible of most of the excitatory postsynaptic potentials recorded in
CA1 area (Beggs et al., 1999; Harnrnond, 1996b; Kandel & Siegelbaum, 2000). Second, shorter and more physiologically relevant levels of presynaptic activity can induce hippocampal LTP by stimulating the NMDA receptor with glutamate, while the postsynaptic ce11 is depolarized via an alternative means such as an input from a second afferent pathway.
Other forrns of LTP, such as that induced in CA3 pyramidal ceUs following mossy fibre tetanization, occur independently of the NMDA receptor and are instead dependent on calcium influx through voltage-gated channels (see Petersen et al., 1998; Maienka &
Nicoll, 1997; Johnston et al., 1992; Malinow, 1998). Whether these channels are located pre- or post-synaptically is still a matter of debate. Even in area CAL, LTP can be induced without the participation of NMDA receptors, provided that the tetanus (or postsynaptic depolarization) is of sufficient intensity to activate voltage-dependent calcium channels
10
F. Role of ATP in neuronal communication
The nucleoside adenosine and its nucleotide, ATP, are well known for their primary
functional rotes, the former as part of the genetic material, the latter as an energy reservoir.
However, these purines also have potent extracellular actions that were first reported by
Dmry and Szent-Gyorgy (1929). Since then, owing to their ubiquitous presence and
potent extracellular activities, the involvement of purines in many physiological and
pathophysiological functions (e.g. neurotransrnission, inflammatory process, and hypoxia)
has been proposed (Barajas-L6pez & Huizinga, 1993; Cook & Karmazyn, 1996; Parkinson
et al., 1996; Cordeiro et al., 1995; Gordon, 1986; White & MacDonald, 1990; Rudolphi
et al,, 1992; Barankiewicz & Cohen, 1990). Thus, besides being released by nerves
(White & MacDonald, 1990), purines are also released by other types of cells such as mast
cells (Marquardt et al., 1984), platelets (Gordon, 1986), lymphocytes (Barankiewicz &
Cohen, 1990), and cardiac tissues (Forrester, 1990).
The role of purines in nerve communication has received particular attention
following the classic review of Burnstock (1972). Experïmental data indicate that
adenosine and ATP work as neurotransrnitters and CO-transmitters in several neuro-
neuronal (Akasu et al., 1984; Edwards et al., 1992; Evans et al., 1992) and neuro- effector synapses (Evans & Surprenant, 1992; Burnstock, 1986; White, 1988). Purines are known to be released from peripheral and central neurons in a calcium-dependent manner and to have potent neuromodulatory effects (Gordon, 1986; White & MacDonald,
1990). In neuro-neuronal synapses it is known that adenosine mediates a slow hyperpolarizing synaptic potential in the cat vesical parasympathetic ganglia (Akasu et al., 1984), and that ATP acts as a fast excitatory synaptic transrnitter in peripherai (Silinsky &
Gerzanich, 1993; Evans et al., 1992) and central neurons (Edwards et al., 1992).
Various actions have been described for the extracellular ATP in hippocampal neurons including the inhibition of synaptic transmission (Cunha et al., 1998; Dunwiddie et al., 1997), opening of ligand-gated channels (Pankratov et al., 1998; Baljit et al.,
1999), and the phosphorylation of ecto-protein kinase proteins involved in LTP (Chen et al., 1996). Recent experimental evidence indicates that ATP might be playing a role as a fast neurotransmitter in the CA1 hippocampal region (Cunha et al., 1998).
Two types of purine extracellular receptors are widely recognized (Burnstock,
1990; houe et al., 1996). Pl sites are adenosine receptors sensitive to theophylline derivatives and P2 sites are nucleotide receptors insensitive to theophylline derivatives.
Nevertheless, nucleotide receptors sensitive to theophylline or its derivatives have been described in peripheral and central neurons (Silinsky & Ginsborg, 1983; Shinozuka et al.,
1988; Forsyth et al., 199 1; Cunha et al., 1994b; Barajas-L6pez et al., 1995). At this receptor: 1) P 1 receptor agonists (adenosine or its analog, 2-chIoroadenosine) and ATP appear to be equipotent (but see Silinsky & Ginsborg, 1983; von Kugelgen et al., 1989;
Barajas-L6pez et al., 1995); 2) a$,-methylene ATP is a weak or an inactive agonist; 3)
ATP actions are blocked by antagonists of Pl receptors (theophylline derivative) rather than antagonists of the P2 receptors (suramin, PPADS, and reactive blue 2). Several studies proposed the name of P3-punnoceptor for this pharmacological profile (Dalziel & Westfall.
1994; Forsyth et al., 1991; Shinozuka et al., 1988). Here, they will be named
P2Y-theophylline sensitive receptors.
