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Noradrenaline-stimulated inositol phospholipid breakdown in rat dorsal lateral geniculate nucleus neurones
314
Brain Research, 371 (1986) 314-318
Elsevier BRE 11626
Noradrenaline-Stimulated Inositol Phospholipid Breakdown in Rat Dorsal Lateral Geniculate Nucleus Neurones JOHN A. KEMP and C. PETER DOWNES MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Cambridge CB2 2QH (U. K.)
(Accepted August 27th, 1985) Key words: noradrenaline- %-receptor- inositol phosphate- lateral geniculate nucleus
Noradrenaline-stimulated inositol phospholipid breakdown in matched vibratome sections through the rat dorsal lateral geniculate nucleus (dLGN). The response was measured as a large accumulation of [3H]inositol labelled inositol monophosphate and was mediated via activation of al-adrenergic receptors. Accumulation of [3H]inositol phosphates was reduced in kainic acid-lesioned animals, indicating that this response occurred within dLGN neurones and not afferent terminals. The results implicate inositol phospholipid breakdown as part of the mechanism of noradrenergic neurotransmission within the dLGN. INTRODUCTION Many neurotransmitters, particularly those with modulatory functions, act through receptors linked to second messenger systems as opposed to receptors that incorporate a specific ion channel as an integral part of their structure. One such signalling system involves cell surface receptors whose activation leads to hydrolysis of a membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to yield diacylglycerol (DG) and inositol 1,4,5-trisphosphate (InsP3)2.4,9. Both products have second messenger functions: D G activates protein kinase C and InsP 3 releases intracellular Ca 2÷ from a membrane-bound, non-mitochondrial store. Protein phosphorylation, alone, or in combination with raised cyctosol Ca 2÷ concentration, can control a variety of cellular responses including secretion of macromolecules and monoamines, alterations in ion fluxes, smooth muscle contraction and glycogenolysis2,4,22. The molecular mechanisms underlying this PIP 2dependent signalling system have mostly been determined using peripheral tissue preparations containing relatively homogeneous cell populations. The
brain contains a variety of receptors whose activation leads to inositol phospholipid breakdown, but the heterogeneity of cell types in the central nervous system has limited the identification of specific neuronal responses that may be attributable to receptor-stimulated PIP 2 hydrolysis. In the rat dorsal lateral geniculate nucleus (dLGN) the majority of the neurones (approximately 94%) 3o project to the striate cortex and are classed as relay or principle cells. The d L G N receives a dense noradrenergic (NA) input from the locus coeruleus 2° and both stimulation of the locus coeruleus and iontophoresis of low doses of N A facilitate responsiveness in the d L G N relay cells 19,24-26. These electrophysiological responses are mediated by al-adrenoceptors 26 which have previously been shown to activate the PIP2-dependent signalling cascade in brain and other tissues 3.5,8. We have developed simple methods that have allowed us to study receptor-dependent changes in inositol phospholipid metabolism in d L G N slices. The results point to an important role for this signalling system as part of the mechanism of noradrenergic transmission within the nucleus.
