- Email: [email protected]
Effects of neuropeptide Y on the electrical properties of neurons
27 Zinsmaier, K. E., Eberle, K. K., Buchner, E., Walter, N. and Benzer, S. (1994) Science 263, 977-980 28 Pierce, J. P. and Lewin, G. R. (1994) Neuroscience 58, 441-446 29 Pierce, J. P. and Mendell, L. M. (1993) J. Neurosci. 13, 4748-4763 30 Robinson, P. J. etaL (1993) Nature 365, 163-165 31 van der Bliek, A. M. and Meyerowitz, E. M. (1991) Nature 351, 411-414 32 Nedivi, E., Hevroni, D., Naot, D., Israeli, D. and Citri, Y. (1993) Nature 363, 718-722 33 Osen-Sand, A. eta/. (1993) Nature 364, 445-448 34 Bray, D. (1991) in The Growth Cone (Letourneau, P. C., Kater, S. B. and Macagno, E. R., eds), pp. 7-17, Raven 35 Pfenninger, K. H. et a/. (1991) in The Growth Cone (Letourneau, P C., Kater, S. B. and Macagno, E. R., eds), pp. 111-123, Raven 36 Pfenninger, K. H. and Friedman, L. B. (1993) Dev. Brain. Res. 71, 181-192 37 Lockerbie, R. 0., Miller, V. E. and Pfenninger, K. H. (1991) J. Ce/IBiol. 112, 1215-1227 38 Bark, C. (1993) J. Mol. Biol. 233, 6 7 - 7 6 39 Catsicas, S. eta/. (1991) Proc. Natl Acad. Sci. USA 88, 785-789 40 Sodhof, T. C. eta/. (1989) Science 245, 1474-1480
41 Trimble, W. S., Gray, T. S., Elferink, L. A., Wilson, M. C. and Scheller, R. H. (1990) J. Neurosci. 10, 1380-1387 42 Mandell, J. W., Czernik, A. J., De Camilli, P., Greengard, P., Townes-Anderson, E. (1992)J. Neurosci. 12, 1736-1749 43 Catsicas, S. etal. (1992)Z Neurosci. Res. 33, I - 9 44 Romano, C. et aL (1987)J. Neurosci. 7, 1294-1299 45 Glanzman, D. L., Kandel, E. R. and Schacher, S. (1990) Science 249, 799-802 46 Schuman, E. M. and Madison, D. V. (1991) Science 254, 1503 - 1506 47 Hess, D. T., Patterson, S. I., Smith, D. S. and Pate Skene, J. H. (1993) Nature 366, 562-565 48 Lohof, A. M., Ip, N. Y. and Poo, M-M. (1993) Nature 363, 350-353 49 Carmignoto, G., Canella, R., Candeo, P., Comelli, M. C. and Maffei, L. (1993) J. Physiol. 464, 343-360 50 Tancredi, V., D'Arcangelo, G., Mercanti, D. and Cafissano, P. (1993) NeuroReport 4, 147-150 51 Lu, B., Yokoyama, M., Dreyfus, C. F. and Black, I. B. (1991) Proc. Natl Acad. 5ci. USA 88, 6289-6292 52 Calakos, N., Bennett, M. K., Peterson, K. E. and Scheller, R. H. (1994) Science 263, 1146-I 149 53 Lynch, M. A., Voss, K. U, Rodriguez, J. and Bliss, T. V. P. (1994) Neuroscience 60, 1 - 5
Acknowledgements Weare grateful to M. Cats~cas,K. Hardy, R. Hvt, J. Knowles, R. A. North and the neuroblo/ogygroup at GIMB for enthusiastic discuss/onsand numerous suggestionson the manuscript, and to C Hebertfor help with the figures.
Effects of neuropeptide g on the electricalproperties of neurons William
F. C o l m e r s a n d D a v i d B l e a k m a n
Neurop@tide Y, one or the scions of the pancreatic" polyp@tide .family, is tbund throughout the nervous system. Based on its abundance alone, one would expect neuropeptide Y to play an important role in the regulation o.f neuronal activity, and indeed many pharmacological studies have demonstrated neuromodulatory e/-fects o/ neuropeptide }7. Here, William F. Colmers and David Bleakman review the known actions of neurop@tide Y on the electrical properties of nerve cells. Neurop@tide Y inhibits Ca e+ currents, and modulates transmitter release in a highly selective manner. Neuropeptide Y might be quite important in the regulation qf neuronal state, as exemplified by its actions in the hippocampus and the dorsal raph? nucleus.
in noradrenergic neurons of the A1 and A., groups in the medulla, and the locus coeruleus (LC). In the hypothalamus, NPY is found predominantly in the arcuate nucleus and lateral hypothalamus (Table I).
Receptors for NPY Neuropeptide Y appears to act upon at least four types of receptors called (at present) YI-Y:~ and an 'atypical Y]' receptor, which mediates the feeding response stimulated by NPY (see Ref. 3). The distinction of receptor subtype is based predominantly on pharmacological profiles of agonist-fragment sensitivity since specific antagonists await development. However, even in the absence of specific pharmacological agents for the purported receptor subtypes, there is convincing evidence emerging supporting the The distribution of neuropeptide Y (NPY) in the CNS existence of these subtypes. Most convincing is the and PNS has been reviewed recently l'z, and will thus recent cloning of the Y] receptor. The contributions not be described extensively here. Briefly, in the that experiments using in vivo antisense knockout of PNS, NPY is found in, for example, the noradrenergic mRNA encoding this receptor I have made to our sympathetic innervation of blood vessels and other knowledge have been summarized recently'~. A pursmooth muscle tissues, and in neurons within the ported clone for the Y:~ receptor ~5was subsequently enteric nervous system. Neuropeptide Y immuno- shown not to encode an NPY receptor 7. Table It reactive fibers also occur in non-vascular smooth shows the rank order of potency for NPY and related muscle, surrounding exocfine glands and surface peptides at the Y]-Y:~ receptor subtypes, and the epithelia. Although NPY is widely distributed, it has tissue in which they have been examined. become apparent from extensive immunocytochemical studies that NPY occurs in subpopulations Actions of NPY in the PNS of neurons (Table I). Neuropeptide Y is generally The effects of NPY have been studied extensively co-localized with other transmitters, particularly on sensory, sympathetic and enteric neurons. noradrenaline. Sensory neurons. Neuropeptide Y has been shown In the CNS, NPY is contained in GABAergic to inhibit the release of both substance P and ACh interneurons in higher centers and in (predominantly from dorsal root ganglion (DRG) neurons in culture. catecholaminergic) cells that project further caudally. How does NPY produce such effects? The effects of For example, NPY is contained in interneurons in NPY on Cae+ influx through voltage-dependent Cae+ cortex, hippocampus, amygdala, basal forebrain and channels (VDCCs) were examined. Such experiments striatum, whereas in the brainstem, NPY is contained were performed on DRG cultured neurons, and NPY
TINS, Vol. 17, No. 9, 1994
© 1994.Elsev,er5c,enceLtd
William £ Colmersis at the Dept of Pharmacology, Universityof Alberta, Edmonton,Alberta, Canada,and David Bleakmanis at the Lilly ResearchCentre Ltd, Er/ WoodManor, Wind/esham, Surrey, UK.