13 ATP can directly activate P2 receptors, P2X and P2Y, in many different types of cells, including neurons. P2X receptors are ligand-gated ion channels whereas P2Y are coupled via G proteins (GTP-binding proteins; Burnstock, 1990; houe et al., 1996). ATP is also known to be hydrolysed extracellularly by nucleotidases to ADP, AMP, and adenosine, and these products influence neurons by interacting with specific membrane receptors (Kreutzberg et al., 1986; Dunwiddie et al., 1997; Burnstock, 1990).
Pl receptors are present in presynaptic nerve terrninals in many central and peripheral synapses and their activation inhibits the release of vanous neurotransmitters including glutamate (Dolphin & Prestwich, 1985; Heron et al., 1993; Fredholm et ai-,
1996), acetylcholine (Barajas-L6pez et al., 199 1; Jin et al,, 1993; Fredholm et al., 1989), noradrenaline (Barajas-L6pez et al., 199 1; Fredholm et al., 1989; Forsyth et al., 199 1), and peptides (Anand-Srivastava et al., 1989; Christofi et al., 1990; Broad et al., 1992).
Presynaptic ATP receptor activation also inhibits neurotransmitter reIease in peripheral
(Silinsky & Ginsborg, 1983; Shinozuka et al., 1988; Silinsky et al., 1990; Forsyth et al.,
199 1) and central neurons (Fredholm & Dunwiddie, 1988; Dunwiddie & Fredholm, 1989;
Santicioli et al., 1993; Cunha et al., 1994a).
In the hippocampus, ATP inhibits the neuronal release of glutamate but it is not clear whether such an action is mediated by the ATP metabolite, adenosine, acting on Pl receptors, or by ATP activation of P2 receptors. A study by Cunha et al. (1998) favours the first hypothesis but at least two of their observations support the hypothesis that P2 receptors are present in nerve terminais and might mediate part of the inhibition. This group observed that the ATP effects on glutamate release are faster than its hydrolysis and
14 that the ATP analog, ATP-y-S, behaves as a full agonist despite its hydrolysis being onIy
marginal. In a recent intracellular study, our laboratory presented experimental evidence
indicating that the ATP presynaptic effects mediated through its metabolite adenosine were
seen only at high concentrations of ATP, and only after pertussis toxin treatrnent
(Mendoza-Femandez et al., 2000). Thus, ATP effects are likely mediated by P2Y
receptors (G-protein-coupled receptors), which are 8-cyclopentyltheophilline (CPT).
Together, these observations support the general hypothesis that ATP is a neurotransmitter
in the hippocarnpus. A Iong-term objective of our laboratory is to understand the
physiological role and rnechanism of action of ATP on the hippocampal synaptic activity.
G. Statement of Purpose
ATP has joined the group of substances that are known to function as fast neurotransmitters
in V~~OUStissues including the hippocarnpus, a structure well known to be involved in
memory and learning. Some studies have indicated that ATP might play a role in the establishment of LTP, which is one of the synaptic modifications proposed to underlie the
memory process. For instance, ATP has been shown to induce a long-lasting enhancement
of the synaptic communication between pyramidal neurons of the CA3 and CA1
hippocampal areas wieraszko & Seyfried, 1989). In this study, we set out to investipate
the modulatorv effects and mechanisrns of action of ATP on synaptic transmission in these
svnames. Our working hypothesis was that activation of different type of P2 receptors
correlates with different modulatory ATP actions on the synaptic transmission of the
hippocampal CA 1 region. 15 VII. MATERIALS AND METHODS
A. Brain slices
Hippocarnpal slices (400 pm) were cut from brains of 4 to 6 week-old male rats (Sprague
Dawley, purchased from Charles River Lab., Montreal' Que, Canada). After decapitation
and cutting the skull with a pair of scissors, the brain was quickly removed and placed in
ice-cold (24°C) artificial cerebrospinal fluid (aCSF). A block of brain, containing the hippocampus, was prepared and glued with cyanoacrylate adhesive to the sectioning charnber of a vibrotome (Campden Instruments, Ltd, Sarasota, FL, USA). Coronal hippocampal slices were cut and placed, until use (for at least 30 min), in a holding chamber containing aCSF at room temperature, A single slice was then transferred to the recording chamber and continuously superfused with aCSF at a rate of 2-4 mL/min. The volume of aCSF in the recording chamber was about - 1 ml and was maintained constant by a needle connected to a vacuum pump. The slice was held subrnerged in the recording chamber with a slice holder. This holder consisted of a platinum "horseshoe" wire with three plastic strings glued to its lateral sides. The strings were positioned parallel to the
Schaffer's collaterals. The aCSF was prepared with nanopure water and had the following composition: 120 mM NaCl, 3.3 rnM KCI, 1.23 mM NaH,PO,,- 1.3 mM MgSO,, 1.8 mM
CaC12, 26 mM NaHCO,, and 11 rnM glucose. aCSF was constantly bubbled with a gas mixture containing 95% O2 and 5% CO,. Application of experirnental substances was achieved by switching the aCSF for one containing the drug(s) using a stopcock.