Correspondence: J.A. Kemp. Present address: Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR, U.K.; or C.P. Downes. Present address: Smith Kline and French Research Ltd., The Frythe, Welwyn, Hertfordshire, U.K.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
315 MATERIALS AND METHODS
t4000T
Slice incubations and measurement of radioactive inositol phosphates Preparation of d L G N slices. Male S p r a g u e - D a w -
1oooo
ley rats (250-300 g) were killed by decapitation and their brains rapidly removed, d L G N were dissected and 300 # m thick slices were cut using an Oxford vibratome as described by Godfraind and Kelly14. Three slices were taken from each nucleus and these were trimmed with fine scissors to remove tissue from surrounding brain regions. The slices were then transferred to separate 5 ml conical flasks containing 2 ml of oxygenated artificial cerebrospinal fluid and incubated at 35 °C for between 1 and 2 h before use. For the kainate lesioned animals a single, 150 # m slice was cut between the second and third slices from both control and lesioned nuclei, and stained with cresyl violet for histological verification of the extent of the lesions. Compared to control d L G N , kainic acid injection caused a marked neuronal cell loss accompanied by glial proliferation, a c o m m o n feature of excitotoxic lesions 7. Three vibratome-cut slices from a single d L G N were transferred to a Beckman Biovial with 0.25 ml of artificial CSF containing 10 m M LiC1 (NaCI reduced in proportion) and 1 #Ci of [3H]inositol (N.E.N.). The vials were gassed with 95% 02: 5% CO2 (v/v) and then capped. After 60 min at 35 °C drugs were added and the incubations continued for a further 30 min. The incubations were terminated by adding 0.25 ml of ice-cold trichloroacetic acid (15%, w/v) and radioactive inositol phosphates were analyzed by anion-exchange chromatography on small Dowex-1 (formate) columns as described previously 11,18. The inositol monophosphate (InsP) fraction was collected directly into a counting vial and radioactivity was determined by scintillation counting. In most experiments one d L G N from each animal served as a control whilst the contralateral d L G N was used to assess the effect of drug additions. The effect of the drug was expressed as the ratio: radioactivity in InsP:with drug radioactivity in InsP:control
Kainic acid lesions Rats were anaesthetized with Equithesin
and
~2000 I aooo-
[__l
Control
6000-
~
+ NA
dpm IOOuM
40002000 i~o-
~[]
(n=i4)
(n 13)
Fig. i. Noradrenaline-stimulated InsP accumulation in dLGN slices. Three slices each from individual dLGN were labelled with [3H]inositol for 60 min. NA (final concentration 10 4 M) or artificial CSF was added and incubations continued for another 30 min. Radioactive InsP was determined as described in the Methods. The number of experiments is indicated in brackets. placed in a David Kopf small animal stereotaxic frame. Unilateral injections were made using a Hamilton syringe into an area immediately ventromedial to the lateral geniculate nucleus. The coordinates according to Paxinos and Watson 23 were: from bregma A -4.0, L 3.0, V 6.0 from skull. Two #1 of a 1 mg/ml solution of kainic acid was injected over a period of 5 min and after a further 5 min the syringe was slowly removed. Lesioned animals were used 7 - 1 0 days later. RESULTS Receptor-stimulated PIP2 breakdown leads to formation of InsP 3. However, this molecule is rapidly degraded by a cascade of phosphatase reactions10, 29 which return inositol phosphates to the free inositol pool of the cell. The final step in this pathway, hydrolysis of InsP to free inositol, is strongly inhibited by Li + (ref. 16) and hence the nlost sensitive assay of receptor-stimulated inositol phospholipid breakdown is the accumulation of InsP when tissue slices are bathed in a medium containing Li + (ref. 3). When d L G N slices were labelled with [3H]inositol and then stimulated with N A for 30 min there was a large (approximately 6-fold) increase in the radioactive InsP peak eluted from Dowex-1 (Fig. 1). In most experiments one d L G N was treated with N A whilst
316 TABLE I
[--]
Control
Effect of adrenergic antagonists" on NA-stimulated lnsP accumulation
~
Kainate
10000
The experiments were carried out exactly as described for Fig. 1. The results are m e a n s _+ S.E.M. for the n u m b e r s of paired d L G N indicated in the brackets. W h e n adrenergic antagonists were present the incubations of the contralateral d L G N contained no drug. Antagonists alone did not affect basal labelling of InsP.