373
TABLE I. Distribution, connectivity and cotransmitters of some representative NPY-containing neurons in the brain
Type of neuron PNS Sympathetic
Location
Cotransmitter(s)
Sympathetic ganglia
Noradrenaline
Otic ganglion Myenteric ganglia
VIP, ACh VIP AChlCCKICGRPI somatostatin
Pyramidal basket cells Short axon cell
Cerebral cortex/ hippocampus Dentate gyrus Olfactory bulb
Medium aspiny cell
Striatum
Projection c e l l s Projection cells
Intergeniculate leaflet Arcuate nucleus
GABA/somatostatin/ NADPH diaphorase GABA SomatostatinlNADPH diaphorase SomatostatinlGABA/ NADPH diaphorase GABA ?
Projection cells
Locus coeruleus
Noradrenaline
Projection cells
C1 adrenergic group
Adrenaline
Parasympathetic Myenteric plexus
CNS Interneuron
Innervating Blood vessels, smooth muscle, organs Salivary gland Smooth muscle, Mucosa
Pyramidal cells Granule cells Granule cells GABAergic interneurons Suprachiasmatic nucleus Paraventricular and other nuclei of the hypothalamus Hypothalamus, entorhinal cortex, not neocortex or spinal cord Spinal cord
Abbreviations: VIP, vasoactive intestinal peptide; CCK, cholecystokinin; and CGRP, calcitonin gene-related peptide. For more details, see Refs I and 2.
was demonstrated to reduce both Ba 2+ and Ca e+ Ca 2+ influx, muscarinic agonists failed to change influx through VDCCs by between 20% and 60% 8'9. [Ca2+]i (Ref. 11). Similar experiments using simulThe inhibition of the VDCCs was often accompanied taneous voltage-clamp measurements of [Cae+]i with by a slowing of the activation kinetics, an effect which the Cae+-sensitive dye fura-2 confirmed the effects of has been described for the action of numerous other NPY in reducing Ica without an effect on basal [Ca2+]i G-protein-coupled receptors (for example, adreno- (Ref. 12). More physiological stimuli, such as trains of ceptors, and somatostatin and opioid receptors) on action potentials, were also used to elicit rises in VDCCs (Ref. 10). The involvement of G proteins in [Ca2+]i. Neuropeptide Y was also effective in reducing this inhibition is supported by experiments in which Ca 2+ influx under these conditions le. The receptor pretreatment of the cultures with pertussis toxin subtype mediating this inhibition of VDCCs appears to prevents the effects of NPY (Ref. 8), whereas be of the Ye subtype, by virtue of its response to addition of the o~ subunit of Go restores NPY NPY, peptide YY (PYY), and C-terminal fragments as receptor-effector coupling following pretreatment short as NPY18-36 (Ref. 12). It should be noted that with pertussis toxin9. a significant proportion (approximately 30%) of DRG One possible explanation for this effect of NPY is neurons, in culture, did not respond to NPY with a that NPY increased [Ca')+]i, and that it was this change in Ca e+ influx. increase in [Ca2+]i that was responsible for the The type of VDCC inhibited by NPY has been reduction in Ca '2+ influx. Such a Cae+-induced examined in acutely isolated vagal afferent neurons reduction in Ca 2+ influx has previously been investi- dissociated from the rat nodose ganglion. Neurogated as a possible mechanism for muscarinic agonist- peptide Y appears to inhibit N- but not T-type VDCCs induced reductions in Ica in sympathetic neurons. (Fig. 1; Ref. 13). The effect of NPY was potent Although Ca 2+ was required per se for the reduction in (EC5o of approximately 6 riM), and maximal concentrations (100nM) blocked about 30% of the peak current 1:~. InterTABLE II. Proposed rank order of potency of NPY and related peptides at the receptors for NPY in estingly, in approximately 5% of prototypical tissues and cultured cells. these neurons, application of NPY increased the influx of Ca e+ Receptor Peptides Tissue through VDCCs by about 20% 1:/ Y1 PYY t> NPY >t [Pro34]NPY* > > NPY13-36 Cloned and expressed Y1 receptor, blood (Fig. 1). In contrast to the invessels, smooth muscle cells, cerebral hibitory effect of NPY which cortex, hippocampal and cortical appeared to be mediated by the neurons, SK-N-MC neuroblastoma cells Ye subtype, the facilitation of PYY > NPY > NPY13-36 > > [Pro34]NPY* Nerve terminals, renal tubules, Y2 VDCC appeared to be a result of hippocampus, SK-N-BE(2) the activation of a Yt receptor. neuroblastoma cells NPY I> [Pro34]NPY* I> NPY13-36 > > PYY Brain stem (NTS), cardiac membranes, Y3 Indeed, application of agonists adrenal medullary chromaffin cells, selective for Y1 and Ye receptors sympathetic neurons could result in the enhancement * [Leu31Pro34]NPY is a widely-used analogue with similar potency and selectivity to [Pro34]NPY.Abbreviations: and inhibition of VDCCs, respectNPY, neuropeptide Y; NTS, nucleus tractus solitarius; and PPY, peptide YY. Modified, with permission, from ively, in the same neuron 1:; Ref. 3. (Fig. 1). 374
TINS, Vol. 17, No. 9, 1994
Although a functional role for NPY in the spinal cord has not yet been established, there are a number of possible physiological functions that NPY could fulfill. Given that NPY is able to modulate transmitter release and Ca2+ influx in sensory neurons, a role for NPY might be anticipated in the control of nociception by the spinal cord. Indeed, intrathecal injections of NPY have been shown to have antinociceptive effects 14. Thermal nociception was inhibited by Y2receptor agonists (consistent with the inhibition, mediated by Y2 receptors, of Ca~+ currents in the DRG). However, mechanical nociception was inhibited only by C-terminal NPY fragments, and not by full-length analogues or even the intact peptide. This latter receptor's agonist profile is not consistent with any known or proposed NPY-receptor subtype 14. Sympathetic neurons. Neuropeptide Y inhibits the release of noradrenaline from sympathetic nerve terminals and, simultaneously, potentiates the effects of noradrenaline postsynaptically3. As in sensory neurons, NPY had been shown to inhibit Ca2+ currents in sympathetic cells 15. Although a Y2 receptor for NPY has been shown to mediate inhibition of noradrenaline release from sympathetic terminals in intact preparations 3, it appears that the inhibition of VDCCs in acutely isolated superior cervical ganglion (SCG) cells is mediated by a different receptor 1~. The agonist profile of this receptor resembles that of the proposed Y3 recepto r17'18 because it is unresponsive to PYY (Ref. 16), unlike the release of noradrenaline, which is very sensitive to PYY (Ref. 