Switching back to standard aCSF washed out substances. Extenal solutions were
16 delivered by gravity. Except where othenvise mentioned. experiments were performed at
34-36°C.
B. Electrophysiological recordings
Electrical field potentials were recorded with glass pipettes filled with 2 M NaCl.
Recording pipettes had a tip resistance of 2-6 MC! and a tip extemal diameter of 3-5 Pm.
These recording electrodes were made from Borosilicate Glass Capillaries (World Precision
Instruments, Inc., Sarasota, FL, USA) using a vertical pipette puller (Narishigue Scientific
Instrument Lab., Tokyo, lapan) and were connected to a Grass (Mode1 PIS; Grass
Intruments Co., Quincy, Mass, USA) amplifier with a silver wire coated with silver
chloride (Figures 3 and 4). The field potentiais were amplified 500 times, digitalized with a
Digidata 1200 (Axon Instrument, Foster City, CA, USA), and stored in the hard drive of a
PC using the software Clampex (Pclamp 6.0. Axon Instruments). Field potentials were
recorded in groups as computer files (six field potentials per file).
At least otherwise stated, these potentials were recorded in the strarwn pyramidale
of CA1 and evoked by electrical stimulation of the Schaffer collateral-commissural afferents
(Figure 1). Field potentials were evoked by electrical pulses (100-200 ps) applied with a bipolar eleceode made by twisting platinum wires (Teflon-coated) of a diameter of 50 pm.
Electrical pulses were delivered at 0.05 Hz and generated by an isolated stimulator
(Digitimer Ltd., Hertfordshire, England) which was controlled by the same PC, software, and Digidata, used to record the field responses (see above). The strength of the stimulation (usually 6 to 20 V) was adjusted to obtain only about 30-50% of the maximal
17 Sprague Dawley
- 4 d Electrical Sti mulator i I ,1 Grass Pl5 1
Hi ppocampal Slices (400 pm)
Maci ntos h
Axograph
00 O O on ,, Iooo aooo ao
Figure 3. Equipment used to record and to analyze the field potentials from rat hippocampal slices. These potentials were evoked by elecaical square pulses applied to the Schaffer colIaterals and recorded in the stratum pyramidale of CAL. The electrical stimulator was controlled by the PC through the A-D converter using the Clampex software @Clamp, Axon Instruments). The programs Axograph (Axon Instruments), Canvas (Deneva Software), and Kaleidagaph (Abelbeck Software), and a Macintosh cornputer were used to prepared al1 Figures. This Ilustration was prepared by Barajas Lopez C. Figure 4. Equipmeot used to superfuse and visualize hippocarnpal süces. The hippocampal slice to be recorded was placed in a plastic chamber and superfused constantly with warmed (34-36OC) adficial cerebrospinal fluid. This fluid was placed in reservoirs and applied by gravity. The fluid level in the recording chamber was rnaintained constant by the suction applied with a needle comected to a vacuum pump. The recording charnber was mounted on a dissecting microscope to visualize the position of the electrodes. A pair of Narishigue micromanipulators (not shown) were used to place these electrodes in the desired positions. This ilustration was drawn by Barajas- Lopez C. spike. The field potential was monitored for 20 min, and expenments were performed only
on slices demonstrating a stable response with the sarne stimulation parameters. The
electrical tetanus used to evoke LïT in CAL consisted of two pulse trains of 100 Hz lasting
1 s, at an intertrain interval of 10 S. In every preparation, these pulses had the same
strength and duration as those used to evoke the field potentids.
The analysis of the evoked field potentials was carried out using the software
CIarnFit (Pclarnp 6.0, Axon Instrument). With the purpose of increasing the signalhoise ratio, the six field potentials of each recorded cornputer file were averaged. The slope of the rising phase of the first positive wave (between 10 and 60% of the peak response) was determined for some of these averaged potentials. For each recorded file, we deterrnined the average amplitude of the population spike, first negative (NI) wave of field potentials
(Figure SA). The magnitude of the population spike was rneasured as the shortest distance between the highest negativity and an irnaginary line drawn between the top of the first (Pl) and second (PZ) positive waves (Figure SB).
C. Drugs
ATP, adenosine, and pyndoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS),
8-cyclopentyltheophylline (CPT), and picrotoxin, were purchased from Research
Biomedical Inc. (Natick, MA, USA). Al1 other substances were purchased from Sigma
(St. Louis, MO, USA). The pH of the aCSF containing 30 mM ATP was always readjusted with NaOH. The addition of the other substances to the extemal solution did not alter its pH.
