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Adrenergic modulation of glutamate induced calcium mobilization in cultured rat visual cortical neurons Yang, Benduan 1994

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ADRENERGIC MODULATION OFG L U T A M A T E INDUCED C A L C I U M M O B I L I Z A T I O N LN C U L T U R E D R A T V I S U A L C O R T I C A L N E U R O N S  by  BENDUAN YANG  B . M e d . , The West China University of Medical Sciences, 1986  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E REQUIREMENTS FOR T H E D E G R E E OF M A S T E R OF SCIENCE in T H E F A C U L T Y O F G R A D U A T E STUDIES (Neuroscience Program)  We accept this as conforming to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A December 1994  ©  Benduan Yang, 1994  In presenting degree at the  this  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department  and study.  publication of  by this  his  or  her  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  that the  representatives.  may be It  thesis for financial gain shall not  permission.  requirements  I further agree  this thesis for scholarly purposes or  the  that  advanced  Library shall make it  by the  understood be  an  permission for extensive  granted  is  for  that  allowed without  head  of  my  copying  or  my written  ABSTRACT  Since Wiesel and Hubel first discovered that the responsiveness of the cortical neurons in the striate cortex to visual stimuli via either eye was subject to postnatal developmental manipulation, three decades have passed in strenuous efforts to unravel the mechanism underlying this ocular dominance plasticity. Among these studies, a variety of neurotransmitters/neuromodulators, including noradrenalin, acetylcholine and serotonin, have been implicated at both system and cellular levels. In view of the facts that noradrenalin changes neuronal excitability and the N-Methyl-D-aspartate (NMDA) receptor plays a crucial role in neuroplasticity, this study was designed to obtain evidence whether adrenergic modification worked through interacting with glutamergic system in terms of calcium dynamics and how this process occurred.  In primary neuronal cultures derived from the visual cortex of embryonic day 16-18 rats, intracellular free calcium concentration, [Ca ]i, was increased by bath application of 2+  glutamate in a dose dependent manner. Noradrenalin applied alone had relatively small effects. However, when glutamate concentrations eliciting modest increases in [Ca ]i 2+  were applied together with 1 |iM noradrenalin, the increase in [Ca ]i could be enhanced 2+  by a factor of up to eight in 147 neurons out of a total of 215 cells observed in 54 experiments. The observed enhancement was much more obvious at low doses of glutamate than with higher doses, augmenting all submaximal calcium responses to similar asymptotic levels. 2-Amino-5-phosphonovalerate (APV), the NMDA receptor antagonist, completely blocked the adrenergic enhancing effect (29/29 cells in 8 experiments). Among the antagonists specific to a l , a2 and p subtypes of adrenoceptors, the (3 antagonist propranolol most completely blocked the enhancing effect (13/14 cells in 4 experiments, by an amplitude of 90%). The involvement of the (3 receptor pathway was further supported by the ability of a cAMP analog to mimic the enhancing effect of noradrenalin. These ii  results suggest that receptors for noradrenalin and glutamate colocalize on postsynaptic cortical cells and that adrenergic modulation of glutamate induced calcium influx most likely work through the (3 receptor pathway. It is further postulated that ocular dominance plasticity may be at least partially implemented via a calcium dependent cascade.  iii  T A B L E OF CONTENTS Abstract  ii  Table of Contents  iv  List of Figures  vii  List of Tables  ix  Acknowledgment  -  x  1  INTRODUCTION  1  1.1 1.1.1  Neuroplasticity and experimental models Ocular dominance plasticity  1 2  1.2 Cellular and molecular mechanisms of ocular dominance plasticity 1.2.1 Effects of neurotransmitters on ocular dominance plasticity in cat visual cortex 1.2.2 Role of NMDA receptors in experience dependent neuroplasticity. 1.3  4 4 6  Effects of neuromodulators on the electrophysiological properties of  neurons 1.3.1 Adrenergic effects 1.3.2 Cholinergic effects  7 7 8  1.4 1.4.1 1.4.2 1.4.3  Morphological implications NMDA receptors Alpha adrenoceptors Beta adrenoceptors  9 9 9 10  1.5  The rationale for this study  11  2  METHODOLOGY  13 iv  2.1 2.1.1 2.1.2 2.1.3 2.1.4  Primary culture of visual cortical neurons Surgical procedures for taking the primary visual cortex Treatment of glass coverslips Dissociation of cortical neurons Characterization of neurons by immunocytochemical staining  13 13 13 17 18  2.2 Calcium imaging study using confocal microscopy 2.2.1 Why confocal microscopy 2.2.2 Microfluorometric measurement of intracellular calcium concentration with confocal microscope  18 19  2.3 Quantitative application of the pharmacological reagents 2.3.1 Specific considerations in the chamber design 2.3.2 Protocol for drug applications  24 24 25  19  3  RESULTS  28  3.1  Experiments were conducted on a pure population of visual cortical neurons  28  3.2  Dynamic changes in [Ca^+Ji were accurately measured by the confocal  system 3.2.1 Sampling rate and the potential problem of aliasing 3.2.2 Calcium increase in the cytoplasm is not less than in the nuclear region  28 28 34  3.3  Glutamate elicited a dose dependent calcium increase  37  3.4  Noradrenalin synergistically enhanced the glutamate induced calcium  3.5  3.6  increase  40  APV blocked the calcium rise caused by both glutamate and by glutamate plus noradrenalin  43  Implication of the (3 adrenoceptor in mediation of the enhancement  v  50  3.7  An analog of cAMP mimics the enhancing effect of noradrenalin  59  3.8  Cholinergic receptor activation also enhanced glutamate response  59  4  DISCUSSION  68  4.1  Enhanced calcium mobilization may be crucial in ocular dominance plasticity  68  4.2  How does noradrenalin enhance calcium mobilization  70  4.3  Is the enhancement unique to the adrenergic system  73  75  References  vi  LIST OF FIGURES Figure 1. A diagram showing the localization of the primary visual cortex (VI) in the rat brain  14  Figure 2. Linearity test of the input-output characteristics of the photomultiplier used in the confocal microscope  22  Figure 3. The perfusion system used for rapid and accurate drug delivery  26  Figure 4. Photomicrographs of immunocytochemical staining against MAP-2 and neuron specific enolase in cells grown for 10 days in vitro  29  Figure 5. The problem of aliasing  32  Figure 6. Comparison of the distribution of calcium concentrations at the peak responses to glutamate and to ionomycin Figure 7. Glutamate induced intracellular free calcium rises at four doses of glutamate  35 38  Figure 8. A pseudocolor illustration of the synergistic enhancement effect of glutamate induced intracellular calcium increase by noradrenalin  41  Figure 9. A typical pattern of adrenergic enhancement of glutamate induced increase in intracellular free calcium  44  Figure 10. Representative plots of the time course of adrenergic enhancement of glutamate induced calcium increase at four different glutamate concentrations  46  Figure 11. APV blockade of glutamate induced calcium rise and synergistic calcium rise caused by glutamate and noradrenalin  48  Figure 12. Effects of adrenoceptor subtype specific antagonists on the synergistic enhancement  51  vii  Figure 13. Summary of the effect of 20 |JM prazosin on adrenergic enhancement of glutamate induced calcium increase in cultured cortical neurons Figure 14. Summary of the effect of 20 |iM rauwolscine on adrenergic enhancement of glutamate induced calcium increase in cultured cortical neurons Figure 15. Summary of the effect of 20 uM propranolol on adrenergic enhancement of glutamate induced calcium increase in cultured cortical neurons Figure 16. The noradrenergic enhancement of glutamate induced intracellular calcium increase can be mimicked by db-cAMP Figure 17. Summary of the effect of db-cAMP mimicking the adrenergic enhancement of glutamate induced calcium increase in cultured cortical neurons Figure 18. A series of pseudocolor microphotographs showing the cholinergic enhancement on glutamate induced calcium increase in cultured neurons  viii  LIST OF TABLES  Table 1. Composition of the brain dissection buffer  16  Table 2. Composition of artificial cerebrospinal fluid  20  Table 3. A summary of the experiments that demonstrated the adrenergic enhancement, the effects of various antagonists and the study of intracellular pathways  67  ix  ACKNOWLEDGMENTS I would like to thank Dr. Max Cynader for having me as one of his students and offering me such a scientifically enriched environment to pursue my graduate study. I have learned and benefited so much from his expertise and profound knowledge in neuroscience. I would also like to thank him for providing me with a research assistantship and for funding my attendance of scientific conferences. Big thanks to Drs. Yihong Wang, Yulin Liu, Qiang Gu, Richard Dyck, William Jia and Yongchang Wang for their generous help and suggestions throughout the years we spent together. Your experience and encouragement are invaluable. Thanks also go to Drs. Paul Finlayson and Gavin Thurston for their technical help and suggestions. A special thanks to Dr. Robert Douglas, our amateur, probably a full-time I should say, system manager. Without you, I would have dozens of more hours of hardtime working out those problems with the computer networking and image processing, etc. I would also like to thank Jiren and Virginia for helping me out around the lab with everything. A special thank-you goes to my mom and dad for their encouragement and love. Last but not the least important, my sincere thanks to Dr. David Mathers for your co-supervising my graduate adventure. You are so generous and patient that I sometimes could not help feeling guilty spending you so many hours on things from note-taking to experiments. I learned so much from you, a true patch-clamper, and wish I could have spent more time in your lab. I will remember those moments spent discussing by the blackboard and fun of talking during coffee time. You have given me so many invaluable suggestions and advises. And at the turning point of my career, thanks seemed not enough for your understanding and encouragement.  