Additions
Ins P accumulation expressed as the ratio NA-stimulated/control
NA (10 -4 M) NA + propranolol (10 -5 M)
6.51 +_ 0.98 (9) 7.46 _+ 0.34 (3)
NA + phentolamine (10 5 M) NA + prazosin (10 5 M)
1.38 _+0.20 (3) 0.98 _+0.09 (3)
9000 8000 7000
1
6000dpm
I I
5ooo4000-
~ x
30002000
0
its contralateral pair served as the control. Throughout the course of these experiments, 9 paired d L G N were used to assess the effect of N A (see Table I). The incorporation of the radioactive tracer into InsP was very consistent whether N A was present or not. This consistency, together with the magnitude of the response to NA, made it unnecessary to correct individual preparations for protein content. N A can act through at least 4 classes of receptors which have been termed cq, a2, fll and f12 receptors, respectively. The accumulation of InsP due to N A was not significantly affected by 10 -5 M propranolol which should block both fll and f12 receptor-mediated responses (Table I). However, the response was almost completely blocked by 10 -5 M phentolamine (a mixed c q , a 2 receptor blocker) and completely blocked by 10-5 M prazosin (a specific a 1 receptor blocker). This result leaves little doubt that NA-stimulated inositol phospholipid breakdown in the rat d L G N is mediated by a I receptors. In the kainate-lesioned d L G N there was a large (greater than 50%) reduction in the accumulation of radioactive InsP compared with the contralateral unlesioned control. The reduction was of a similar magnitude whether or not N A was included in the incubations (Fig. 2). These data suggest that a substantial portion of the [3H]inositol incorporated into InsP is in neurones whose cell bodies lie in the dLGN. Furthermore,' a similar proportion of the [3H]InsP that accumulates during a I receptor stimulation with N A appears to be located in d L G N neurones. A n alternative explanation that cannot readily be discounted is that kainate treatment, in addition to its specific toxic
lesioned
CONTROL
+
NA
[t00 ~JM)
Fig. 2. Effect of kainate lesioning on InsP accumulation in dLGN slices. The experiments, using slices prepared from control and kainate lesioned dLGN, were carried out exactly as for Fig. 1. The results are means + S.E.M. for n = 4 separate dLGN for each bar on the figure.
effects on neurones, can also lead to changes in inositol lipid metabolism or uptake of [3H]inositol into nerve terminals or non-neuronal cells. However, in a similar series of experiments designed to determine the cellular location of muscarinic-agonist stimulated inositol lipid metabolism in hippocampus m e m b r a n e vesicles, Fisher et al. ;3 have used their results, with the excitatory neurotoxin, ibotenate, to pinpoint such responses to membranes derived from neuronal cell bodies or dendrites. DISCUSSION
All of the components of the inositol lipid-dependent signalling system are present in the brain. PIP2 is concentrated in brain and nerves and protein kinase C was first described in brain where it is widely distributed and highly active 31. Furthermore, stimulation of a variety of neurotransmitter receptors in brain slice preparations, brain membrane vesicles and sympathetic ganglia, leads to enhanced metabolic turnover of inositol phospholipids and accumulation of inositol phosphatesS. These responses (at least via activation of muscarinic receptors), also occur in vivo, since Li + treatment of rats leads to atropineblockable accumulation of InsP in many brain re-
317 gions 27. Our results, and those of Fisher et al.12,13, demonstrate that receptor-stimulated inositol lipid breakdown can occur in neurones, though it is to be expected that some receptors on glial cells may also function this way. Intracellular recording techniques have made an important contribution to understanding the ionic mechanisms of neurotransmitter action, but a complete understanding of the molecular events between receptor occupation and altered cellular response requires a combination of electrophysiological and biochemical methods. We chose the rat d L G N for these studies because it contains a dense population of