20 Stimulus
Figure 5. Typical field potential recorded in the stratum pyramidale of CA1 hippocampal area. A) Three waves were usually observed, P 1, N 1, and P2. The area under these waves is shadowed. Pl and P2 correspond to the postsynaptic potential and N1 corresponds to the population spike. B) The amplitude of this spike was calculated using the ClarnpFit software (pCIamp, Axon Instruments). A macro was created to trace a straight line as indicated by "a". The amplitude of the population spike was calculated as indicated. D. Statistical analysis
Results were expressed as rnean + S.E.M. and the number of slices are indicated as n number. A single slice was recorded per rat. The paired Student's t-test was used to evaluate differences between mean values obtained from the same slices and the unpaired
Student's t-test was used for data obtained from different groups of slices. The ANOVA
(one factor) was used before the unpaired Student's t-test to test the uniformity of the variances. P values of 0.05 or Iess were considered statistically significant. Statistical analysis was carried out with the software Excel (Microsoft@, Santa Rosa, CA, USA).
Preparation of the graphs and printing of the field potentials were done with the software
Kaleidagraph (Synergy Software, PA, USA), Axograph (Axon Instruments), and
CANVAS (Deneva Software, Miami, FL, USA) softwares in a Macintosh cornputer (Apple
Computer Inc. CA, USA). VIII. RESULTS
A, Field potentials in the straturn pyramidale and molecular layer
Low intensity stimulation induced a field potential with a single wave, which was either negative (downward deflexion) or positive (upward deflexion) when recorded in the molecular layer (Figure 6A) or in the straturn pyramidale (Figure 6B), respectively.
According to previous literature (Lyubkin er al., 1997; Cunha et al., 1994a; Nishimura et al., 1990; Hammond, 1996b) these potentials correspond to the fast excitatory postsynaptic potentials (EPSPs), and when they are recorded extracellularly these are called field EPSPs (fEPSPs) or field potentials.
As shown in Figure 7, the shape of the fEPSPs recorded in the stratum pyramidale of the CA1 hyppocampal region was stimulus dependent. lncreasing the stimulus strength changed the field potential into a multiphasic response. The stimulus intensity used in rnost experiments evoked field potentials with three typical waves. The initial positive wave (P 1) was followed by a relativety faster negative wave (Nl), and then by a second positive one
(P2). Pl and P2 are the fEPSP and N1 is the supenmposed action potential, known as population spike. This spike corresponds to the action potential generated by many neurons that reach the threshold dunng the EPSP. The average amplitude of Pl, NI, and
P2 was, 0.55+0.1, -2.4110.36, and 1.92-r-0.16 mV in ten analyzed slices. The time between the stimulus and the beginning of Pl (latency) was 2.9410.12 ms. Higher stimulus intensity increased the amplitude of these waves and induced the appearance of multiple population spikes (Figure 7). Molecular Layer
Figure 6. Field excitatory postsynaptic potentials (fEPSPs) recorded in the molecular layer (A) and straturn pyramidale (B) of the CA1 have opposite polarities. Notice the absence of population spikes as those seen in Figures 5 and 7. These potentials were evoked by application of single electrical pulses of 200 ms duration, at 0.05 Hz to the Schaffer collaterals. Recordings shown in panels A and B are from two different slices. A small potential was some times recorded preceding the EPSP, particularly when recardings were made in the rnolecular layer. This potential is called presynaptic volley and corresponds to the depolarization of the Schaffer collaterals. A small wave was noticed in the field potentials of some slices, in particular in
those recorded in the molecular layer (Figure 6), immediately after the stimulus artifact.
This wave is known as the presynaptic volley and it is produced by the depolxization of
presynaptic fibers arriving to CA1 (Lyubkin et al., 1997). The average time between the
top of this volley and the beginning of Pl was 1.17-eO.12ms.
B. LTP in the stratum pyramidale
High frequency tetanic stimulation of the Schaffer colIaterals increased the population spike
(NI) amplitude and the slope of Pl of field potentials recorded in the strarurn pyramidale.
These two parameters have been widely used to assess changes in synaptic strength. An
increase in the value of these parameters in indicative of a higher synaptic strength whereas
a decrease indicates lower strength. Figure 8 shows the average changes in these
parameters after three successive tetanus of six experiments. As it is shown in Figure 8A, the first tetanic stimulation induced a significant increase in the slope of Pl to an average value of 129512% (P50.05; n=6). A marginal additional increased in this slope was observed with the second and third tetanus, up to steady state values of 139112% and
144112%, respectively. A much larger increase was noticed in the population spike amplitude to an average value of 233+-51% (PIO.OS), 268+86% (P10.05), and 269159%
(P10.05)for the first, second, and third tetanus, respectively (Figure 8B; n=6). Therefore, changes in this parameter were used to assess the synaptic strength in the following experirnents. Figure 7. Typical field potentials recorded in the stratum pyramidale of CA1 hippocampal is stimulus dependent. Field potentials were evoked by application of single electrical pulses of 200 ms duration, 0.05 Hz, and at the indicated intensities (Voltage). With the lowest intensity (6 V), a very small population spike is observed on the top of an excitatory postsynaptic potentials. Field potentials were increased when larger stimulation voltages were used. The field potentials recorded in the following figures were evoked by adjusting the stimulus intensity to induce population spikes of about -40% of its maximal amplitude, considering field potentials with only one spike. This prevented an over stimulation of the slice and also the generation of a secondary spike. In this example, the maximal amplitude of the population spike was reached with 36 V. A 1st LTP Tetanus 200 - n œ g g 150 - U * O y 100 - -w & 3rd LTP t-i 2nd LTP 0 50 er, - Tetanus Tetanus -O m I.,,I.,,I,l.#!I, 020 O 20 40 60 80 Time (min)
B 1st LTP - Tetanus
- I 2nd LTP 3rd LTP Tetanus - -' Tetanus
Time (min)
Figure 8. Application of a high frequency tetanic stimulation (Tetanus) increased both the population spike amplitude (A) and the slope of initial wave (Pl) of the field potentials (B). Three tetanus were given at 30 min intervals. The first tetanus induced the largest change in the population spike and Pl slope. The change in these parameters are known to last for hours and are known as long-terrn potentiation (LTP, see Introduction). The third tetanus correLates with the smallest change in these parameters, indicating a saturation of LTP. Squares and bars represent the mean and S.E.M. of six slices. C. ATP modulation of synaptic transmission
The following studies were designed to investigate the modulatory actions of ATP on the hyppocampal synaptic transmission between the pyramidal neurons of CA3 and CA1 regions. First, it was investigated if low concentrations of ATP can induce LTP as suggested by a previous report (Wieraszko & Seyfried, 1989).