3). The type of VDCC inhibited by NPY appears to be the to-conotoxin GVIA sensitive N-type VDCC, and this NPY receptor is also coupled to the Ca2+ channels by a substrate that is sensitive to pertussis toxin. Interestingly, SCG cells also possess a separate receptor for the related molecule, pancreatic polypeptide (PP). Activation of this receptor by PP results in the inhibition of Ca ~+ influx, even in neurons that are unresponsive to NPY (Ref. 16). Bullfrog sympathetic ganglion 'C' cells that innervate blood vessels, contain NPY, as do the noradrenergic neurons that innervate mammalian blood vessels. Bullfrog 'B' fibers and sympathetic neurons innervating skin in mammals tend not to contain NPY (for example, see Ref. 3). Neuropeptide Y reduces a Ca ~+ current in C cells also 19. In addition, NPY, like noradrenaline, activates an inwardly rectifying K + current in these cells 2°. Currently, this is the only description of NPY activating voltage-gated currents other than Ca2+ currents, although it has been demonstrated that NPY can potentiate, but not activate, the inwardly rectifying K + current activated by o~2-adrenoceptors in LC neurons 21. Physiologically, NPY could be important in regulation (by autoreceptors) of the excitability of the sympathetic neurons that innervate blood vessels. Mechanism of presynaptic action Several potential mechanisms are known whereby NPY might inhibit release of noradrenalfile from sympathetic nerve terminals, including an increase in K ÷ conductance, a decrease in Ca 2+ conductance, or an action on the release mechanism. The majority of studies performed using NPY have examined effects on neuronal cell bodies. The disadvantage in using such preparations is that the techniques used, such as TINS, Vol, 17, No. 9, 1994
+10
(mY) -70
0.25 nA 20 ms
Fig. 1. Neuropeptide Y (NPY) receptors have differential effects on Ca 2÷ currents in single acutely dissociated nodose ganglion neurons. Calcium currents were evoked from V~ = - 7 0 m V by an 8 0 m V voltage step defivered via the patch pipette. Middle traces, control and washout; bottom, in the presence of 100 nM of the Y1-receptor-selective agonist [Pro34]NPY; and top, in the presence of lOOnM of the Y2-receptor-selective agonist NPY13-36. Activation of neither receptor affected sustained, high-threshold Ca2+ currents eficited from Vh = --40mY in these cells. Reproduced, with permission, from
Ref. 13.
whole-cell voltage-clamp electrophysiology, require adequately sized structures. How tenable, then, is it to extrapolate observations made on the cell soma to nerve terminals, the sites of transmitter release? One way to justify such extrapolations is to correlate results from studies of the cell soma with synaptic events. Unfortunately, in such systems, it is often difficult to establish sites of action unequivocally. However, imaging methods, in combination with electrophysiology, have provided clues to the mechanism of action of NPY. Sympathetic cells grown in culture with atrial myocytes form synaptic connections between axon terminals and target cells. In these cultures, the spontaneous activity of the muscle cells is inhibited by stimulation of the presynaptic neuron (in this case, trains of action potentials are induced by stimulation with a patch pipette). Bath application of NPY blocks the effect of neuronal activation 22. To identify synaptic terminals, the styryl dye, RH414, can be used, which partitions into the outer leaflet of the membrane, and stains the interior of synaptic vesicles upon recyclinge3. By recording from the neuron using a patch pipette fried with the Ca2+-sensitive dye fura-2, intracellular Ca2+ concentrations in proximal, RH414positive, presynaptic terminals can be measured. Trains of action potentials elevate [Ca2+]i in the terminal region, but not in the axon. Application of NPY reversibly reduces the rise in intraterminal [Ca2+] elicited by the action potential (Fig. 2). When ¢o-conotoxin GVIA is applied to block N-type Ca 2+ channels, the stimulated rise in [Ca2+]i is reduced irreversibly by approximately 60%, but subsequent 375
or both, during elevated or prolonged activity. It might be specu600 NPY lated that such effects will result in less noradrenaline release with a concomitant increase in postsynaptic-receptor sensitivity, thus maintaining tone with reduced adrenoceptor activation (and presumably desensitization). A proper c- 400 test of such a hypothesis awaits the availability of effective, sub..ICgTX type-specific, antagonists to NPY NPY receptors. Moreover, it is un0 certain what the Ya-receptormediated inhibition of somatic Ica 200 means for sympathetic physiology. Myenteric plexus neurons. Actions of NPY have also been studied in myenteric plexus neurons in primary cell culture. Similar results to S 10 S S S S S S S S S S S S those obtained in autonomic neur1540 1840 2140 ons were found, that is, NPY 1240 HO 940 inhibits VDCCs in myenteric plexus neurons also. The current inhibited Time (s) by NPY was insensitive to dihydropyridines, and was characterized as Fig. 2. Neuropeptide Y (NPY) inhibits an w-conotoxin GVIA (CgTx)-sensitive Ca 2+ current at identified synapses between sympathetic nerve terminals and atrial myocytes in culture. Nerve an N-type VDCC (Ref. 27). Again terminals were identified both by staining with the styryl dye RH414, and by the response to action the actions of NPY were sensitive potentials elicited in the cell soma by a patch pipette filled with fura-2. Trains of action potentials to pretreatment with pertussis (20 Hz, 2 s), elicited approximately every 70-100s, elicited simultaneous increases in [Ca2+], in toxin. Hirning and colleagues z7 also three separate presynaptic regions (traces shown superimposed). Application of 100n44 NPY demonstrated that NPY did not act reduced the Ca 2+ influx by 38%. Washout of NPY reversed the inhibition. Application of CgTx to via an easily diffusible second the preparation reduced the influx of Ca2+ by 6_6% (the response labeled 10 is to a lOHz, 4 s train messenger, as the activity of single of action potentials). After application of conotoxin, NPY reduced the influx of Ca2+ only by 9% of N-type VDCC recorded in the cellthe first response, and had no effect on the second response. Figure courtesy of V. P. Bindokas, attached mode was unaffected by data reported in Ref. 22. bath application of NPY.