neurones which respond in a similar fashion to the application of N A 24 26. Such homogeneity is a prerequisite for biochemical experiments in which the measured response represents an average of the events occurring in all cells present in the preparation. Furthermore, the vibratome slice preparation is suitable for both biochemical and electrophysiological experiments. Stimulation of a 1 receptors on d L G N cells facilitates their responsiveness to synaptic excitation and iontophoresis of L-glutamate 25. A similar action for N A has been reported for hippocampal CA1 pyramidal cells, but in this case the effect is mediated by fladrenoceptors, mimicked by 8-bromo cyclic A M P and results from inhibition of a Ca2+-activated K + conductance 21. However, acetylcholine, in addition to inhibiting a voltage-dependent K+ conductance17, also inhibits a Ca2+-activated K ÷ conductance in hippocampal pyramidal cells due to activation of muscarinic receptors 6 and such receptors in the hippocampus have been shown to enhance inositol lipid breakdownl2,18. In the d L G N acetylcholine also facilitates
REFERENCES 1 Aghajanian, G.K., Modulation of a transient outward current in serotonergic neurones by al-adrenoceptors, Nature (London), 315 (1985) 501-503. 2 Berridge, M.J., Inositol trisphosphate and diacylglycerol as second messengers, Biochem. J., 220 (1984) 345-360. 3 Berridge, M.J., Downes, C.P. and Hanley, M.R., Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands, Biochem. J., 206 (1982) 587-595. 4 Berridge, M.J. and Irvine, R.F., Inositol trisphosphate, a novel second messenger in cellular signal transduction, Nature (London), 312 (1984) 315-321. 5 Brown, E., Kendal, D.A. and Nahorski, S.R., Inositol
synaptic responsiveness28 and produces a marked hydrolysis of inositol phospholipids (Jakobson et al., unpublished observations). Thus, one possibility is that the actions of N A and acetylcholine in the d L G N could (like their actions in the hippocampus) involve effects on K + conductances and this is further supported by a recent study which showed that activation of a I receptors inhibits a K + current (IA) in serotonergic raphe neurones 1. However, these assumptions remain to be verified by the appropriate intracellular studies. Several lines of evidence suggest that NA-stimulated InsP formation occurs in the relay or principle cells of the rat dLGN. Firstly, the response to N A is large in magnitude and readily detectable, suggesting that the response resides in a dominant cell population in the slice preparation. Secondly, the response is clearly mediated by al-adrenoceptors, as is the electrophysiological response to N A applied directly to relay cell bodies. Finally, the accumulation of radioactive InsP is greatly diminished in slices prepared from kainate-lesioned nuclei. These experiments, therefore, point to an important role for inositol phospholipid-derived signals in noradrenergic transmission within the rat dLGN. Future experiments will need to detect rapid formation of inositol trisphosphate (the primary product of PIP2 breakdown) and to examine the electrophysiological response to intracellular injection of inositol trisphosphate and/or exogenous application of synthetic diacylglycerol. These results are the first step towards determining a neuronal response in brain that can be directly linked to increased inositol phospholipid turnover.
phospholipid hydrolysis in rat cerebral cortex slices: 1. Receptor characterisation, J. Neurochem., 42 (1984) 1379-1387. 6 Cole, A.E. and Nicoll, R.A., Acetylcholine mediates a slow synaptic potential in hippocampal pyramidal cells, Science, 221 (1983) 1299-1301. 7 Coyle, J.T. and Schwarcz, R., Lesion of striatal neurones with kainic acid provides a model for Huntington's chorea, Nature (London), 263 (1976) 244-246. 8 Downes, C.P., Inositol phospholipids and neurotransmitter-receptor signalling mechanisms, Trends Neurosci., 6 (1983) 313-316. 9 Downes, C.P. and Michell, R.H., Phosphatidylinositol 4phosphate and phosphatidylinositol 4,5-trisphosphate: lipids in search of a function, Cell Calcium, 3 (1982) 467-502.