1. Effects of nanomolar concentrations of ATP
Figure 9 shows the effect of 300 nM ATP on field potentials recorded in the strahfm pyramidale of CAI. ATP induced a statistically significant (P< 0.05) increase of the spike population amplitude (Figure 9). This effect, however, was transient and the spike amplitude returned to its control values about 40 min after removing the ATP. Tetanic electrical stimulation can still induce a LTP after ATP treatment, As it is shown in Figure
10, this ATP effect was prevented by the presence of 300 nM CPT in the superfusion media. CPT by itself significantly increased the amplitude of the population spike. The same concentration of adenosine (300 nM) failed to induce any change in the amplitude of the population spike (Figure 11).
Phmacological blockage of GABA, receptors has been demonstrated to modify an NMDA-independent form of LTP, which depends on postsynaptic, voltage-dependent calcium channels (Grover & Yan, 1999). Opposite to what it was found here, in the granular ce11 layer of the guinea-pig hippocampus, nanomolar concentrations of adenosine enhanced synaptic transmission (Nishimura et al., 1990). Therefore, it was investigated whether picrotoxin, a GABAA receptor antagonist (Otani & Connor, 1996), could enhance
28 300 r ATP 300 nM
LTP Tetanus 200
100
y20 O 20 40 60 80 100 120 Time (min)
Figure 9. A low ATP concentration (300 nM) transiently increases the population spike amplitude in the CA1 area. The plot shows the popuIation spike amplitude (% of control values) as a function of time. ATP was applied as indicated by the bar. A tetanic stimulation (see Methods) was applied as indicated by the arrow. This tetanic stimulation increased the synaptic strength. Squares and bars represent the mean and S.E.M. of four slices. Lower traces are representative field potentials taken from one hippocampal slice at the tirnes indicated by letters. Figure 10. 8-Cyclopentyltheophyline (CPT) blocks the effects of tow ATP concentrations (300 nM) but it increases the amplitude of the population spike by itself. The plot shows the population spike amplitude (% of control) against time. CPT and ATP were applied as indicated by bars. CPT by itself potentiated synaptic transmission in a nonreversibie manner. Squares and bars represent the mean and S.E.M. seven slices.
Adenosine 300 nM -
Figure 11. Low adenosine concentration (300 nM) fails to modify the evoked population spike. The plot shows the population spike amplitude (% of control) against time. Adenosine was applied as indicated by the bar. Squares and bars represent the mean and S.E.M. of four slices. a sirnilar adenosine effect, In our experiments, picrotoxin (30 PM) increased the number of population spikes and prolonged the field potentids in al1 tested slices (n=10; Figures 12A-
B ). This treatment, however, failed to rnodify the effects of adenosine (Figure 12C).
2. Effects of micromolar concentrations of ATP
Figure 13 shows the effect of superfusing 100 FM ATP during 14 min on the field potentials recorded in the stmtum pyramidale of CA1. ATP induced a statistically significant (P< 0.05) decrease of the spike population. This effect was almost reverted 6 min after ATP removal from the bath. This inhibitory ATP action and the purinoceptor involved have been previously characterized using intracellular recordings
(Mendoza-Fernandez et al., 2000). Here it was investigated if a similar effect can also be seen in newly pofentiated synapses.
ATP (100 PM) completely inhibited the population spikes and almost completely inhibited the fEPSPs when given immediately after application of the tetanic stimulation to the Schaffer collaterals (Figure 14). Such an effect was transient and the amplitude of the spike potentials increase to a stable level about 8 min after beginning ATP removal from the recording chamber. At this steady state the amplitude of these potentials was significantly larger (P10.05) than their control amplitude reflecting the presence of LTP despite the ATP treatment. The graph shown in Figure 15 is a zoom in of the data near the time of ATP application of Figure 14. This plot depicts the kinetics of ATP's inhibitory actions.