x  1 1.1  INTRODUCTION  Neuroplasticity and experimental models  In the odyssey of understanding how learning and memory occurs, it has been speculated for decades that the efficiency of synaptic communication between neurons changes when information is stored in the brain. This synaptic plasticity hypothesis was substantiated when experimental evidence emerged to show the existence of a synapse with modifiable transmission efficiency. It was found that brief trains of high-frequency stimulation to hippocampal excitatory monosynaptic pathways resulted in an abrupt and sustained increase in the efficiency of synaptic transmission (Bliss 1973). This effect, called long-term potentiation (LTP), was later found to exist also in the neo-cortex of kitten and rat visual cortex (Aroniadou and Teyler, 1992; Artola and Singer, 1987; Komatsu, 1983), although it is still disputable whether memory is stored in the cortex. On the contrary, an inverse phenomenon called long-term depression (LTD) was recently identified in the hippocampus. In case of LTD, synaptic strength could be lessened after a prolonged low frequency (1 Hz) stimulation (Dudek and Bear, 1992; Mulkey and Malenka, 1992). The highly malleable nature of the brain was further reflected in the finding that LTP can be reversed after subjecting the preparation to a stimulation paradigm for eliciting LTD (Mulkey and Malenka, 1992). While LTP has long been extensively studied as a potential biological substrate of memory in the hippocampus, an earlier cortical model of neuroplasticity with many features in common, the ocular dominance plasticity (ODP) discovered in early 1960's, also stimulated a large amount of  1  investigation, leading to many parallel and comparable discoveries in the understanding of neuroplasticity (see below and also (Stent, 1973)).  1.1.1  Ocular dominance plasticity  Following their pioneering work to describe the physiology of cells in the visual cortex of the adult cat (Hubel and Wiesel, 1962), Wiesel and Hubel moved on to examine the role of visual experience in normal development in the early 1960's, a question raised and discussed by philosophers since the time of Descartes. They initially found that kittens with an eye temporarily deprived of vision by lid suturing during the first 3 months in early development were blind in the closed eye (Wiesel and Hubel, 1963). This was observed by recording the responsiveness of the cortical neurons to inputs from the two eyes. In normal conditions, most neurons in kitten primary visual cortex could be activated via either eye (Hubel and Wiesel, 1963). But under the experimental condition in which one eye was deprived of contour vision, most cortical neurons in the striate visual cortex responded only to stimulation of the normal eye (Wiesel and Hubel, 1963). The ability of the striate cortex to undergo a shift in ocular preference following monocular deprivation is called ocular dominance plasticity (ODP).  Ocular dominance plasticity was best seen only when the deprivation was done during a short period of time in postnatal development, namely, the critical period. The ocular preference in monkeys is most susceptible to such manipulation during the first 6 weeks of life and there was no shift in ocular dominance when the closure was done in the adult (LeVay et al., 1980). The discovery of a temporal window of ocular dominance plasticity has an important clinical significance in therapeutic strategy of congenital cataract. If the cataract is not removed early after birth, a permanent loss of vision in  2  these patients ensues. On the contrary, adult cataract patients will have normal vision when the cataracts are removed. It is very likely that the mechanisms of these two conditions are common in nature.  It was also observed that the highly malleable connectivity of cortical neurons with peripheral projections was determined as a result of competition rather than a consequence of disuse (Wiesel and Hubel, 1965). There was experimental evidence suggesting that this binocular competition occurs at the orientation-selective cortical neuron level between terminals of the lateral geniculate nucleus (LGN) cells connected to the two eyes (Cynader and Mitchell, 1977). An anatomical basis for the functional shift of ocular dominance was provided by results of transneural autoradiographic studies showing that the cortical territory in layer IVc occupied by LGN terminal fibers from the deprived eye considerably compromised in amount in comparison with that from the open eye (Hubel et al., 1977; LeVay et al., 1980; Shatz et al., 1977). Studies employing anterograde labeling of LGN projection fibers revealed that the compromise in territory was accompanied with a dramatic reduction in complexity of geniculocortical axonal arbors from the deprived eye as compared to that of the nondeprived eye (Antonini and Stryker, 1993). However, the effect on axonal arbor complexity could be observed in animals monocularly deprived of vision for at least 6 days (Antonini and Stryker, 1993), but the physiological effects of monocular deprivation are detectable after only 2 to 3 days of deprivation (Malach et al., 1984; Mioche and Singer, 1989; Movshon and Dursteler, 1977; Olson and Freeman, 1975; Olson and Freeman, 1978; van, 1978). The inconsistency between these two results suggests a functional base, such as inhibition of the input or a physiological down-regulation of the efficacy of existing synapses from the deprived eye, playing at least a partial role (Blakemore et al., 1982; Burchfiel and Duffy, 1981; Duffy et al., 1976; Mioche and Singer, 1989). A functional base for the plasticity of connectivity in the central nervous system is also reconcilable with the finding that eye  3  preference of the cortical neurons can be reversed if the once-deprived eye was opened and the once-open eye was sutured within the early part of the critical period (LeVay et al., 1980). These findings have been widely replicated and have since served as a reliable cortical model for neuroplasticity studies.  1.2  Cellular and molecular mechanisms of ocular dominance plasticity  1.2.1  Effects of neurotransmitters on ocular dominance plasticity in cat visual  cortex  In many studies endeavoring to unravel the mechanisms underlying this experience dependent plasticity, increasing evidence has implicated several neuromodulators. Among a variety of modulatory agents studied so far, noradrenalin was first implicated by the findings that destruction of noradrenergic projection fibers induced either by intraventricular or intracortical injection of 6-hydroxydopamine (6-OHDA) reduced ocular dominance plasticity (Kasamatsu and Pettigrew, 1976; Kasamatsu and Pettigrew, 1979). Since then many experiments have further elaborated this concept, but the degree and mechanisms of noradrenalin effects on neuroplasticity remain controversial (for a review see Gordon et al., 1988). Consistent results were obtained by other labs using the intracortical approach to deliver 6-OHDA (Bear et al., 1983; Daw et al., 1983; Paradiso et al., 1983) but not intraventricular injection (Adrien et al., 1985, Daw, 1985 #1069; Trombley et al., 1986). Furthermore, the doses of 6-OHDA necessary to affect plasticity were much higher than the minimal dose for maximal noradrenalin depletion (Trombley et al., 1986). In addition, systemic administration of 6-OHDA or disruption of the dorsal noradrenergic pathway, both of which caused significant  4  noradrenalin depletion in visual cortex, also failed to reduce ocular dominance plasticity (Bear and Daniels, 1983; Daw et al., 1984). These results casted doubts on the early assertion that 6-OHDA prevented plasticity by depleting noradrenalin alone.  A theory to explain these conflicting results was proposed in a report showing that only combined depletion of adrenergic and cholinergic innervation could reduce ocular dominance plasticity while blockade of either system alone was not effective (Bear and Singer, 1986). In these experiments, combined cholinergic depletion by N M D A neurotoxic lesion in the basal forebrain and adrenergic depletion by 6-OHDA lesion in the dorsal noradrenergic bundle blocked plasticity as effectively as surgical lesion of cingulate gyrus through which both cholinergic and adrenergic fibers project to the neocortex. This study further showed that 6-OHDA could, in addition to disrupting noradrenergic projection fibers, suppress cholinergic transmission, suggesting that intracortical 6-OHDA prevents plasticity by simultaneously suppressing both adrenergic and cholinergic modifications (Bear and Singer, 1986). Later on, it was found blocking cholinergic innervation by intracortical infusion of scopolamine can also disrupt ocular dominance plasticity (Gu and Singer, 1993; Kasamatsu et al., 1989).  Targeting of receptor subtypes responsible for modifying the plasticity initially fell on the P adrenoceptor, because (3 or (31 receptor antagonists not a antagonists were found capable of preventing plasticity, although to an extent hardly as great as 6-OHDA (Kasamatsu and Shirokawa, 1985; Shirokawa and Kasamatsu, 1986; Shirokawa et al., 1989). This was supported by the report that infusing dibutyryladenosine-cyclic monophosphate (db-cAMP), an analog of the second messenger cAMP used in the (3 receptor pathway, could restore the usual blockade of plasticity by 6-OHDA (Kasamatsu, 1980). Later, it was also reported that systemic or intracortical delivery of lithium effectively blocked ocular dominance plasticity (Kasamatsu et al., 1991), suggesting that  5  lithium sensitive processes, for example phosphatidyl inositides (PI) turnover which is the intracellular signal transduction pathway of many receptors including a l , M l and M3, are also responsible for mediating plasticity. New evidence supports this conclusion. It was found that blockade of M l receptors, but not M2 or nicotinic receptors, indeed prevented ocular dominance plasticity (Gu and Singer, 1993).  