318 10 Downes, C.P., Mussat, M.C. and Michell, R.H., The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane, Biochem. J., 203 (1982) 169-177. 11 Downes, C.P. and Wusteman, M.M., Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands, Biochem. J., 216 (1983) 633-640. 12 Fisher, S.K., Boast, C.A. and Agranoff, B.W., The muscarinic stimulation of phospholipid labelling in hippocampus is independent of its cholinergic input, Brain Research, 189 (1980) 284-288. 13 Fisher, S.K., Frey, K.A. and Agranoff, B.W., Loss of muscarinic receptors and of stimulated phospholipid labelling in ibotenate-treated hippocampus, J. Neurosci., 1 (1981) 1407-1413. 14 Godfraind, J.W. and Kelly, J.S., Intracellular recordings from thin slices of the lateral geniculate nucleus of rats and cats. In G.A. Kerkut and H.V. Wheal (Eds.), Electrophysiology of Isolated Mammalian CNS Preparations, Academic Press, London, 1981, pp. 257-284. 15 Goedert, M., Pinnock, R.D., Downes, C.P., Mantyh, P.W. and Emson, P.C., Neurotensin stimulates inositol phospholipid hydrolysis in rat brain slices, Brain Research, 323 (1984) 193-197. 16 Hallcher, L.M. and Sherman, W.R., The effects of lithium ion and other agents on the activity of myo-inositol-l-phosphatase from bovine brain, J. Biol. Chem., 255 (1980) 10896-10901. 17 Halliwell, S.V. and Adams, P.R., Voltage clamp of muscarinic excitation in hippocampal neurons, Brain Research, 250 (1982) 71-92. 18 Jacobson, M.D., Wusteman, M. and Downes, C.P., Muscarinic receptors and hydrolysis of inositol phospholipids in rat cerebral cortex and parotid gland, J. Neurochem., 44 (1985) 465-472. 19 Kayama, Y., Negi, T., Sugitani, M. and Iwama, K., Effects of locus coeruleus stimulation on neuronal activities of dorsal lateral geniculate nucleus and perigeniculate reticular nucleus of the rat, Neuroscience, 7 (1982) 655-666. 20 Kromer, L.F. and Moore, R.Y., A study of the organisation of the locus coeruleus projections to the lateral geniculate nuclei in the albino rat, Nearoscience, 5 (1980) 255-271.
21 Madison, D.V. and Nicoll, R.A., Noradrenaline blocks accommodation of pyramidal cell discharge in the hippocampus, Nature (London), 299 (1982) 636-638. 22 Nishizuka, Y., The role of protein kinase C in cell surface signal transduction and tumour promotion, Nature (London), 308 (1984) 693-698. 23 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1982. 24 Rogawski, M.A. and Aghajanian, G.K., Norepinephrine and serotonin: opposite effects on the activity of lateral geniculate neurons evoked by optic pathway stimulation, Exp. Neurol., 69 (1980) 678-694. 25 Rogawski, M.A. and Aghajanian, G.K., Modulation of lateral geniculate neurone excitability by noradrenaline microiontophoresis or locus coeruleus stimulation, Nature (London), 287 (1980) 731-734. 26 Rogawski, M.A. and Aghajanian, G.K., Activation of lateral geniculate neurones by locus coeruleus or dorsal noradrenergic bundle stimulation: selective blockade by the alphal-adrenoceptor antagonist prazosin, Brain Research, 250 (1982) 31-39. 27 Sherman, W.R., Leavitt, A.L., Honchar, M.P., Hallcher, L.M. and Phillips, B.E., Evidence that lithium alters phosphoinositide metabolism: chronic administration elevates primarily o-myo-inositol-l-phosphate in cerebral cortex of the rat, J. Neurochem., 36 (1981) 1947-1951. 28 Sillito, A.M., Kemp, J.A. and Berardi, N., The cholinergic influence on the function of the cat dorsal lateral geniculate nucleus (dLGN), Brain Research, 280 (1983) 299-307. 29 Storey, D.J., Shears, S.B., Kirk, C.J. and Michell, R.H., Stepwise enzymatic dephospharylation of inositol 1,4,5trisphosphate to inositol in liver, Nature (London), 312 (1984) 374-376. 30 Sumitomo, I. and Iwana, K., Some properties of intrinsic neurons of the dorsal lateral geniculate nucleus of the rat, Jap. J. Physiol., 27 (1977) 717-730. 31 Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y., Calcium-dependent activation of a multifunctional protein kinase by membrane phospholipids, J. Biol. Chem., 254 (1979) 3692-3695. 32 Uchida, T., Ito, H., Baum, B.J., Roth, G.S., Filburn, C.R. and Sacktor, B., Alphal-adrenergic stimulation of phosphatidylinositol phosphatidic acid turnover in rat parotid cells, Mol. Pharmacol., 21 (1982) 128-132.