Maximal ATP effects occur in less than 140 seconds. Control
Picrotoxin 30 pM
Adenosine 300 nM - -
Y 0 tL 1 ma -10 O 10 20 30 40 50 60 Time (min)
Figure 12. Adenosine also bils to change the population spike after inhibition of GABAA receptors with picrotoxin. Field potentials recorded before (A) and in the presence of picrotoxin (B) are shown. Picrotoxin, as expected, increases the duration of the field potentials and the numbers of population spikes. C)The population spike amplitude (% of control) as a function of time is shown in this plot. Adenosine was applied as indicated by the bar. Squares and bars represent the mean and S.E.M. of eight slices. 150 - ATP 100 pM -n 2 Eza5Qra YC U
w aa,
.CI
6 a, 25a V1 CL
-20 -10 O 10 20 30 40 Time (min)
Figure 13. A micromolar concentration (100 pM) of ATP reversibly blocks synaptic transmission. The plot shows the population spike amplitude (% of control) as a function of time. ATP was applied as indicated by the bar. Squares and vertical bars represent the mean and S.E.M. of four slices. 0- 300 YLi ATP 100 pM 6E: t - - 1st LTP
2nd LW Tetanus
1...1...I...I O 20 40 60 80 Time (min)
Figure 14. Electrically potentiated field potentials were also inhibited by a micromolar concentration (100 PM) of ATP but did not prevent the subsequent appearance of long-term potentiation (LTP). The plot shows the population spike amplitude (% of control) against time. A tetanic stimulation was given just before ATP application (as indicated by arrow). ATP application almost completely inhibited the field potentials. After ATP removal, the population spike amplitude was Iarger than the initial control values thus, indicating the presence of LTP. A second tetanic stimulation is correlated with a further increase in the population spike amplitude. Squares and bars represent the mean and S.E.M. of seven slices. Lower traces are representative field potentials taken from one hippocampal slice at the times indicated by the letters. ATP 100 pM ///////////////////MA L t
-100 O 100 200 Time (s)
Figure 15. ATP-induced inhibition of electrically potentiated field potentials occurs very rapidly (< 140 seconds). This plot is a zoom-in of the data presented in Figure 14, near the time of ATP application. Squares and bars represent the mean and S.E.M. of seven slices. Lower traces are represe~tstivefieid potentials taken from one hippocarnpal siice at the times indicated by letters. In another series of experiments, ATP (100 PM) was applied after the LTP response had been saturated by the appkation of three periods of tetanic stimulation. In such experirnents, ATP totally inhibited the population spikes and almost completely inhibited the fEPSPs (Figure 16). Such an effect was transient and the amplitude of the spike potentiais increase to a stabIe Ievel -10 min after the ATP removai from the recording chamber. At this steady state the ampIitude of these potentials was not signifîcantly different than their amplitude before ATP application. A fourth tetanic stimulation of the
Schaffer collaterals produced only a transient increase in the population spike amplitude, indicating LTP was still saturated.
3. Effects of millimolar concentrations of ATP
Figure 17 shows the effect of superfusing 30 MM ATP for 30 seconds on the population spike amplitude of fEPSPs recorded in the straarm pyramidale of CAL. ATP induced a statistically significant (PI 0.05) decrease of the spike amplitude. This effect persisted, at least partially, -40 min after ATP application. This inhibition was partially blocked by the presence of the P2X receptor antagonist, PPADS (Figure 17). Thus, the area under the curves between tirne zero and 43 min was significantly different (P<0.05), the mean area for control slices was 3868+121%/43 min and in presence of PPADS the area was
2995+.550%/43 min. 1st LTP Tetanus ATP 100 pM 1- i 5
1 1 Tetanus a / 2nd LTP -b Tetanus
I III. I II, 01III ! I O 50 100 150 200 Time (min)
Figure 16. ATP also inhibits the field potentials after total saturation of the long-term potentiation (LTP). The plot shows the population spike amplitude (% of control) against time. Three tetanic stimulations were given to saturate LTP and then ATP was applied (as indicated by the bar). ATP application virtually inhibited al1 synaptic communication. After ATP was removed from the bath, the amplitude of the population spike almost compIetely recovered. A fourth tetanic stimulation only induced a transient potentiation, indicating saturation of this synaptic response. Squares and bars represent the rnean and S.E.M. of six slices. -r- Control lso r PPADS (100 pM) ATP 30 mM
-20 -10 O 10 20 30 40 Time (min)
Figure 17. A short application of a high ATP concentration (30 mM) induces a long-term synaptic depression which is partially blocked by the P2X receptor antagonist, PPADS. The plot shows the population spike amplitude (% of control) of two diFferent experimental groups against time. Control experiments (M) were done in "normal" artificial cerebrospinal fluid whereas the other experimencs were perfonned in the presence of PPADS (O),a P2X receptor antagonist. PPADS (100 pM) was added ten minutes before and during ATP application. This nucleotide was applied for 30 seconds from time zero, as indicated by arrow. Symbols and bars represent the means and S.E.M., respectively, of seven (l)and four (0)slices. IX. DISCUSSION
The results described here indicate that there are at least three different effects of ATP on the hippocampal synaptic transmission, which rnight be mediated by different purinoceptors.