1.2.2  Role of N M D A receptors in experience dependent neuroplasticity  Glutamate has been widely accepted as an excitatory neurotransmitter in the CNS (Headley and Grillner, 1990). Its receptors occur as at least four major subtypes, namely the ionotropic category including NMDA,  AMPA  and kainate receptors and the  metabotropic receptor group. It has now become clear that the NMDA type of glutamate receptors play an important role in mediating experience dependent neuroplasticity (for review see (Bliss and Collingridge, 1993; Fox and Daw, 1993)). It was found that minipump infusion of the NMDA receptor specific antagonist APV into the cortex reduced or abolished ocular dominance shift (Bear et al., 1990; Kleinschmidt et al., 1987; Rauschecker et al., 1990). In the hippocampus, APV can effectively prevent induction of LTP (Collingridge et al., 1983). One important consequence of NMDA receptors activation is an influx of extracellular calcium into the cytoplasm in a voltage dependent manner (conferred by M g  2 +  inhibition) (Mayer and Miller, 1990). Extensive studies  show that calcium triggered intracellular events are involved in producing LTP (Regehr and Tank, 1990) and that blockade of this process by intracellular calcium chelation adversely affects production of synaptic plasticity (Brocher et al., 1992; Kimura et al., 1990).  6  It is an important characteristic of NMDA receptors that the functional state of the glutamate-NMDA-receptor-calcium axis is highly dependent on the excitability of the postsynaptic neurons. A strong calcium influx is achieved only if the depolarization of the postsynaptic neuron is large enough to remove the M g  2 +  ion inhibition at the same  time that glutamate activates NMDA receptors. This property of NMDA receptor makes it particularly susceptible to the hyperpolarizing influence of synaptic inhibition (Collingridge et al., 1988) and accounts for the frequency-dependence of the induction of LTP (Davies et al., 1991). Therefore, it is conceivable that any factor that can modify the resting potential of a neuron or its excitability may more or less change the efficacy of glutamate-NMDA-receptor-calcium axis.  1.3  Effects of neuromodulators on the electrophysiological properties of neurons  Among all the neuromodulators, adrenergic and cholinergic systems are two of the more extensively studied in terms of their effects on the electrophysiological properties of postsynaptic neurons. This literature is so extensive that only a few relevant papers are reviewed here.  1.3.1 Adrenergic effects  The locus coeruleus (LC) nucleus sends projection fibers to virtually all structures in the mammalian central nervous system (Itakura et al., 1981). In the adult rat hippocampal slice, either exogenous or endogenous noradrenalin was found to be able to initiate both a short- and a long-term potentiation (LTP) of the dentate gyrus response to perforant path input (Harley, 1991). This potentiating effect depends on beta receptor  7  activation and does not require a high-frequency stimulus. In the visual cortical slice, noradrenergic activation raised the probability of tetanic stimuli induced LTP, also via beta receptors (B roc her et al., 1992). Noradrenalin enhanced the depolarizing response to the tetanus and increased NMDA receptor-gated conductance during this response (Brocher et al., 1992). In other studies, it was found that activation of beta -adrenergic receptors resulted in an enhanced excitability and responsiveness of cortical pyramidal cells to depolarizing inputs (Dodt et al., 1991; McCormick et al., 1991). This effect was found to be mediated either by an increased amplitude of the APV sensitive late component of the excitatory postsynaptic potential (EPSP) (Dodt et al., 1991) or by a reduced spike frequency adaptation by a marked suppression of a slow C a  2 +  activated  potassium current known as I (AHP) (McCormick et al., 1991).  1.3.2  Cholinergic effects  The cholinergic system in the mammalian central nervous system originates from the basal forebrain and projects widely to almost all areas of the brain. By recording intracellular responses of CA1 neurons in the hippocampal slice to iontophoretic application of NMDA, it was found that this response was potentiated by acetylcholine (Markram and Segal, 1990). In the LTP model of the rat visual cortical slice, activation of the cholinergic system was found to increase the chance to induce LTP by raising the excitability of the neurons (Brocher et al., 1992). Further studies showed that acetylcholine acted synergistically with noradrenalin in terms of the enhancing effect (Brocher et al., 1992). In both studies, the muscarinic subtype of cholinergic receptor was targeted as mediating this potentiating effects. Activation of muscarinic receptors can either activate PI turnover or decrease intracellular cAMP levels depending on whether M l , M3 or M2, M4 subclasses of muscarinic receptors were involved. From evidence  8  obtained by manipulating the intracellular cascades related to PI turnover, it was proposed that activation of the inositol trisphosphate (InsP-3) branch of the phosphoinositide pathway potentiated responses to NMDA and that InsP-3 exerted this effect by elevating [Ca ]i (Markram and Segal, 1992). This was in line with data 2+  obtained using calcium imaging, showing that noradrenalin potentiated an NMDAevoked calcium rise in hippocampal neurons (Segal, 1992).  1.4  Morphological implications  1.4.1  NMDA receptors  Using immunocytochemical and receptor autoradiographic studies, Cynader and colleagues have systematically studied the localization during postnatal development of a variety of neurotransmitters and neuromodulators. In the kitten, NMDA receptors showed a changing laminar distribution during the peak period for visual cortex plasticity (Cynader et al., 1991). NMDA receptors predominated in layers IV and V during postnatal day 0-10. But by postnatal day 20-30, the dominance shifted to layers IV. By 40 days of age through adulthood, their peak concentration was in layers I to III. The coincidence of the highest NMDA receptor concentration in layer IV during the peak of the kitten's critical period implies a crucial role of the receptor in visual cortical plasticity.  1.4.2 Alpha adrenoceptors  9  The developmental profiles of the a l and a2 receptor binding patterns in kitten are similar and also undergo remarkable laminar changes (Jia et al., 1994). Binding sites for both receptors were scarce throughout all six cortical layers at birth. While an overall increase in their binding densities was observed with age in all layers, cortical layers I and IV were much more densely labeled at 10 days of age for a l and a few days later for a2 receptors. This laminar pattern became more clear at postnatal days 30-40 and the peak densities in superficial and middle layers were observed at postnatal days 60-75. Thereafter, a gradual overall reduction in binding densities occurred with the middle layers showing the greatest decline. As a result, the superficial layers became the most densely labeled ones in adulthood. In addition, the susceptibility of this developmental patterns to input deprivation was also studied by making lateral geniculate nucleus (LGN) lesions, severing the dorsal bundles, or by making optic tract lesions (Jia et al., 1994). It was found that a more substantial reduction in binding densities was observed of a2 than a l receptors after these surgical manipulations. Unlike a l sites, where the reduction appeared homogeneous across the cortical laminae following the lesions, the most affected layer in case of a2 receptors was layer IV.  1.4.3  Beta adrenoceptors  The developmental localizations of (31 and 02 adrenoceptors in kitten visual cortex were similar and are therefore summarized here collectively as [3 receptors (Liu et al., 1993). From birth through postnatal days 20, binding sites of (3 receptors were mainly located in the middle and deep cortical laminae, namely layers IV-VI. Between days 2030, binding densities began to increase dramatically in both superficial and deep layers, especially in the superficial ones. The increase in layers I and II was so great that the density in these layers became comparable to that in layer V and VI at days 30-40. In  10  contrast, the binding in layers III and IV became less and less during this period of development. From day 40 onward, the pattern of high binding densities in superficial and deep layers remained similar through adulthood. In contrast to the case with a adrenoceptors, development of the (3 adrenoceptors seemed much less vulnerable to either L G N lesions or to adrenergic deprivation (Liu et al., 1993). The hemisphere undergoing such surgical manipulations showed a similar pattern of receptor distribution as the control side, with two dense bands marking the superficial and deep layers of the visual cortex, separated by a band of lower binding in layer IV. However, a reduction in the binding densities was noted on the lesion side compared to the contra-lateral side.  Immunocytochemical staining against p adrenoceptors revealed morphological information at a much higher spatial resolution than receptor autoradiography. The laminar distribution seen in immunostaining basically paralleled that found by receptor autoradiography, with immunoreactivity mainly in superficial and deep layers (Liu et al., 1993; Liu et al., 1992). In both kitten and adult cat visual cortices, the immunoreactive elements were mostly neurons with a few astrocytes positively stained. From postnatal days 24 to 40, most of the positively stained neural cells were pyramidal cells. In addition, the immunoreactivity was found to be associated with both neuronal perikarya and neuronal processes, with the most dense staining being found at cell surface membranes, in the cytoplasm and along the dendrites of neurons.  