Brain Research, 371 (1986) 314-318
Elsevier BRE 11626
Noradrenaline-Stimulated Inositol Phospholipid Breakdown in Rat Dorsal Lateral Geniculate Nucleus Neurones JOHN A. KEMP and C. PETER DOWNES MRC Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Cambridge CB2 2QH (U. K.)
(Accepted August 27th, 1985) Key words: noradrenaline- %-receptor- inositol phosphate- lateral geniculate nucleus
Noradrenaline-stimulated inositol phospholipid breakdown in matched vibratome sections through the rat dorsal lateral geniculate nucleus (dLGN). The response was measured as a large accumulation of [3H]inositol labelled inositol monophosphate and was mediated via activation of al-adrenergic receptors. Accumulation of [3H]inositol phosphates was reduced in kainic acid-lesioned animals, indicating that this response occurred within dLGN neurones and not afferent terminals. The results implicate inositol phospholipid breakdown as part of the mechanism of noradrenergic neurotransmission within the dLGN. INTRODUCTION Many neurotransmitters, particularly those with modulatory functions, act through receptors linked to second messenger systems as opposed to receptors that incorporate a specific ion channel as an integral part of their structure. One such signalling system involves cell surface receptors whose activation leads to hydrolysis of a membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to yield diacylglycerol (DG) and inositol 1,4,5-trisphosphate (InsP3)2.4,9. Both products have second messenger functions: D G activates protein kinase C and InsP 3 releases intracellular Ca 2÷ from a membrane-bound, non-mitochondrial store. Protein phosphorylation, alone, or in combination with raised cyctosol Ca 2÷ concentration, can control a variety of cellular responses including secretion of macromolecules and monoamines, alterations in ion fluxes, smooth muscle contraction and glycogenolysis2,4,22. The molecular mechanisms underlying this PIP 2dependent signalling system have mostly been determined using peripheral tissue preparations containing relatively homogeneous cell populations. The
brain contains a variety of receptors whose activation leads to inositol phospholipid breakdown, but the heterogeneity of cell types in the central nervous system has limited the identification of specific neuronal responses that may be attributable to receptor-stimulated PIP 2 hydrolysis. In the rat dorsal lateral geniculate nucleus (dLGN) the majority of the neurones (approximately 94%) 3o project to the striate cortex and are classed as relay or principle cells. The d L G N receives a dense noradrenergic (NA) input from the locus coeruleus 2° and both stimulation of the locus coeruleus and iontophoresis of low doses of N A facilitate responsiveness in the d L G N relay cells 19,24-26. These electrophysiological responses are mediated by al-adrenoceptors 26 which have previously been shown to activate the PIP2-dependent signalling cascade in brain and other tissues 3.5,8. We have developed simple methods that have allowed us to study receptor-dependent changes in inositol phospholipid metabolism in d L G N slices. The results point to an important role for this signalling system as part of the mechanism of noradrenergic transmission within the nucleus.
Correspondence: J.A. Kemp. Present address: Merck, Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR, U.K.; or C.P. Downes. Present address: Smith Kline and French Research Ltd., The Frythe, Welwyn, Hertfordshire, U.K.