The first effect was obsewed at a nanomolar ATP concentration and it consisted in a transient enhancement of the glutaminergic synaptic transmission. This ATP action rnight be mediated by a P2Y purinoceptor on which CPT appears to work as an antagonist. The second action was observed at a micromolar concentration and was characterised by inhibition of the synaptic transmission. According to a recent study from our laboratory
(Mendoza-Fernandez et al., 2000), such an effect is mediated by a presynaptic P2Y receptor, which is CPT sensitive. Our present observations indicate that such purinoceptor is incorporated in the presynaptic terminals right after the induction of LTP. The third effect was observed by a short superfusion of a millirnolar concentration of ATP and was characterised by inhibition of synaptic transmission. This inhibition appeared to have two different phases, a short-lasting and a long-lasting. The latter resembles long-term depression.
A. The synaptic potentiation induced by a nanomolar concentration
of ATP appears to be mediated by ATP itself
The synaptic transmission enhancement induced by 300 nM ATP could be mediated by
ATP itself or by its metabolites ADP or AMP (e-g. Cunha et al., 1998) but very unlikely by adenosine because the latter substance (300 nM) failed to produce a similar effect as
39 ATP. These observations, however, do not give any insight regarding the site of action of
ATP. In other words, this ATP effect could be mediated by an increase in transmitter release or by augmenting the postsynaptic sensitivity to glutamate or by both actions. The putative postsynaptic action could be ruled out by measuring the effects of this concentration of ATP on the postsynaptic response to the local application of glutamate.
These experiments could be carried out by intracellular recordings (patch clamp or sharp electrodes).
B. The synaptic potentiation induced by a nanomolar concentration
of ATP appears to be mediated by P2Y-CPT sensitive receptors
The synaptic transmission enhancement induced by 300 nM ATP could be mediated by activation of a P2Y purinoceptor sensitive to theophylline derivatives. In various preparations, including hippocarnpal slices, CPT (at the concentration used here, 300 nM) has been shown to be an antagonist of either Al (a Pl receptor subtype) or a subgroup of
P2Y purinoceptors (Barajas-Lope2 et al., 1995; Mendoza-Fernandez et al., 2000). In this study, it was found that this antagonist also blocks the enhancement induced by ATP on synaptic transmission. CPT by itself also produced an enhancement of synaptic transmission sirnilar to that induced by 300 nM ATP. This finding is in agreement with the fact that endogenous release of adenosine has been demonstrated to constantly occur in these brain slices (Cunha et al., 1998). Therefore, it is likely that CPT is blocking the effects of endogenously released adenosine on Al receptors. Activation of Al receptors is known to inhibit synaptic release of glutamate (the neurotransrnitter responsible of the fEPSPs recorded here) in hippocarnpal slices (Mendoza-Femandez et al,,2000).
C . The synaptic potentiation induced by a nanomoIar concentration
of ATP is different than LTP
Nanomolar ATP concentrations (300 rèM) only transitorily increased the amplitude of the population spike and it is therefore different than LTP, which last for hours. A similar but long-lasting synaptic potentiation induced by nanomolar concentrations of ATP was described before by Wieraszko and Seyfried (1989) in the mouse hippocarnpus. In such an study, this potentiation resembles LTP and the same group had previously shown that ATP is released during high frequency stimulation of Schaffer collaterals (Wieraszko er al.,
1989), for which this group proposed that this nucleotide plays a role in LTP. The experiments descnbed by these authors were performed in a non-circulating charnber and
ATP was not removed from the chamber during the experïment. In the present study, the synaptic potentiation induced by ATP reverted a few minutes &ter removing this substance from the external medium and therefore, it does not resemble LTP. The difference in ATP application is likely the explanation for these apparently contradictory observations. The hypothesis that these ATP actions are independent from LTP could be further studied by analyzinp the effects of ATP after LTP saturation (as shown in Figure 16). Ef ATP further potentiates synaptic transmission then such a finding would support this hypothesis. D. PZY-CPT receptors are rapidly incorporated in the putative new
synapses formed during the LTP
The inhibitory effect of 100 pM AT"on synaptic transmission has been characterised previously in Our laboratory using intracellular recordings (Mendoza-Fernandez er al.,
2000). Various observations indicate that this inhibitory effect is rnediated by activation of presynaptic P2Y receptors (G-protein-coupled receptors) that are CPT sensitive
(Mendoza-Femandez et al., 2000). Here, these effects were corroborated using an extracellular recording technique. It was found that this effect was transitory and that the amplitude of the population spike returned to its control values &ter washing out ATP from the extemal medium.