1.5  The rationale for this study  In view of the findings that 1) ocular dominance plasticity is modifiable by adrenergic manipulation, 2) noradrenalin changes neuronal excitability, 3) the NMDA receptor plays a crucial role in neuroplasticity, 3) calcium influx through NMDA receptor  11  triggers important intracellular cascades, 4) the dynamic laminar redistribution of adrenoceptors coincides with that of NMDA receptors during the critical period, the following questions are addressed in this study: l)Does the adrenergic system interact with glutamergic system in terms of calcium dynamics ? 2)What is the nature of these potential interaction ? 3) What intracellular pathways are responsible for the potential modifiability ?  12  2  METHODOLOGY  2.1  P r i m a r y culture of visual cortical neurons  2.1.1  Surgical procedures for dissecting a n d dissociating the p r i m a r y visual cortex  A tissue culture model system of dissociated cortical neurons was used in this study. 16-18 day pregnant Long Evans rats (Charles River) were decapitated under halothane anesthesia. Standard aseptic procedures were exercised in all later surgical steps. Rat embryos were removed from the uterus and securely positioned on a sterile wax plate. Under a dissection microscope, both hemispheres were completely exposed. The primary visual cortex (VI) was located as shown in Figure 1. An approximately 2X3 mm piece of visual cortex was removed with a scalpel blade and kept in ice cold C a and M g  2 +  2 +  free Hank's balanced salt solution (HBSS, Gibco) with composition as shown  in Table 1. The pia mater of an embryonic brain at this age was easily peeled off with a pair of fine forceps. Removal of the pia mater effectively reduces the amount of cell types other than neurons in the cultures.  2.1.2  Treatment of glass coverslips  Glass coverslips were chosen as the substrate upon which to grow neurons, as these 80 micron thick coverslips fitted optimally with the working distance of the lens 13  Figure 1. A diagram showing the localization of the primary visual cortex (VI) in the rat brain. At gestation day 17 and thereafter, both hemispheres are well exposed when the skull is opened. A small block of tissue from the primary visual cortex can be readily removed with a small scalpel blade. The tissue block prepared for dissociation measures approximately 2X3 mm in size.  14  15  Table 1 Composition of the brain dissection buffer (mM). pH = 7.4.  NaCl  138  KC1  15.7  KH2PO4  0.3 4  0.3  Dextrose  18  NaHC0  4  Na HP0 2  3  15.7  HEPES  16  used in the imaging system. Cleanliness is an important factor in culturing neurons, so the following procedures were strictly followed. Coverslips (Biophysica Technologies) 1 inch in diameter were mounted on porcelain rack and washed in distilled water. Then racks of coverslips were immersed in concentrated nitric acid for at least 48 hours. Thereafter, a two hour wash in running water was followed by two washes of one hour each by immersion in distilled water in order to thoroughly clean the coverslips. Then the coverslips were dried and autoclaved before use. Truly clean coverslips were identified by the fact that water did not build up at the center but spread easily around.  Coating of the coverslip with poly-L-lysine was essential for neurons to adhere properly to the coverslip. Before each experiment, coverslips were placed in a 35 mm petri dish and covered with 0.5 ml of poly-L-lysine (0.1 mg/ml) for 2 hours. Then the coverslips were washed twice with distilled water and allowed to dry immediately before plating cells onto them.  2.1.3  Dissociation of cortical neurons  On the average, the brain tissue obtained from 10 hemispheres were enough to plate 20 coverslips 1 inch in diameter. The small pieces of tissue were minced with iris scissors before being transferred into a centrifuge tube containing 2 ml of 0.25% trypsin (Sigma) and 1 ml of 0.1% DNAse I (Boehringer Mannheim). The tissue fragments were incubated at 37 °C for 5 minutes and then 2 ml of ice cold fetal bovine serum (Gibco) was added to stop enzyme action. Gentle trituration with a plastic pipette could bring cells into complete dissociation . The cell suspension was subjected to centrifugation at 1500 g for 2 minutes. The pellet was resuspended in 3 ml of minimum essential medium (MEM, Gibco) supplemented with (in mM): L-glutamine 4, NaHC03 16, HEPES 20 and  17  10% fetal bovine serum, pH 7.4. After two washes with M E M , a suspension of approximately one million cells per ml M E M was made and one 0.5 ml of the cell suspension was plated on to each poly-L-lysine coated 1 inch glass coverslip. The density of cells plated on the coverslip was a crucial factor in obtaining good quality neuronal cultures. Cell suspensions with the above concentration gave optimal cell density when the 0.5 ml suspension was plated to cover an area approximately 15-18 mm in diameter on the coverslips. The dishes were filled with additional M E M to a 2 ml final volume about 30 minutes after plating and placed in a 5% CO2 incubator at 37 °C. One or two days after plating, cells were exposed to M E M containing 20 | i M cytosine arabinofuranoside (Sigma) for 24 hours to inhibit the proliferation of glial cells. The medium was then changed twice a week and cells were grown for a total of 10-14 days before experimentation.  2.1.4  Characterization of neurons by immunocytochemical staining  In order to characterize the nature of the cells used in this study, conventional immunocytochemical staining against microtubule associated protein (MAP-2) and neuron specific enolase (NSE) was applied to distinguish between neurons and nonneuronal cells. Anti-MAP-2 and anti-NSE antibodies were obtained from Sigma and Polysciences respectively. Standard A B C immunocytochemical procedures were followed and photomicrographs were taken on an inverted Nikon Optiphot-2 microscope.  2.2  Calcium imaging study using confocal microscopy  18  2.2.1 Rationale for use of confocal microscopy  The long-wavelength calcium indicator fluo-3 undergoes greater enhancement of emission intensity upon calcium binding than fura-2. This superior photon efficiency of fluo-3 allowed us to use very low light intensity for imaging. Moreover, the confocal microscope employs high speed scanning of a single laser beam to reconstruct a 2-D image and therefore effectively reduces exposure time to the light source as compared with the global illumination used in a fura-2 system. As the nature of our experiments required repetitive recording, we utilized these advantages of fluo-3 to significantly reduce the phototoxicity otherwise encountered with more intense UV light as in fura-2 system. In addition, the autofluorescence associated with biological samples is often less of a problem if the preparation is excited at longer wavelengths. Although the lack of spectral shift of fluo-3 upon calcium binding eliminated the possibility of ratiometrical measurement, the calibration discussed below, if used properly, proved to be consistently reliable especially in terms of intra-experimental error.  2.2.2 Microfluorometric measurement of intracellular calcium concentrations with the confocal microscope  For imaging studies, a coverslip with a high density of healthy neurons was chosen and gently washed with artificial cerebral spinal fluid (ACSF) bubbled with 5%95% carbogen to bring the pH to 7.4. The composition of ACSF was as shown in Table 2. Cells were then loaded with the calcium indicator fluo-3 by immersion in ACSF containing 1 pJVI acetoxymethyl ester fluo-3 (Molecular Probes) at room temperature for 30 minutes. The coverslip was mounted on a chamber (Bionique Laboratory) modified to accommodate one inlet and two outlets for continuous perfusion (see details below).  19  Table 2. Composition of artificial cerebrospinal fluid (mM). pH = 7.4 when bubbled with 5%-95% carbogen.  NaCl  124  KC1  3  CaCl  1.6  2  KH P0 2  MgS0  0.75  4  1.2  4  NaHC0  24  3  glucose  10  glycine  0.005  20  Microfluorometric measurements were performed on a Bio-Rad MRC-600 laser scanning confocal system configured with a Nikon DIAPHOT-TMD inverted microscope, using a 60X, 1.4 numeric aperture Nikon PlanApo oil objective. Neurons were identified in'the epi-fluorescence mode and then imaged with the confocal system using excitation at 488 nm and signal collection through a 515 nm long pass filter. Fluorescent signals amplified through the photomultiplier tube were sent to a frame grabber card installed in an IBM 80486 computer to reconstruct a 2-D image. The black level, gain, pinhole aperture, and neutral density filter value were carefully set each time in order to utilize only the linear segment of the photomultiplier's input-output characteristic curve shown in Figure 2.  The application software was configured so that a time series of pictures could be taken at the best possible temporal resolution without sacrificing spatial resolution. This procedure eliminated any possibility of temporal aliasing, as confirmed by using an extremely high temporal resolution line scanning mode in some experiments (see details in section 3.2.1). At the end of each experiment, fluorescent signals were calibrated using a method introduced by Kao et al (Kao et al., 1989) and converted into absolute calcium ion concentrations. Further data analyses were done after the original image files were transferred onto a Macintosh Ilfx and imported into an image processing software program NIH Image 1.51. The calibration method is briefly described as follows. The calcium ionophore ionomycin was added into the chamber to a final concentration of 2 |iM and cells quickly became saturated with extremely high intracellular calcium levels. MnSOu (2 uM final concentration) was added to quench the fluorescence and the pixel values within the contour of a cell were determined by NIH Image, i.e., FMn. Then digitonin (80 uM final concentration) was added to release fluo-3 from the cells. This allowed determination of background fluorescence by measuring the cell residues, i.e.  21  Figure 2. Linearity test of the input-output characteristics of the photomultiplier used in the confocal microscope. Readings of fluorescence intensity from the photomultiplier were plotted as a function of FITC dextran concentrations. In all experiments, the system was configured as such that the fluorescent calcium signals were measured within the area of the curve far below pixel value of 200, where best linear correlation was achieved by the system.  