]
A
v
|
|
|
I I
|
|
•
f
•
I •
"
applications of NPY have no effect on the remainder of the stimulated rise in [Ca2+]i (Fig. 2). Therefore, NPY causes presynaptic inhibition at terminals of sympathetic neurons in culture largely by inhibiting N-type VDCCs at the nerve terminal 2e. The receptor subtype mediating this effect remains to be determined. Inclusion of GTPyS in the patch pipette renders the effects of NPY on Ca 2+ influx at these presynaptic terminals irreversible, suggesting that the receptor mediating this inhibition is coupled by a G protein 22. The coupling might be direct, as N-type VDCCs have recently been demonstrated to bind the a subunit of G,, (Ref. 24). The actions of NPY at the sympathetic neuroeffector junction are twofold: a presynaptic inhibition mediated by the Y2 receptor, and a potentiation of the postsynaptic actions of noradrenaline and other excitatory transmitters via a YFreceptor-mediated mechanism (see Ref. 3). The effect of NPY via the Y1 receptor correlates well with the potentiation of Ltype VDCCs by NPY observed in smooth muscle cells, which results from a shift in the steady-state activation curve of the channel e5. It has been suggested that this potentiation provides for an increase in 'synaptic gain'26, by amplifying postganglionic output. The inhibition of noradrenaline release that is mediated by Y2 receptors might be important in limiting the amplitude of the postsynaptic response or the desensitization of the adrenoceptors, 376
*•
I
Central nervous system Effects of NPY on the electrophysiology of neurons of the CNS have been examined in several brain regions. To date, most observed effects of NPY on central neurons are inhibitory. Although most work has been done in acutely prepared hippocampal slices and hippocampal neurons in culture, other areas of the CNS have also been studied. Hippocampus. Many GABAergic interneurons in hippocampus contain NPY and also somatostatin~; electron microscopy shows immunoreactivity for NPY in varicosities near to putative glutamatergic terminals in areas CA1 and CA3 (Ref. 29). When NPY is applied to a hippocampal slice, the result is a reduction in elicited synaptic excitation of pyramidal cells in areas CA1 and CA3 (Refs 30-32). One possible explanation for this response is that NPY has effects on some of the conventional electrical properties of neurons. However, in vitro, neither the resting membrane potential, conductance, axonal excitability nor the action-potential parameters are affected by NPY (Refs 30 and 33). Furthermore, NPY does not affect Ba 2+dependent action potentials in hippocampal pyramidal cells in slices 3a, or their Ca ~+ currents in primary culture a4, nor does it affect their responses to glutamate aa-aa neither in culture nor in slices (but see Refs 36 and 37). Neuropeptide Y had no effect on synapfically mediated GABAergic inhibition 32. Having excluded all these possibilities, the most parsimonious explanation for the observed reduction in excitability TINS, Vol. 17, No. 9, 1994
A
krnV
B
2s
/
100 ms
Fig. 3. Presynaptic inhibition by neuropeptide Y, but not postsynaptic inhibition by 5-HT, inhibits stimulus-tram-induced bursting (STIB) in rat hlppocampal area CA3 neurons in vitro. Extracellular chart recording from the pyramidal ceil layer in area CA3B of a rat hippocampal slice. A stimulus train (600 ms, 60Hz) is applied to stratum radiatum of CA3 (Ref. 40), resulting in the prolonged afterdischarge in CA3. (A) STIB response in control (left) and during application of 1i~44 NPY (right). Remaimng response represents discharge of a single (unidentified) extraceflular unit. (B) STIB response in the same slice in control (left) and during application of 3 l~M 5-carboxamidotryptamine (right), which acts at 5-HT1A receptors to activate an inwardly rectifying K ÷ current in the postsynaptic pyramidal cells. Expansions, below, show digital oscilloscope traces of representative regions of the afterdischarge on a faster timescal¢. Note that the morphology of the afterdischarge components is altered by 5-carboxamidotryptamine, although the afterdischarge is as prolonged as in control. (Ref. 40 and unpublished data.) would be an action of NPY at a presynaptic site, most likely the nerve terminal, to selectively inhibit excitatory synaptic transmission. As with the studies performed on peripheral neurons, it seems that the Y2 receptor mediates this inhibitory action of NPY, since NPY and PYY are equipotent agonists and C-terminal fragments as short as NPY16-36 are agonists with lower potency (although efficacy was not assessed34'38). In a few hippocampal neurons in cell culture, excitatory responses were produced by NPY via a Y1 receptor ~4, a situation reminiscent of excitatory effects seen in nodose ganglion neurons (see above). The selective suppression of excitatory transmission by NPY in the hippocampus suggests that its physiological rote in hippocampus might relate to the control of excitability. The prime example of uncontrolled excitability in hippocampus is epileptic seizure. Indeed, there are models of epileptiform activity induced in in vitro slices in which such a hypothesis can be tested. If hippocampal slices are exposed to saline containing nominally zero Mg ~+, epileptiform activity results, often resembling interictal discharges 3"~. Neuropeptide Y potently inhibits the frequency of these discharges, without affecting their waveform 4°. In another such model, stimulus trains applied to area CA3 result in afterdischarges that closely resemble electrographic seizures recorded in vivo41. When applied to such a preparation, NPY causes cessation of the elicited responses 4° TINS, Vol. 17, No. 9, 1994