0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
315 MATERIALS AND METHODS
t4000T
Slice incubations and measurement of radioactive inositol phosphates Preparation of d L G N slices. Male S p r a g u e - D a w -
1oooo
ley rats (250-300 g) were killed by decapitation and their brains rapidly removed, d L G N were dissected and 300 # m thick slices were cut using an Oxford vibratome as described by Godfraind and Kelly14. Three slices were taken from each nucleus and these were trimmed with fine scissors to remove tissue from surrounding brain regions. The slices were then transferred to separate 5 ml conical flasks containing 2 ml of oxygenated artificial cerebrospinal fluid and incubated at 35 °C for between 1 and 2 h before use. For the kainate lesioned animals a single, 150 # m slice was cut between the second and third slices from both control and lesioned nuclei, and stained with cresyl violet for histological verification of the extent of the lesions. Compared to control d L G N , kainic acid injection caused a marked neuronal cell loss accompanied by glial proliferation, a c o m m o n feature of excitotoxic lesions 7. Three vibratome-cut slices from a single d L G N were transferred to a Beckman Biovial with 0.25 ml of artificial CSF containing 10 m M LiC1 (NaCI reduced in proportion) and 1 #Ci of [3H]inositol (N.E.N.). The vials were gassed with 95% 02: 5% CO2 (v/v) and then capped. After 60 min at 35 °C drugs were added and the incubations continued for a further 30 min. The incubations were terminated by adding 0.25 ml of ice-cold trichloroacetic acid (15%, w/v) and radioactive inositol phosphates were analyzed by anion-exchange chromatography on small Dowex-1 (formate) columns as described previously 11,18. The inositol monophosphate (InsP) fraction was collected directly into a counting vial and radioactivity was determined by scintillation counting. In most experiments one d L G N from each animal served as a control whilst the contralateral d L G N was used to assess the effect of drug additions. The effect of the drug was expressed as the ratio: radioactivity in InsP:with drug radioactivity in InsP:control
Kainic acid lesions Rats were anaesthetized with Equithesin
and
~2000 I aooo-
[__l
Control
6000-
~
+ NA
dpm IOOuM
40002000 i~o-
~[]
(n=i4)
(n 13)
Fig. i. Noradrenaline-stimulated InsP accumulation in dLGN slices. Three slices each from individual dLGN were labelled with [3H]inositol for 60 min. NA (final concentration 10 4 M) or artificial CSF was added and incubations continued for another 30 min. Radioactive InsP was determined as described in the Methods. The number of experiments is indicated in brackets. placed in a David Kopf small animal stereotaxic frame. Unilateral injections were made using a Hamilton syringe into an area immediately ventromedial to the lateral geniculate nucleus. The coordinates according to Paxinos and Watson 23 were: from bregma A -4.0, L 3.0, V 6.0 from skull. Two #1 of a 1 mg/ml solution of kainic acid was injected over a period of 5 min and after a further 5 min the syringe was slowly removed. Lesioned animals were used 7 - 1 0 days later. RESULTS Receptor-stimulated PIP2 breakdown leads to formation of InsP 3. However, this molecule is rapidly degraded by a cascade of phosphatase reactions10, 29 which return inositol phosphates to the free inositol pool of the cell. The final step in this pathway, hydrolysis of InsP to free inositol, is strongly inhibited by Li + (ref. 16) and hence the nlost sensitive assay of receptor-stimulated inositol phospholipid breakdown is the accumulation of InsP when tissue slices are bathed in a medium containing Li + (ref. 3). When d L G N slices were labelled with [3H]inositol and then stimulated with N A for 30 min there was a large (approximately 6-fold) increase in the radioactive InsP peak eluted from Dowex-1 (Fig. 1). In most experiments one d L G N was treated with N A whilst
316 TABLE I
[--]
Control
Effect of adrenergic antagonists" on NA-stimulated lnsP accumulation
~
Kainate
10000
The experiments were carried out exactly as described for Fig. 1. The results are m e a n s _+ S.E.M. for the n u m b e r s of paired d L G N indicated in the brackets. W h e n adrenergic antagonists were present the incubations of the contralateral d L G N contained no drug. Antagonists alone did not affect basal labelling of InsP.