22  300  i-  >  250  \-  (D X •H  200  r-  Ul i—I  150 100  h  50  \-  0  •  0  100  200  •  »  •  300  •  •  • • ' • »  400  i • • • • I • • • •  500  600  I  700  FITC d e x t r a n J i g / m l  23  Fbkg. The absolute calcium concentrations, [Ca ]i, were estimated using following 2+  equations. Fmax = (FMn- Fbkg)/0.2 + Fbkg Fmin = (Fmax-Fbkg)/40 + Fbkg [Ca ]i = Kd(F-Fmin)/(Fmax-F) 2+  Here, Fmax is the maximum fluorescence, Fmin is the minimum fluorescence, Kd is dissociation constant of fluo-3 and F is the average fluorescence of the cell in study.  2.3  Quantitative application of pharmacological reagents  The quantitative nature of this study required us to design a drug application system that would allow exactly known concentrations of reagents to reach the target neurons. This was achieved by two types of technical procedures.  2.3.1 Specific considerations in the chamber design  Direct dropping of concentrated reagents into the perfusion chamber was initially attempted. In this approach, one faced two difficulties. First is the complete uncertainty of the drug concentration around observed neurons. The other is the unpredictability of the pattern of drug distribution in the chamber, resulting in inconsistent responses of neurons to the same dosage of drugs used in different trials. Finally, we came to a solution by making a chamber with the dual outlet perfusion system shown in Figure 3. As illustrated in the figure, one outlet allowing a sufficient pool of ACSF to cover the cells could be switched with another outlet that could suck away most perfusion fluid over the cells gently but rapidly. Thereafter, the chamber could be refilled with ACSF containing known concentrations of one or a combination of drugs of interest. Extreme caution had  24  to be exercised to avoid complete exposure of neurons to air by allowing a thin layer of fluid to still cover the neurons when switching between the two outlets. Exposure to air inevitably introduced an artifact by causing a massive rise in intracellular calcium levels in the neurons.  2.3.2 Protocol for drug applications  By using the specially designed chamber discussed above, one could quantitatively apply pharmacological reagents at accurate concentrations as desired. In all experiments, monosodium glutamate of the desired final concentration was prepared in the artificial cerebrospinal fluid (ACSF) used to perfuse the neurons. Then perfusion was stopped and excessive ACSF was drained by switching the outlet to the lower one. Then glutamate was added into the chamber in replacement of the drained ACSF. On the other hand, noradrenalin and/or the antagonists of either NMDA receptors or adrenoceptor subtypes were added into the perfusing ACSF (therefore exact concentrations were known) and continuously applied to the neurons over a 3 minute time window during which glutamate was applied at the beginning of the third minute, mimicking background adrenergic stimulation.  25  Figure 3. A: Schematic drawing of the perfusion system designed for rapid and accurate drug delivery. When switched to outlet A, a high fluid level was maintained, which was desirable during the long interval between stimulations. When switched to outlet B, most fluid was drained, leaving a thin layer of perfusing fluid just covering the neurons. The volume of the remaining fluid was too small to significantly dilute the bath, which contained one or a combination of reagents at known concentrations. B: a photograph of the perfusion chamber used in these experiments.  26  A  27  3 3.1  RESULTS  Experiments were conducted on a pure population of visual cortical neurons Under normal conditions, over 90% of the originally plated cells could survive  until the time of the experiment. At this stage, the processes of these phase bright neuronlooking cells extended exuberantly to form an extensive mesh-like network. These cells assumed a mostly pyramidal configuration, with bipolar and round neurons also in evidence. Neurons could be readily distinguished by morphology from the underlying large and polygonal fibroblasts and glioblasts. Immunocytochemical staining showed that cells assuming a pyramidal profile were 100% MAP-2 and NSE positive, while only some of the bipolar and round cells were positively stained (Figure 4). Therefore, by preferentially studying the cells assuming a pyramidal configuration, only cortical neurons were included in our experiments .  3.2  Dynamic changes in [Ca ] were accurately measured by the confocal system 2+  3.2.1 Sampling rate and the potential problem of aliasing  Emission intensities from fluo-3 loaded cells were sampled at 0.33 Hz. This means one image was captured in every 3 seconds, a rate capable of giving a satisfactory combination of spatial and temporal resolution. This sampling rate was determined by the 28  Figure 4. Photomicrographs of immunocytochemical staining against MAP-2 and neuron specific enolase in cells grown for 10 days in vitro. Al: phase contrast picture of the cells in culture; A2: immunostaining against MAP-2; Bl: phase contrast picture of the cells in culture; B2: immunostaining against neuron specific enolase. Note that all the pyramid shaped cells were positively stained for both markers, indicating that the pyramidal cells chosen for study in these experiments were neurons.  29  30  31  Figure 5. The problem of aliasing during sampling was ruled out by an experiment using line scan mode at 15 times (5 Hz) the sampling rate used in all experiments (0.33 Hz). A: an illustration showing the image of two pyramidal cells included in this study. The green line marks where the scanning laser beam is parked to repeatedly sample fluorescence intensity during a time span of 30 seconds; B: a pseudocolor digital photomicrograph of a series of  150 scanning lines taken at  0.2 second intervals. Calibrated calcium  concentrations were color coded as shown in the underlying color spectrum; C: a plot of fluorescence intensities for the right hand cell shown in B, as a function of time; Note the noise level is relatively high in this plot, derived from single line results. Under real experimental conditions, a 2-D image consisting of multiple lines should yield a much smoother graph. This was confirmed by the plot averaged from 4 lines of data as shown in D; E: a plot of a typical calcium response curve versus time measured at the standard sampling rate of 0.33 Hz. Note that this graph quite faithfully resembles the one shown in D.  32  33  following procedure. Firstly, the line scan mode of the confocal system was chosen to sample the fluorescence intensities over a fixed line crossing the cell body of a neuron (Figure 5 A). Because the laser beam scans at the same place over and over again, the sampling rate can be as high as 100 Hz. As a result, a high temporal resolution series of fluorescence signals at that line can be constructed against time. As shown in Figure 5 B, a series of 150 lines with a 0.2 second interval were scanned to cover a 30 second period of time at 5 Hz sampling rate. Then signal intensities averaged from each line can be plotted against time as shown in Figure 5 C. Secondly, series of 2-dimensional images were taken of the same neuron under exactly the same stimulation conditions. As shown in Figure 5 E, a typical response of neuron to glutamate can be faithfully recorded at 0.33 Hz without aliasing. This sampling rate can be readily achieved on the confocal system at the second best spatial resolution. Neither higher sampling rates at the expense of spatial resolution nor the best image quality at the expense of much lower sampling rate was chosen in our experiments.  3.2.2  Calcium increase in the cytoplasm was not less than in the nuclear region  When the intracellular calcium level of neurons was elevated upon glutamate stimulation, a bigger increase in fluorescence was observed in the nuclear region than in the remainder of the cell (Figure 6 A). To test whether this truly reflected a larger increase of calcium concentration at the nuclear region, images taken at the peak responses to either glutamate or ionomycin were compared. As ionomycin causes a global increase of intracellular calcium by opening pores on the cell membrane, increases in calcium concentration should be of the same amplitude in both the nucleus and the cytoplasm. However, as shown in Figure 6, ionomycin elicited a similar calcium concentration pattern as seen with glutamate. Since cells on the coverslips have a bulging  34  Figure 6. Comparison of the distribution of calcium concentrations measured at the peak of responses to glutamate and to ionomycin. The upper panels showed digital photomicrographs taken on the confocal microscope. The lower panels are 3-dimensional representions of the intracellular calcium concentrations shown in the corresponding photos, with vertical heights proportional to local calcium concentrations. Note the distribution patterns in A and B were not significantly different. The scale bar is 5 microns in length.  35  A. glutamate  B. ionomycin  36  nuclear region, much thicker than the rest of the cells, it was suspected that this nuclear preponderance in calcium increase was probably an artifact resulting from cell geometry.  