(Fig. 3). Interestingly, activation of dendritic K + currents by an agonist acting at the 5-HT1A receptors did not suppress the afterdischarges 4° (Fig. 3). In cultures of hippocampal pyramidal neurons, networks of excitatory synaptic connections are formed which cause resonating synaptic potentials resembling epileptiform discharges. Neuropeptide Y, by activating a Y2 receptor, potently inhibits these resonating synaptic potentials 34. Finally, there are several key experimental observations from studies in vivo that point to NPY having effects in models of epilepsy. For example, the release of NPY is enhanced following kindling 42, and the expression of NPY is increased 43 or even following several following convulsions', repetitions of relatively brief periods of intense activity 44. N P Y and the sigma (a) receptor site. Several reports have suggested that NPY might be a natural ligand for the putative 0 receptor site in the hippocampus. Experiments have shown that NPY was able to displace ligands for this receptor, and the iontophoretic application of NPY and related peptides onto CA3 pyramidal cells of rat dorsal hippocampus in vivo potentiated the extracellularly recorded responses of these neurons to iontophoretic pulses of N-methyl-Daspartate (NMDA)'36"'"37.45 ' . In the same cells, NPY did not potentiate either quisqualate [acting at D,L-O~amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors] or kainate responses 36'37. However, this response is difficult to fit to any of the
377
known binding profiles for NPY receptors, and other workers have been unable to reproduce the competitive displacement of NPY binding by ligands for the 0 receptor 46'47. Furthermore, responses of rat dorsal hippocampal CA3 neurons in brain slices to iontophoretic applications of NMDA were insensitive to NPY, although the presynaptic actions on mossyfiber responses described above were observed 35. Interestingly, binding studies in the mouse in vivo show that a certain percentage of binding to 0 receptors is displaced by NPY and related peptides in a pattern consistent with a Y~ receptor 4s. The failure of the results obtained in vivo to be reproduced in vitro is puzzling, and suggests that further work is needed to confirm the possibility of interactions between NPY receptors and o ligands. Dorsal raphd nucleus. The dorsal raph6 nucleus of the brainstem comprises the largest group of 5-HTcontaining neurons in the CNS. The 5-HT that these neurons release has been associated with the control of many behaviors, including mood, aggression, food intake and reproductive behavior, the last two of which have been associated with NPY also. Stimulation of the synaptic inputs to the 5-HT-containing neurons in the dorsal raph6 nuclei reveals complex responses, comprising sequentially, rapid, excitatory (AMPA/NMDA) and inhibitory (GABAA) receptormediated potentials, a 5-HTaa-receptor-mediated IPSP, and an oq-adrenoceptor-mediated slow EPSP (Ref. 49). When a Y2 receptor is activated, there is a selective reduction of the 5-HT- and adrenoceptormediated responses, without any change in the potentials mediated by amino acids 5°. As with the hippocampal cells, there was no evidence for an effect of NPY on postsynaptic-membrane properties, nor did it affect the conductance changes induced in the dorsal raph6 cells by activation of either 5-HT~A or oqadrenoceptors, suggesting a selective, presynaptic site of action to inhibit release of noradrenaline and 5-HT (Ref. 50). The actions of NPY at first appear paradoxical in that both excitation and inhibition are reduced. The tonic firing activity observed in dorsal raph6 neurons in vivo results from tonic noradrenergic input, while the IPSP results from activation of an autoreceptor by 5-HT. Therefore, release of NPY in the dorsal raph6 nucleus would probably reduce the tonic activity of dorsal raph~ neurons by suppressing the release of noradrenaline. At the same time, it would enable the dorsal raph6 neurons to fire for longer periods of time in response to the activation of their excitatory amino acid receptors, since it will also reduce the autoinhibition, by reducing the local release of 5-HT. A similar change of neuronal state, termed 'quiet readiness '51, is induced by the actions of noradrenaline on hippocampal pyramidal neurons, albeit by a postsynaptic mechanism52. Locus coeruleus. Many noradrenergic fibers of the brain arise from neurons of the brainstem nucleus LC. Innervation from this group of cells extends to the hippocampus and the surface of the cerebral cortex. Many LC cells that contain noradrenaline express NPY also. Neuropepfide Y hyperpolarizes these neurons by increasing a K + conductance. However, it only does so when o~2-adrenoceptors are activated. Thus, NPY potentiates the K+-channel response to o~2-adrenoceptor agonists'~1. This is specific to the o~2378
adrenoceptor-effector pathway, since NPY has no further effect when the same K + channel is activated via the ~ opioid receptor instead21. In several smooth muscle preparations, NPY potentiates contractile responses mediated by oq-adrenoceptors in smooth muscle via a Y1 receptora; indeed all reported receptor-receptor interactions have been via the Y1 receptor. Surprisingly, therefore, the NPY effect in the LC was consistent with the involvement of a Y2 receptor 53. The release of NPY in the LC could possibly contribute to activity dependent inhibition of the neurons; if NPY is released locally from LC cells it could help potentiate the inhibitory o~2-autoreceptor response 54, and thereby help limit the firing of those LC cells that are strongly stimulated, in another form of the synaptic gain theory26. Concluding remarks Neuropeptide Y has been shown to have numerous actions on the electrophysiology of neurons and their connections in both the peripheral and CNSs. In some cases, we can speculate as to the task this extensively distributed neuromodulator might play in a given part of the nervous system. However, a vast amount of work remains to be carried out. For example, there is, at present, no documentation of the nature or mechanism of action of the Y1 receptor on neurons of the neocortex or other forebrain areas, yet this is potentially of great importance to the understanding of mechanisms of anxiety4. The apparent differential distribution of NPY receptors on sympathetic neurons (Y3 receptors at the soma, Y2 receptors at the terminal) is also at present unexplained. The site and mechanism of action of NPY at the atypical Y1 receptor, which mediates the feeding response, is also not yet known but will also be central to the understanding of, and potential therapeutic intervention at, feeding mechanisms that are mediated centrally. Finally, many questions will remain open until appropriate antagonists become available, although the use of antisense oligonucleotides directed against either the receptors' or the peptide's mRNA might assist in the absence of the appropriate pharmacological tools 3'4. In any case, the pace of the field is likely to remain as brisk as it has been in the decade or so since the discovery of NPY.