Additions
Ins P accumulation expressed as the ratio NA-stimulated/control
NA (10 -4 M) NA + propranolol (10 -5 M)
6.51 +_ 0.98 (9) 7.46 _+ 0.34 (3)
NA + phentolamine (10 5 M) NA + prazosin (10 5 M)
1.38 _+0.20 (3) 0.98 _+0.09 (3)
9000 8000 7000
1
6000dpm
I I
5ooo4000-
~ x
30002000
0
its contralateral pair served as the control. Throughout the course of these experiments, 9 paired d L G N were used to assess the effect of N A (see Table I). The incorporation of the radioactive tracer into InsP was very consistent whether N A was present or not. This consistency, together with the magnitude of the response to NA, made it unnecessary to correct individual preparations for protein content. N A can act through at least 4 classes of receptors which have been termed cq, a2, fll and f12 receptors, respectively. The accumulation of InsP due to N A was not significantly affected by 10 -5 M propranolol which should block both fll and f12 receptor-mediated responses (Table I). However, the response was almost completely blocked by 10 -5 M phentolamine (a mixed c q , a 2 receptor blocker) and completely blocked by 10-5 M prazosin (a specific a 1 receptor blocker). This result leaves little doubt that NA-stimulated inositol phospholipid breakdown in the rat d L G N is mediated by a I receptors. In the kainate-lesioned d L G N there was a large (greater than 50%) reduction in the accumulation of radioactive InsP compared with the contralateral unlesioned control. The reduction was of a similar magnitude whether or not N A was included in the incubations (Fig. 2). These data suggest that a substantial portion of the [3H]inositol incorporated into InsP is in neurones whose cell bodies lie in the dLGN. Furthermore,' a similar proportion of the [3H]InsP that accumulates during a I receptor stimulation with N A appears to be located in d L G N neurones. A n alternative explanation that cannot readily be discounted is that kainate treatment, in addition to its specific toxic
lesioned
CONTROL
+
NA
[t00 ~JM)
Fig. 2. Effect of kainate lesioning on InsP accumulation in dLGN slices. The experiments, using slices prepared from control and kainate lesioned dLGN, were carried out exactly as for Fig. 1. The results are means + S.E.M. for n = 4 separate dLGN for each bar on the figure.
effects on neurones, can also lead to changes in inositol lipid metabolism or uptake of [3H]inositol into nerve terminals or non-neuronal cells. However, in a similar series of experiments designed to determine the cellular location of muscarinic-agonist stimulated inositol lipid metabolism in hippocampus m e m b r a n e vesicles, Fisher et al. ;3 have used their results, with the excitatory neurotoxin, ibotenate, to pinpoint such responses to membranes derived from neuronal cell bodies or dendrites. DISCUSSION
All of the components of the inositol lipid-dependent signalling system are present in the brain. PIP2 is concentrated in brain and nerves and protein kinase C was first described in brain where it is widely distributed and highly active 31. Furthermore, stimulation of a variety of neurotransmitter receptors in brain slice preparations, brain membrane vesicles and sympathetic ganglia, leads to enhanced metabolic turnover of inositol phospholipids and accumulation of inositol phosphatesS. These responses (at least via activation of muscarinic receptors), also occur in vivo, since Li + treatment of rats leads to atropineblockable accumulation of InsP in many brain re-
317 gions 27. Our results, and those of Fisher et al.12,13, demonstrate that receptor-stimulated inositol lipid breakdown can occur in neurones, though it is to be expected that some receptors on glial cells may also function this way. Intracellular recording techniques have made an important contribution to understanding the ionic mechanisms of neurotransmitter action, but a complete understanding of the molecular events between receptor occupation and altered cellular response requires a combination of electrophysiological and biochemical methods. We chose the rat d L G N for these studies because it contains a dense population of neurones which respond in a similar fashion to the application of N A 24 26. Such homogeneity is a prerequisite for biochemical experiments in which the measured response represents an average of the events occurring in all cells present in the preparation. Furthermore, the vibratome slice preparation is suitable for both biochemical and electrophysiological experiments. Stimulation of a 1 receptors on d L G N cells facilitates their responsiveness to synaptic excitation and iontophoresis of L-glutamate 25. A similar action for N A has been reported for hippocampal CA1 pyramidal cells, but in this case the effect is mediated by fladrenoceptors, mimicked by 8-bromo cyclic A M P and results from inhibition of a Ca2+-activated K + conductance 21. However, acetylcholine, in addition to inhibiting a voltage-dependent K+ conductance17, also inhibits a Ca2+-activated K ÷ conductance in hippocampal pyramidal cells due to activation of muscarinic receptors 6 and such receptors in the hippocampus have been shown to enhance inositol lipid breakdownl2,18. In the d L G N acetylcholine also facilitates
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