3.3  Glutamate elicited a dose dependent rise i n [ C a ] i 2 +  The basal level of [Ca ]i in neurons was 70±12 nM and spontaneous fluctuations 2+  usually did not exceed 10 nM. Occasionally, dramatic spontaneous increases in [Ca ]i 2+  were noted when cells were exposed to excessive excitation UV light during a cell search session. The spatial distribution of intracellular calcium at rest appeared homogeneous within most neurons. However, some cells did show slightly lower or higher fluorescent signals at the cell center, delineating a clear nuclear contour, especially in cases of lower fluorescence in the nuclear region. Upon application of glutamate, a dramatic increase in [Ca ]i was elicited in the large majority of neurons studied. Intracellular calcium levels 2+  reached their peak within 2 to 4 seconds (Figure 5 C). In most cases, [Ca ]i levels could 2+  rise to hundreds of nM, with occasional overshoots above mM levels. The glutamate induced increase in [Ca ]i was obviously dose dependent, as shown in Figure 7. Higher 2+  doses resulted in larger increases in [Ca ]i, with a plateau observed when saturating 2+  concentrations of glutamate were used. The threshold concentration for glutamate to elicit a calcium response was constant for individual groups of cells studied in particular experiments. It did vary however considerably between batches of cells, ranging from 1 |iM to 15 uM. Usually a higher threshold was observed in coverslips with higher cell densities. This might be a result of a facilitated interneuronal communication with inhibitory neurons coactivated by glutamate.  37  Figure 7. Glutamate induced intracellular free calcium increases (nM) at four doses of glutamate (|J.M). High doses of glutamate elicited larger calcium increases, a plateau being reached at 25-30 uM levels in this case. Data represent values averaged from seven cells in one experiment, and are expressed as mean plus one standard error. Similar results were obtained in three more experiments with slight changes in threshold and plateau concentrations of glutamate. Between glut 15 and glut20 p = 0.006, between glut20 and glut25 p = 0.059, between glut25 and glut30 p = 0.064.  38  glut 15  glut 20  glut 25  39  glut 30  3.4  Noradrenalin synergistically enhanced the glutamate induced calcium  increase The natural neuromodulator noradrenalin (NA) was used to modulate glutamateevoked calcium responses in these experiments. Noradrenalin-HCl (RBI) was always freshly prepared and kept from light and air to avoid oxidation. Neurons were continuously exposed to N A about 2 minutes before, during, and after transient glutamate stimulation. This stimulation paradigm was achieved by switching between ACSF solutions containing exactly known concentrations of NA alone or N A plus glutamate. In our early experiments, a concentration of 50 pM NA was chosen. When neurons were subjected to this concentration of NA alone, a very small increase (less than 20% of the resting calcium level) in [Ca ]i was elicited in the majority of neurons. 2+  Greater increases could be observed in cell types other than neurons, such as the large underlying fibroblasts. However, when glutamate concentrations eliciting a small increase in [Ca ]i if given alone were added to a background level of 50 (xM NA, a 2+  synergistic increase in [Ca ]i was observed (Figure 2D). We later found that N A 2+  concentrations as low as 1 |LiM were effective in enhancing the glutamate induced increase in [Ca ]i, with no difference between the amplitudes of enhancement at the two 2+  concentrations (data not shown). This synergistic interaction was observed in a total of 147 neurons investigated in 54 different experiments (see Table 3, P < 0.001). Figure 8 shows a digital pseudocolor photomicrograph of a typical case of enhancement. Here, a moderate response (peak [Ca ]i at 265 nM) elicited by glutamate was enhanced (peak 2+  [Ca ]i at 676 nM) by 50 pM noradrenalin. 2+  When lower concentrations of glutamate were used to elicit a smaller calcium response, the enhancement effect of noradrenalin was much more evident. In Figure 9, enhancement at lower glutamate doses was plotted from 14 neurons studied in 5 separate  40  Figure 8. A pseudocolor illustration of the synergistic enhancement of glutamate induced intracellular calcium increase by noradrenalin. In the left column, two neurons were stimulated with 30 |iM glutamate alone. The middle column showed the effect of noradrenalin alone on the resting level of intracellular calcium. The right column showed a enhanced response of calcium concentrations to coapplication of glutamate and noradrenalin. Note the neuron on the left hand side showed an elevated resting calcium level even before stimulation because it had become sick after multiple stimulation with glutamate, but the neurons on the right hand side was responding in excellent condition. Only every other frames were presented here, with therefore each frame 4 seconds apart from each other. The color code was as same as the one used in Figure 5.  41  glutamate 30 fjM  noradrenalin 50 IJM  glutamate 30/vM noradrenalin 50  ft  12 sec  .C.  %  18 sec  24 sec  0  30 sec peak [Ca ]i = 265 nM  peak [Ca ]|= 676 nM  2+  2+  42  experiments. Here 1.25 pM glutamate alone elicited a nominal increase of [Ca ]i. While 2+  l p M NA yielded a negligible increase in [Ca ]i, the same concentration of glutamate 2+  given together with 1 pM NA resulted in an increase in [Ca ]i of almost an order of 2+  magnitude.  To further characterize the nature of this enhancement, experiments shown above were repeated at four different glutamate concentrations. As shown in Figure 10, noradrenalin enhanced the glutamate induced increase in intracellular calcium concentrations much more significantly at lower glutamate levels than at higher ones, ranging from hundreds of percent increases at 15 pM glutamate to none at 30 p M glutamate. In another words, adrenergic enhancement was capped when glutamatergic transmission reached certain defined levels. This result was in line with the saturability of the intraneuronal increase in calcium upon glutamate stimulation, illustrated in Figure 7.  3.5  APV blocked the calcium rise caused by both glutamate and by glutamate  plus noradrenalin  2-amino-5-phosphonovalerate (APV), an efficient antagonist of NMDA subtype of glutamate receptors, was found capable of completely blocking the glutamate induced calcium rise in neurons at a 25 pM concentration (Figure 11), suggesting the glutamate induced calcium rise was mediated by calcium influx through NMDA receptors. When 25 pM APV was given together with NA and glutamate, the synergistic rise of [Ca ]i 2+  was also completely blocked (Figure 11 and Table 3). These consistent findings observed in many experiments indicated that the adrenergic enhancement of glutamate induced calcium rise was indeed receptor mediated, with NMDA receptors being a crucial factor for triggering the process (see discussion later). It was also noted in a few experiments  43  Figure 9. A typical pattern of adrenergic enhancement of glutamate induced increase in intracellular free calcium. A: resting calcium concentration; B: small response induced by a low concentration of glutamate, 1.25 uM; C: calcium concentration two minutes after 1 uM noradrenalin in bath, D : greatly enhanced calcium response induced by 1.25 uM glutamate in combination with 1 |iM noradrenalin. Data were averaged from 14 neurons studied in five experiments and expressed as mean plus one standard error.  44  p < 0.01.  500 "  +  450 -  co 400 A  P  CM  CJ  350 300 250 200 150 100  H  50 0  D  B  45  Figure 10. Representative plots of the time course of adrenergic enhancement of glutamate induced calcium increase at four different glutamate concentrations. Cells were exposed to glutamate with specified concentrations at the time as indicated by the arrow.. Note that the amplitudes of adrenergic enhancement of glutamate induced increase in intracellular free calcium were more significant when low dose of glutamate was used. Open squares: glutamate at specified concentration plus 50 u M noradrenalin. Filled squares: glutamate at specified concentrations alone.  46  47  Figure 11. A representative plot of one of eight experiments, showing that A P V completely blocked the glutamate induced calcium increase as well as the synergistic calcium rise caused by glutamate and noradrenalin. The number following each reagent indicated the |iM concentrations of that reagent. Abbreviation: glut = glutamate, N A = noradrenalin, APV = amino-phosphonovalerate. Data were expressed as mean plus one standard error, and n=4. t p < 0.05, * p < 0.01.  48  c  225  1 4-  ^7  200  U  S  1 5 0  o c 125  100  75  H  50  H  25  H  glut. 2.5  glut. 2.5 NA 50  glut. 2.5 APV 25  49  glut. 2.5 NA 50 APV 25  that a lower concentration of APV (12.5 uM), just sufficient to block glutamate induced calcium rise, failed to completely block the synergistically enhanced calcium rise (data not shown). The fact that a higher level of APV was required to block the calcium rise implied that an increased sensitivity of NMDA receptor-channel complex might be a contributing mechanism.  3.6  Implication of the (3 adrenoceptor in mediation of the enhancement  Adrenoceptors have been pharmacologically classified into three major subtypes, namely a l , a 2 and p\ each of which mediates different cascades of intracellular biochemical events. In an attempt to single out the specific receptor subtype (s) responsible for the enhancing effect of noradrenalin, specific antagonists of a l (prazosin), a2 (rauwolscine) and p (propranolol) subtypes of adrenoceptors were used. Antagonist concentrations of about 20 times noradrenalin concentrations were used. It was found that prazosin ( a l receptor blocker) could block the enhancing effect in only a few of the neurons studied (see Figures 12 and 13, Table 3). Rauwolscine (a2 receptor blocker) showed a limited ability to inhibit the noradrenergic effect, causing a 30-40% reduction in two thirds of cells observed (Figures 12 and 14, Table 3). The most potent antagonist was propranolol (P receptor blocker) which could inhibit the synergistic enhancement by more than 95% in almost all neurons that showed a positive enhancement effect of noradrenalin (Figures 12 and 15, Table 3). A typical example of the inhibitory effects evoked of these three antagonists is shown in Figure 12. Because all the antagonists exhibit only partial specificity, it is hard to rule out nonspecific effects of one antagonist on another subtype of adrenoceptor. That being noted, the results showing that propranolol blocked the enhancing effect with much greater efficacy in more neurons than the other two antagonists suggested that p receptor activation is the most likely  50  Figure 12.  