Selected references 1 Hendry, S, H. C. (1993)in The Biology of Neuropeptide Yand Related Peptides (Colmers, W. and Wahlestedt, C., eds), pp. 65-156, Humana Press 2 Sundler, F., B6ttcher, G,, Ekblad, E. and H,~kanson, R. (1993) in The Biology of Neuropeptide Y and Related Peptides (Colmers, W. and Wahlestedt, C., eds), pp. 157-196, Humana Press 3 Wahlestedt, C. and Reis, D. J. (1993)Annu. Rev. PharmacoL ToxicoL 32, 309-352 4 Wahlestedt, C., Merlo Pich, E., Koob, G. F., Yee, F. and Heilig, M. (1993) Science 259, 528-531 5 Heilig, M., Koob, G. F., Ekman, R. and Britton, K. T. (1994) Trends Neurosci. 17, 80-85 6 Rimland, J. R. etal. (1991) Mol. Pharmacol. 40, 869-875 7 Jazin, E. E. et aL (1993) Regul. Peptides 47, 247-258 8 Walker, M. W., Ewald, D. A., Perney, T. M. and Miller, R. J. (1988) J. NeuroscL 8, 2438-2446 9 Ewald, D. A., Pang, I-H., Sternweis, P. C. and Miller, R. J. (1989) Neuron 2, 1185-1193 10 Bean, B. P. (1989) Nature 340, 153-156 11 Beech, D. J., Bernheim, L., Mathie, A. and Hille, B. (1991) Proc. Natl Acad. 5ci. USA 88, 652-656 12 81eakman, D., Colmers, W. F., Fournier, A. and Miller, R. J. (1991) Br. J. Pharmacol. 103, 1781-1789
TINS, Vo/. 17, No. 9, 1994
13 Wiley, J. W., Gross, R. A. and Macdonald, R. L. (1993) J. Neurophysiol. 70, 324-330 14 Hua, X-Y. et al. (1991) J. Pharmacol. Exp. Ther. 258, 243 -248 15 Plummer, M. R., Rittenhouse, A., Kanevsky, M. and Hess, P. (1991) J. Neurosci. 1 I, 2239-2248 16 Foucart, S., Bleakman, D., Bindokas, V. P. and Miller, R. J. (1993) J. Pharmacol. Exp. Ther. 265, 903-909 17 Balasubramaniam, A., Sheriff, S., Rigel, D. F. and Fischer, J. E. (1990) Peptides 1 I, 545-550 18 Grundemar, L., Wahlestedt, C. and Reis, D. J. (1991) J. Pharmacol. Exp. Ther. 258, 633-638 19 Schofield, G. G. and Ikeda, S. R. (1988) Eur. J. Pharmacol. 151,131-134 20 Zidichouski, J. A., Chen, H. and Smith, P. A. (1990) Neurosci. Left. 117, 123-128 21 liles, P. and Regenold, J. T. (1990) Nature 344, 62-63 22 Toth, P. T., Bindokas, V., Bleakman, D., Colmers, W. F. and Miller R. J. (1993) Nature 364, 635-639 23 Betz, W. and Bewick, G. S. (1992) Science 255, 200-203 24 McEnerny, M. W., Snowman, A. M. and Snyder, S. H. (1994) J. Biol. Chem. 269, 5 - 8 25 Xiong, Z., Bolzon, B. and Cheung, D. W. (1993) Pfl6gers Arch. 423,504-510 26 Horn, J. P. (1992) Can. J. Physiol. Pharmacol. (Suppl.) 70, 19-26 27 Hirning, L. D., Fox, A. P. and Miller, R. J. (1990) Brain Res. 532, 120-130 28 Kohler, C., Eriksson, L., Davies, S. and Chan-Palay, V. (1987) Neurosci. LetL 78, 1 - 6 29 Milner, T. A. and Veznedaroglu, E. (1992) Hippocampus 2, 107-126 30 Colmers, W. F., Lukowiak, K. D. and Pittman, Q. J. (1987) J. PhysioL 383,285-299 31 Haas, H. L., Hermann, A., Greene, R. W. and Chan-Palay, V. (1987) J. Comp. Neurol. 257, 208-215 32 Klapstein, G. J. and Colmers, W. F. (1993) Hippocampus 3, 103-112 33 Colmers, W. F., Lukowiak, K. D. and Pittman, Q. J. (1988) J. Neurosci. 8, 3827-3837 34 Bleakman, D., Harrison, N. L., Colmers, W. F. and Miller, R. J.
(1992) Br. J. PharmacoL 107, 334-340 35 McQuiston, A. R. and Colmers, W. F. (1992) Neurosci. Lett. 138, 261-264 36 Monnet, F. P., Debonnel, G. and de Montigny, C. (1990) Eur. J. Pharmacol. 182, 207-208 37 Monnet, F. P., Fournier, A., Debonnel, G. and De Montigny, C. (1992)J. PharmacoL Exp. Ther. 263, 1212-1218 38 Colmers, W. F., Klapstein, G. J., Fournier, A., St-Pierr e, S. and Treherne, K. A. (1991) Br, J. Pharmacol. 102, 41-44 39 Coan, E. J. and Collingridge, G. L. (1985) Neurosci. Lett. 53, 21-26 40 Klapstein, G. J. and Colmers, W. F. (1993) Soc. Neurosci. Abstr. 19, 1869 41 Stasheff, S. A., Bragdon, A. and Wilson, W. A. (1985) Brain Res. 344, 296-302 42 Rizzi, M., Monno, A., Samanin, R., Sperk, G. and Vezzani, A. (1993) Eur. J. Neurosci. 5, 1534-1538 43 Wahlestedt, C. et aL (1990) Brain Res. 507, 65-68 44 Goodman, J. H. and Sloviter, R. S. (1993) Brain Res. 616', 263-272 45 Roman, F. J. etaL (1989) Eur. J. PharmacoL 174, 301-302 46 Tam, S, M. and Mitchell, K. N. (1991) Eur. J. Pharmacol. 193, 121-122 47 Quirion, R., Mount, H., Chaudieu, I., Dumont, Y. and Boksa, P. (1991)in NMDA Receptor Related Agents: Biochemistry, Pharmacology and Behavior (Kamayama, T., Nabeshima, T. and Domino, E. F., eds), pp. 203-210, NPP 48 Bouchard, P., Dumont, Y., Fournier, A., St-Pierre, S. and Quirion, R. (1993) J. Neurosci. 13, 3926-3931 49 Williams, J. T., Colmers, W. F. and Pan, Z. Z. (1988) J. Neurosci. 8, 3499-3506 50 Kombian, S. B. and Colmers, W. F. (1992) J. Neurosci. 12, 1086-1093 51 Livingstone, M. S. and Hubel, D. H. (1981) Nature 291, 554-561 52 Madison, D. V. and Nicoll, R. A. (1982) Nature 299, 636-638 53 Illes, P., Finta, E. and Nieber, K. (1993) Naunyn-Schmied. Arch. PharmacoL 348, 546-548 54 Williams, J. T., Henderson, G. and North, R. A. (1985) Neuroscience 14, 95-101
Acknowledgements We would like to thank our co-authors on our individualand collaborativestudies on neuropeptide Y, mostnotably R.J.Mi//er, V. P. Bindokas, P. Toth, N. Harrison, £ B. Kombian, A. R.McQuiston and G.3. Klapstein,many of whomgenerously supp/iedunpublished data orfigures. Researchin W F.C's laboratory was supportedby the MedicalResearch Councilof Canada. W.E C isa Seholarof the AIberta Heritage Foundation for MedicalResearch.