As it was mentioned in the Introduction, the cellular mechanisms for LTP are still a matter of debate. Recently, it has been reported that a fast delivery and redistribution of
AMPA receptors to the dendritic spines in pyramidal neurons might underlie the LTP induced by NMDA receptor activation (Shi et al., 1999). Despite the fact that these changes have been demonstrated only at the postsynaptic level, they suggest that LTP is mediated by the formation of new functional synapses. It is not known what happens at the presynaptic level. In the present study it was showed that ATP (100 pM) can completely inhibit the population spike in less than two hundred seconds after LTP induction. It was also demonstrated that ATP can completely inhibit the population spike even after complete saturation of LTP. These observations suggest that if new synapses are formed during
LTP they already have P2Y receptors at their presynaptic terminais or that these receptors are rapidly incorporated into newly forrned nerve terrninals. These receptors would mediate the ATP inhibitory actions observed here.
E. Activation of P2X receptors might mediate long-term
depression (LTD)
A 30 s superfusion of a 30 milimolar ATP concentration induced an inhibitory effect that resembles the long-term depression (LTD) in synaptic transmission reported by other manoeuvres (Bear & Abraham, 1996; Nicoll et al., 1998; Bear & Abraham, 1996). For instance, LTD can be induced by low frequency electrical stimulation (eg. 1 Hz dunng 15 min) of the Schaffer collaterals (Nicoll et al., 1998; Bear & Abraham, 1996). It is not yet clear, however, whether the synaptic depression induced by ATP is LTD or if it sirnply resembles LTD. This question will be addressed in future expenments.
It is Likely that the synaptic depression induced by 30 rnM ATP could be the result of the erevation of intracellular calcium. In support to this interpretation, LTD also requires a rïse in the intracellular calcium concentration (Bear & Abraham, 1996)- LTD has a lower calcium threshold than LTP and it is, therefore, seen at lower calcium concentrations than
LTP (Bear & Abraham, 1996; Beggs et al., 1999). ATP has been shown to elevate the intracellular calcium concentration in hippocampal neurons (Inoue et al., 1995). This effect appears to be due to a direct action on purinergic receptors located in the postsynaptic membrane because it is not prevented by substances that block synaptic transmission.
Furtherrnore, these ATP effects were blocked by suramin, a P2 receptor antagonist. It is difficult to know if such an ATP action is mediated by P2X or by P2Y receptors because 43 suramin blocks various types of these receptors, although not al1 of them (Barajas-Lope2 et
al., 1993; Barajas-L6pez et al.. 1995; Mendoza-Fernandez et al., 2000). P2Y-CPT
sensitive receptors mediating the presynaptic effects of ATP on glutamate release are
suramin and PPADS resistant (Mendoza-Fernandez et al., 2000) and they are therefore,
different than those receptors mediating the increase in intracellular calcium.
It is very likely that the initial inhibitory effect of 30 mM ATP is mediated by a
decrease in glutamate release and the P2Y receptors sensitive to CPT. This hypothesis is
supported by the fact that PPADS (a P2X receptor antagonist; Barajas-Lopez et al., 1996;
Cunha et al., 1998) does not completely prevent ATP effects. P2Y receptors that mediate
the inhibitory effects of ATP on the synaptic release of glutamate are insensitive to PPADS, and suramin but CPT sensitive (Mendoza-Femandez et al., 2000). This hypothesis will be further analyzed in experiments performed in the presence of CPT in order to block ATP actions at the presynaptic levet.
At least part of the synaptic depression observed with 30 m.ATP might be mediated by P2X receptors. These receptors are ligand-gated channels known to be calcium permeable, activated with rnicromolar ATP concentrations, and desensitized relatively rapidly during the continuous presence of ATP (North, 1996; Barajas-Lopez et al., 1996; Cunha et al., 1998). These are some of the reasons why a relatively high concentration of ATP and a short-time application was chosen in the present study.
PPADS blocks ATP actions on various P2X receptors including on those present in the hippocampus (Cunha et al., 1998). In the present study, it was shown that PPADS blocks part of the long-term synaptic depression induced by ATP, which suggests the involvement of P2X receptors in this effect. X. CONCLUSIONS
In surnmary, it was found that ATP potentiates synaptic transmission at nanomolar concentrations by acting on P2Y receptors sensitive to CPT. This effect however, is different than the classical LTP induced by an electrïcal tetanic stimulation. A miUimo1ar concentration of ATP inhibits synaptic transmission that lasts for at least 40 min and resembles LTD. ATP (100 PM) appears to equally modulate glutamate synaptic release before and after LTP and the P2Y receptors mediating these ATP actions appear to be aiready present (or to be rapidly incorporated) in the putative new synapses formed during
LTP. Altogether, these observations indicate that ATP actions on hippocampai synapses are complex and that a diverse population of purinoceptors mediate its effects. XI. REFERENCES
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