Effects of adrenoceptor subtype specific antagonists on the synergistic  enhancement of glutamate response. A: 2.5 pM glutamate, a subthreshold stimulation, B: 2.5 p M glutamate and 1 pM noradrenalin, a strong enhancement, C: "B" plus 20pM rauwolscine, D: "B" plus 20 pM prazosin, E: "B" plus 20 pM propranolol. This was a representative plot taken from several experiments showing similar response patterns (see Figures 13, 14 and 15; also see Table 3 for summary).  51  0  3  6  9  12  15  18  21  24  Time  52  27  30  (sec)  Figure 13. Summary of the effect of 20 pM prazosin on adrenergic enhancement of glutamate induced calcium increase in cultured cortical neurons. A weak, but statistically significant, blocking effect was observed in six out of twelve cells in three experiments, indicating a relatively small contribution of prazosin on the enhancement effect by noradrenalin. Data were expressed as mean plus one standard error, n = 6, ^t' p = 0.03.  53  700  +  CNJ  600  OS CJ  500  400  H  300  H  200  H  100  Glut  Glut+NA  54  Glut+NA+Praz  Figure 14. Summary of the effect of 20 pM rauwolscine on adrenergic enhancement of glutamate induced calcium increase in cultured cortical neurons. A moderate blocking effect was observed in ten out of fifteen cells studied in three experiments. Data were expressed as mean plus one standard error, n = 10, -I' p < 0.01.  55  800  .=  700  600  H  500  H  400  H  300  H  200  100  H  Glut  Glut+NA  56  Glut+NA+Rauw  Figure 15. Summary of the effect of 20 pM propranolol on adrenergic enhancement of glutamate induced calcium increase in cultured cortical neurons. A dramatic blocking effect was observed in thirteen out of fourteen cells studied in four experiments, indicating the great blocking effect of propranolol in both amplitude and prevalence. Data were expressed as mean plus one standard error, n = 13, % p < 0.01.  57  r-^  800  i  700  i  600  i  500  H  400  H  300  H  200  H  100  H  CM CO CJ 1 — 1  Glut  Glut + NA  58  Glut + NA+Prop  mechanism by which adrenergic input enhances the glutamate induced intraneuronal calcium rise.  3.7  An analog of cAMP mimicked the enhancing effect of noradrenalin Activation of p (either p i or p2 subtypes) adrenergic receptors results in a G  protein mediated increase of intracellular cyclic adenosine monophosphate (cAMP). To confirm the involvement of the P adrenoceptor in the enhancement effect, dibutyryladenosine-cyclic monophosphate (db-cAMP), a cell permeant analog of cyclic AMP, was added into the bath to mimic effects consequent to p receptor activation. It was found that db-cAMP coapplied with glutamate could also synergistically enhance the calcium response in the same neurons in which such enhancement was observed with noradrenalin (Figure 16 and Table 3). This phenomenon was consistently observed in three experiments summarized in Figure 17. The mimicking effect of db-cAMP was repeatedly found to be dose dependent. For example, in the case illustrated in Figure 16, a dose of 10 pM db-cAMP failed to elicit the enhancing effect observed with 40 pM dbcAMP.  3.8  Cholinergic receptor activation also enhanced glutamate response  Since the cholinergic system has also been found to modulate cortical ocular dominance plasticity, the cholinergic receptor specific agonist carbachol was tested as to its ability to modulate glutamate elicited calcium increase. Figure 17 shows that carbachol could also enhance the glutamate elicited calcium influx. This effect was observed in 11 out ofT8 cells studied in five experiments (see also Table 3). This result is  59  in agreement with the report that an NMDA-evoked calcium increase in hippocampal cells could be enhanced by acetylcholine (Segal, 1992).  60  Figure 16. The noradrenergic enhancement of glutamate induced intracellular calcium increase can be mimicked by db-cAMP, an analog of cyclic AMP. A : 2.5 uM glutamate alone; B: 2.5 uM glutamate plus 10 uM noradrenalin; C : 2.5 u M glutamate plus 10 u M noradrenalin and 200 U.M propranolol; D : 2.5 jiM glutamate plus 10 jiM db-cAMP; E : 2.5 u M glutamate plus 40 |XM db-cAMP.  61  1000  -r-  900  800  700  +  600  -4-  500  400  +  300  +  200  +  1 0 0 + 5 ••••  0  D  B  62  Figure 17. Summary of db-cAMP mimicry of the adrenergic enhancement of glutamate induced calcium increase in cultured cortical neurons. This effect was observed in eleven out of twelve cells studied in three experiments. Data were expressed as mean plus one standard error, n= 11..Note that db-cAMP was as effective as noradrenalin in increasing intracellular calcium concentrations, further suggesting that the beta receptor mediated cAMP activation pathway is an underlying mechanism of the adrenergic enhancement effect. tp<0.01,*p<0.01.  63  1000  H  900  H  800  H  700  H  600  H  500  H  400  300  H  200  H  100  H  Glut  Glut + NA  64  Glut+db-cAMP  Figure 18. A series of pseudocolor photomicrographs  showing the cholinergic  enhancement on glutamate induced calcium increase in cultured neurons. Cholinergic agonist carbachol was used in the experiments and concentrations used were as shown in the figure. This effect was observed in eleven out eighteen cells studied in five experiments.  65  66  T3 <D £ O XI  o  00  X! O  O  T5 3  x  oo  I  3  o  ON  2  ON  00  1  <  3 ID 3 oo  (D  W> 03  4>  e  <D O  r-H  ^  O  Ul  ID CU <D  ON  X  2  2  ^  ^  to ©  3 00  x  •s u <D  03  o  c o  a  3 O  3  oo  *> ,  a X!  -»-» s 03  .<D T3 <D > <U oo  X 03  X) O (D X  o t-  £  z e  >o 3  .2  ca •+ -tH -» a  O  a, <D 3 <D T3  3 O  2 •3  a  ID -»-> O  »  3 <D  o  ID T3  <D T3 03  <  67  3 C <D  4•—»  o CL,  (D T3 c3  o  -5  X> (D 3  o  •§ 3  >  -£3  M O  03  M O  3 O  133  o i-l  •«  ~03 3 03  O  '*3 3 ID  o  — 4- >  <D  3 O  3  4-—*  o a, 3 o o -a  o  o o -° N a3  S-i  3 ID O  •*—»  a,  03  M  -s o  c3  s  •—.  (D .3  O i  "o  T3  U  X  X!  4 4.1  DISCUSSION  Enhanced calcium mobilization may be crucial in ocular dominance  plasticity  Results obtained from this study show that 1) receptors for glutamate and noradrenalin colocalize postsynaptically on cortical neurons, 2) the presence of noradrenalin tends to augment submaximal excitatory calcium responses to glutamate to an asymptotic level. A hypothesis on the significance of this phenomenon is proposed as follows. Intracellular free calcium has been well characterized as a second messenger, in addition to its charge carrier role in excitable cells and its ability to modulate bursting activity, action potential shape, and other aspects of electrical activity. Among a variety of cellular events, including triggering of secretion and muscle contraction, activation of protein kinase and calcium dependent ion channels, some calcium induced changes are thought essential to bring about structural and functional changes in neurons, for example, gene expression and changes of enzyme efficiency through protein phosphorylation (Bading et al., 1993; Bading and Greenberg, 1991; Morgan and Curran, 1986). In the central nervous system, dramatic increases in [Ca ]i can be triggered by 2+  synaptically-released glutamate (Regehr and Tank, 1990). Glutamate increases the opening probability of the NMDA subtype of glutamate receptors which are permeable to divalent cations including calcium (Mayer and Miller, 1990). As a result, dynamic changes in the concentration of intracellular free calcium during interneuronal communication may play a key role in defining the fate of synapses. This is in line with  68  previous studies of the role of NMDA receptors in neuroplasticity, as discussed in the Introduction. Despite the experimental difficulty of directly proving that calcium dependent mechanisms are critically involved in cortical ocular dominance plasticity, recent evidence has strongly implicated calcium in NMDA receptor mediated plastic changes in other experimental settings such as LTP (Malenka, 1991).  