Columnarorganizationin the midbrainperiaqueductalgray: modulesfor emotionalexpression? R i c h a r d B a n d l e r a n d M i c h a e l T. S h i p l e y
Independent discoveries in several laboratories suggest that the midbrain periaqueductal gray (PAG), the celldense region surrounding the midbrain aqueduct, contains a previously unsuspected degree of anatomical and functional organization. This organization takes the form of longitudinal columns of afferent inputs, output neurons and intrinsic interneurons. Recent evidence suggests: that the important functions that are classically associated with the PAG - defensive reactions, analgesia and autonomic regulation - are integrated by overlapping longitudinal columns of neurons; and that different classes of threatening or nociceptive stimuli trigger distinct co-ordinated patterns of skeletal, autonomic and antinociceptive adjustments by selectively targeting specific PAG columnar circuits. These findings call for a fundamental revision in our concept of the organization of the PAG, and a recognition of the special rolesplayed by different longitudinal PAG columns in co-ordinating distinct strategies for coping with different types of stress, threat and pain.
havioral responses to threatening or stressful stimuli. Traditionally, there was little interchange between these research areas. However, a meeting held during 1990 brought together scientists working on the PAG from a variety of perspectives. A number of important points of consensus emerged from this meeting and a subsequent symposium at the Annual Meeting of the Society for Neuroscience in 1992. First, that the nocicepfive inhibition and behavioral responses elicited from the PAG were best conceptualized as components of co-ordinated reactions important for survival. Second, that functional specificity within the PAG was represented in the form of distinct, longitudinal neuronal columns extending, for varying distances, along the rostrocaudal axis of the PAG 1-3. In this article, the evidence for longitudinal columnar organization within the PAG is reviewed, and the mechanisms by which PAG columns coordinate distinct patterns of behavioral and physiological reactions critical for survival are considered.
RichardBandleris at the Dept of Anatomy and Histology, Universityof Sydney, Sydney, New South Wales2006, Australia, and Michael T. Shipleyis at the Oept of Anatomy, University of Maryland, 655 WestBaltimore Street, Baltimore,MD 21201-1559, USA.
During the past two decades, two largely independent Historical perspective themes have dominated research on the PAG, namely Early studies reported that defensive or 'aversive' inhibition of nociception, and the integration of be- reactions, hypertension and, later, antinociception, TINS, Vol. 17, No. 9, 1994
© 1994, Elsevier ScienceLtd
379
41 Trimble, W. S., Gray, T. S., Elferink, L. A., Wilson, M. C. and Scheller, R. H. (1990) J. Neurosci. 10, 1380-1387 42 Mandell, J. W., Czernik, A. J., De Camilli, P., Greengard, P., Townes-Anderson, E. (1992)J. Neurosci. 12, 1736-1749 43 Catsicas, S. etal. (1992)Z Neurosci. Res. 33, I - 9 44 Romano, C. et aL (1987)J. Neurosci. 7, 1294-1299 45 Glanzman, D. L., Kandel, E. R. and Schacher, S. (1990) Science 249, 799-802 46 Schuman, E. M. and Madison, D. V. (1991) Science 254, 1503 - 1506 47 Hess, D. T., Patterson, S. I., Smith, D. S. and Pate Skene, J. H. (1993) Nature 366, 562-565 48 Lohof, A. M., Ip, N. Y. and Poo, M-M. (1993) Nature 363, 350-353 49 Carmignoto, G., Canella, R., Candeo, P., Comelli, M. C. and Maffei, L. (1993) J. Physiol. 464, 343-360 50 Tancredi, V., D'Arcangelo, G., Mercanti, D. and Cafissano, P. (1993) NeuroReport 4, 147-150 51 Lu, B., Yokoyama, M., Dreyfus, C. F. and Black, I. B. (1991) Proc. Natl Acad. 5ci. USA 88, 6289-6292 52 Calakos, N., Bennett, M. K., Peterson, K. E. and Scheller, R. H. (1994) Science 263, 1146-I 149 53 Lynch, M. A., Voss, K. U, Rodriguez, J. and Bliss, T. V. P. (1994) Neuroscience 60, 1 - 5
Acknowledgements Weare grateful to M. Cats~cas,K. Hardy, R. Hvt, J. Knowles, R. A. North and the neuroblo/ogygroup at GIMB for enthusiastic discuss/onsand numerous suggestionson the manuscript, and to C Hebertfor help with the figures.
Effects of neuropeptide g on the electricalproperties of neurons William
F. C o l m e r s a n d D a v i d B l e a k m a n
Neurop@tide Y, one or the scions of the pancreatic" polyp@tide .family, is tbund throughout the nervous system. Based on its abundance alone, one would expect neuropeptide Y to play an important role in the regulation o.f neuronal activity, and indeed many pharmacological studies have demonstrated neuromodulatory e/-fects o/ neuropeptide }7. Here, William F. Colmers and David Bleakman review the known actions of neurop@tide Y on the electrical properties of nerve cells. Neurop@tide Y inhibits Ca e+ currents, and modulates transmitter release in a highly selective manner. Neuropeptide Y might be quite important in the regulation qf neuronal state, as exemplified by its actions in the hippocampus and the dorsal raph? nucleus.
in noradrenergic neurons of the A1 and A., groups in the medulla, and the locus coeruleus (LC). In the hypothalamus, NPY is found predominantly in the arcuate nucleus and lateral hypothalamus (Table I).