The finding that noradrenalin enhances glutamate induced increase in [Ca ]i has 2+  led us to postulate that adrenergic modifiability of ocular dominance plasticity is at least partially mediated through a calcium dependent process. This was indirectly suggested by various observations including 1) increased calcium influx through NMDA receptors into the cytoplasm ensues in the wake of enhanced depolarization and 2) adrenergic enhancement of neuronal depolarization/excitability has been noted in a variety of experimental settings (see below). In vivo studies show that iontophoretically delivered noradrenalin does not affect spontaneous activity of neurons in rat and kitten visual cortices (Azizi et al., 1983; Videen et al., 1984). However, noradrenalin does enhance both excitatory and inhibitory responses in rat visual cortical cells (Azizi et al., 1983; Waterhouse et al., 1991). The situation in cat was more complex, with varying percentages of cells showing either enhanced or inhibited responses to visual stimulation (Kasamatsu and Heggelund, 1982; Videen et al., 1984). Another line of evidence comes from the effects of anesthesia on ocular dominance plasticity. Under these conditions, cortical arousal was prevented by blockade of several chemically defined ascending pathways, including the adrenergic system. Ocular dominance plasticity in this situation was found to be greatly reduced. This might result from the loss of a combined activating contribution from cholinergic ascending reticular activating system, from adrenergic systems and from serotonergic systems. While cholinergic facilitation of calcium mobilization in visual cortical neurons has been observed in preliminary experiments in this lab, a similar enhancement of calcium influx through NMDA receptors after  69  acetylcholine treatment has been reported in cultured hippocampal neurons (Segal, 1992). These results are consistent with the view of a combined contribution involving both adrenergic and cholinergic systems in modulation of ocular dominance plasticity (Bear and Singer, 1986). Electrophysiological studies in brain slice preparations also support our results. It has been shown that noradrenalin can enhance the excitability and responsiveness of cortical neurons to depolarizing inputs by suppressing a slow calcium activated potassium current known as I (AHP) (McCormick et al., 1991). Additionally cortical neurons showed robust discharges to otherwise subthreshold iontophoretic doses of glutamate in the presence of noradrenalin (Mouradian et al., 1991). Therefore an enhanced calcium response, along with a variety of calcium dependent intracellular events such as activation of calcium/calmodulin kinase II (Ca/CaM-KII) and protein kinase C, may serve to translate the enhanced postsynaptic reactivity into intracellular consequences important in determining the fate of synapses.  4.2  How does noradrenalin enhance calcium mobilization  Activation of different subtypes of adrenoceptors causes different and even opposing intracellular consequences. It has been well demonstrated that activation of a l adrenoceptors has as its predominant effect the initiation of PI turnover, while a2 receptor activation results in the decrease of intracellular cAMP levels. Activation of (3 receptors results in an increase in intracellular cAMP levels (for a full summary and classification see (Anonymous, 1994)). The result that prazosin almost completely failed to block the enhancement effect of noradrenalin suggests that a l adrenoceptors are unlikely to mediate the effect observed. The observation that noradrenalin alone did not elicit significant increase in [Ca ]i in cells also supported this point of view. Therefore, 2+  70  the mechanism underlying the adrenergic enhancement is probably not simply additional calcium mobilization from internal stores following activation of IP3 receptors.  The strongest and most prevalent blocking effect was observed with P adrenoceptor antagonist propranolol. This is of great interest because 1) it coincides with the electrophysiological data that p activation increases neuronal excitability (see section 1.3.1 and Dodt et al., 1991; McCormick et al., 1991); 2) there has been several reports that blockade of the p pathway was involved, at least in part, with ocular dominance plasticity (Kasamatsu and Shirokawa, 1985). With the predominant effect of beta activation in neurons being an increased level of cAMP (Anonymous, 1994), the finding that cAMP analog, db-cAMP, mimicked the enhancing effect of noradrenalin further supports involvement of the P receptor pathway in the mediating mechanism.  However, the specific events which follow cAMP activation and results in modulation of glutamate induced calcium increase remain unknown. A few speculative molecular mechanisms are discussed here. Elevation of cAMP activates cAMP dependent protein kinase which can in turn phosphorylate a series of target proteins. The way a particular neuron responds to an elevation of cAMP depends on its particular spectrum of cell-specific substrate proteins which can be phosphorylated by this kinase. For example, in heart muscle cells, P-adrenergic agonists increase the duration of the cardiac action potential via a cAMP-dependent protein kinase mediated modulation of voltagedependent calcium channels (Tsien, 1987). This effect could be mimicked by intracellular injection of cAMP (Tsien, 1987). By analogy, noradrenalin triggered activation of cAMP-dependent protein kinase may also act on the voltage dependent calcium channel in the cortical neurons. If this is the case, then the initial depolarization caused by glutamate may regeneratively activate the facilitated voltage dependent calcium channel, resulting in an enhanced calcium influx. However, this mechanism seems unlikely  71  because it is in contradiction with the result that APV can completely block the enhancement. APV does not block the non-NMDA type of glutamate receptors which mediate the initial depolarization crucial to this hypothesis. On the contrary, the fact that APV can completely block the enhancement effect strongly suggests a direct modulation of the NMDA receptor itself. As a consequence of receptor modulation by cAMP dependent protein kinase, the NMDA receptors may become more permeant to the divalent cations including calcium ions, resulting in an enhanced calcium influx. A third possibility may also exist. A calcium-releasing mechanism from internal calcium store following an initial calcium increase has been described in a variety of cell types including neurons (Galione et al., 1991; Ivanenko et al., 1993; Petersen et al., 1991; Stein et al., 1992; White et al., 1993). This calcium-dependent calcium release is mediated by the ryanodine receptor, which provides a positive feedback step in calcium mobilization. If the ryanodine receptor happens to be a natural substrate of cAMP dependent protein kinase, it is conceivable that an enhanced positive feedback mechanism might be part of the mechanism underlying the synergy observed here. This is also reconcilable with the observed APV block, because blocking the initial calcium increase through NMDA receptor would abolish the whole positive feedback loop.  The result that antagonists of a2 receptors can also block the enhancement, although to a much less extent, is in obvious contradiction with the possible mechanisms discussed above, because the predominant effect of a2 activation is a decrease of intracellular cAMP level. The developmental studies of the receptor ontogeny in kitten revealed that the amount of binding sites of a2 in layer IV is more vulnerable to input deprivation than either a l or (3 adrenoceptors (see section 1.4.2 and 1.4.3), suggesting its potential role in cortical development during the critical period. Possible explanations of this apparent contradiction are 1) the modest block of a2 antagonist might result from non-specific effects on the (3 adrenoceptor; 2) the significance of a2 receptor at this  72  developmental stage may undergo a process not detectable by calcium imaging, 3) and species difference between kitten and rat may exist.  4.3  Is the enhancement unique to. the adrenergic system  Increasing evidence has indicated that enhancement of glutamate induced calcium increase in neurons is not a unique property of adrenergic system. Other systems may exert similar effects, but probably through different mechanisms. Enhancing effects of cholinergic system on NMDA conductance and the glutamate induced calcium rise (see section 1.3.2) all suggest a PI turnover pathway mediated mechanism (Markram and Segal, 1992; Segal, 1992). This is in line with the report that anticholinergic agents reduce ocular dominance plasticity through actions on M l receptors which activate PI turnover pathway, but not through M2 receptors which decreases cAMP levels (Gu and Singer, 1993). The preliminary results showing that carbachol could aiso enhance glutamate induced calcium rise in cultured visual cortical neurons further supports the existence of multiple modulatory mechanisms for the regulation of intracellular calcium concentration. Whether this is also mediated through a PI turnover pathway has yet to be tested. In addition, modulatory effects of serotonergic system have also been addressed in a few electrophysiological studies. Previous studies have found that cortical neurons in the cat fired more robustly to NMDA when 5-HT was present than when absent (Nedergaard et al., 1986; Nedergaard et al., 1987). In rat cortical slices, both voltage- and current-clamp recordings showed that neuronal responses to NMDA were increased by 5HT and a long-lasting effect of such enhancement was also reported (Reynolds et al., 1988). More recent in vivo recordings in kittens showed that blockade of the 5-HT2C (previously classified as 5-HTic)receptor could prevent ocular dominance plasticity (Wang et al., 1993). However, the question of whether and how the results of this PI  73  turnover linked 5-HT2C receptor could be related to potential modulating effects on NMDA responses remains to be answered.  The presence of multiple modulatory systems is consistent with the finding that the synergistic interaction with either adrenergic or cholinergic systems was always seen in a fraction of the neurons studied. Some cells responded only to glutamate and some cells showed synergistic enhancement with either adrenergic or cholinergic systems, but not both. It will be interesting to test the modulatory pattern on glutamate induced calcium influx in future studies when both adrenergic and cholinergic systems are activated simultaneously. This heterogeneity in response patterns may reflect the existence of different populations of neurons which are subject to preferential modulation by different inputs. At a molecular level, this phenomenon likely reflects the complexity of combinations of receptors and their subtypes in neurons grown in the mixed culture environment. Although it is not yet known to what extent this resembles the diversity of receptor phenotypes in vivo , the difference in the calcium responses among neurons must have significance in implementing the functionality of an intact neural circuitry.  74  References Adrien J, Blanc G, Buisseret P, Fregnac Y, Gary-Bobo E, Imbert M, Tasson JP, Trotter Y (1985) Noradrenaline and functional plasticity in kitten visual cortex: a re-examination.. 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