Receptors for NPY Neuropeptide Y appears to act upon at least four types of receptors called (at present) YI-Y:~ and an 'atypical Y]' receptor, which mediates the feeding response stimulated by NPY (see Ref. 3). The distinction of receptor subtype is based predominantly on pharmacological profiles of agonist-fragment sensitivity since specific antagonists await development. However, even in the absence of specific pharmacological agents for the purported receptor subtypes, there is convincing evidence emerging supporting the The distribution of neuropeptide Y (NPY) in the CNS existence of these subtypes. Most convincing is the and PNS has been reviewed recently l'z, and will thus recent cloning of the Y] receptor. The contributions not be described extensively here. Briefly, in the that experiments using in vivo antisense knockout of PNS, NPY is found in, for example, the noradrenergic mRNA encoding this receptor I have made to our sympathetic innervation of blood vessels and other knowledge have been summarized recently'~. A pursmooth muscle tissues, and in neurons within the ported clone for the Y:~ receptor ~5was subsequently enteric nervous system. Neuropeptide Y immuno- shown not to encode an NPY receptor 7. Table It reactive fibers also occur in non-vascular smooth shows the rank order of potency for NPY and related muscle, surrounding exocfine glands and surface peptides at the Y]-Y:~ receptor subtypes, and the epithelia. Although NPY is widely distributed, it has tissue in which they have been examined. become apparent from extensive immunocytochemical studies that NPY occurs in subpopulations Actions of NPY in the PNS of neurons (Table I). Neuropeptide Y is generally The effects of NPY have been studied extensively co-localized with other transmitters, particularly on sensory, sympathetic and enteric neurons. noradrenaline. Sensory neurons. Neuropeptide Y has been shown In the CNS, NPY is contained in GABAergic to inhibit the release of both substance P and ACh interneurons in higher centers and in (predominantly from dorsal root ganglion (DRG) neurons in culture. catecholaminergic) cells that project further caudally. How does NPY produce such effects? The effects of For example, NPY is contained in interneurons in NPY on Cae+ influx through voltage-dependent Cae+ cortex, hippocampus, amygdala, basal forebrain and channels (VDCCs) were examined. Such experiments striatum, whereas in the brainstem, NPY is contained were performed on DRG cultured neurons, and NPY
TINS, Vol. 17, No. 9, 1994
© 1994.Elsev,er5c,enceLtd
William £ Colmersis at the Dept of Pharmacology, Universityof Alberta, Edmonton,Alberta, Canada,and David Bleakmanis at the Lilly ResearchCentre Ltd, Er/ WoodManor, Wind/esham, Surrey, UK.
373
TABLE I. Distribution, connectivity and cotransmitters of some representative NPY-containing neurons in the brain
Type of neuron PNS Sympathetic
Location
Cotransmitter(s)
Sympathetic ganglia
Noradrenaline
Otic ganglion Myenteric ganglia
VIP, ACh VIP AChlCCKICGRPI somatostatin
Pyramidal basket cells Short axon cell
Cerebral cortex/ hippocampus Dentate gyrus Olfactory bulb
Medium aspiny cell
Striatum
Projection c e l l s Projection cells
Intergeniculate leaflet Arcuate nucleus
GABA/somatostatin/ NADPH diaphorase GABA SomatostatinlNADPH diaphorase SomatostatinlGABA/ NADPH diaphorase GABA ?
Projection cells
Locus coeruleus
Noradrenaline
Projection cells
C1 adrenergic group
Adrenaline
Parasympathetic Myenteric plexus
CNS Interneuron
Innervating Blood vessels, smooth muscle, organs Salivary gland Smooth muscle, Mucosa
Pyramidal cells Granule cells Granule cells GABAergic interneurons Suprachiasmatic nucleus Paraventricular and other nuclei of the hypothalamus Hypothalamus, entorhinal cortex, not neocortex or spinal cord Spinal cord
Abbreviations: VIP, vasoactive intestinal peptide; CCK, cholecystokinin; and CGRP, calcitonin gene-related peptide. For more details, see Refs I and 2.
was demonstrated to reduce both Ba 2+ and Ca e+ Ca 2+ influx, muscarinic agonists failed to change influx through VDCCs by between 20% and 60% 8'9. [Ca2+]i (Ref. 11). Similar experiments using simulThe inhibition of the VDCCs was often accompanied taneous voltage-clamp measurements of [Cae+]i with by a slowing of the activation kinetics, an effect which the Cae+-sensitive dye fura-2 confirmed the effects of has been described for the action of numerous other NPY in reducing Ica without an effect on basal [Ca2+]i G-protein-coupled receptors (for example, adreno- (Ref. 12). More physiological stimuli, such as trains of ceptors, and somatostatin and opioid receptors) on action potentials, were also used to elicit rises in VDCCs (Ref. 10). The involvement of G proteins in [Ca2+]i. Neuropeptide Y was also effective in reducing this inhibition is supported by experiments in which Ca 2+ influx under these conditions le. The receptor pretreatment of the cultures with pertussis toxin subtype mediating this inhibition of VDCCs appears to prevents the effects of NPY (Ref. 8), whereas be of the Ye subtype, by virtue of its response to addition of the o~ subunit of Go restores NPY NPY, peptide YY (PYY), and C-terminal fragments as receptor-effector coupling following pretreatment short as NPY18-36 (Ref. 12). It should be noted that with pertussis toxin9. a significant proportion (approximately 30%) of DRG One possible explanation for this effect of NPY is neurons, in culture, did not respond to NPY with a that NPY increased [Ca')+]i, and that it was this change in Ca e+ influx. increase in [Ca2+]i that was responsible for the The type of VDCC inhibited by NPY has been reduction in Ca '2+ influx. Such a Cae+-induced examined in acutely isolated vagal afferent neurons reduction in Ca 2+ influx has previously been investi- dissociated from the rat nodose ganglion. Neurogated as a possible mechanism for muscarinic agonist- peptide Y appears to inhibit N- but not T-type VDCCs induced reductions in Ica in sympathetic neurons. (Fig. 1; Ref. 13). The effect of NPY was potent Although Ca 2+ was required per se for the reduction in (EC5o of approximately 6 riM), and maximal concentrations (100nM) blocked about 30% of the peak current 1:~. InterTABLE II. Proposed rank order of potency of NPY and related peptides at the receptors for NPY in estingly, in approximately 5% of prototypical tissues and cultured cells. these neurons, application of NPY increased the influx of Ca e+ Receptor Peptides Tissue through VDCCs by about 20% 1:/ Y1 PYY t> NPY >t [Pro34]NPY* > > NPY13-36 Cloned and expressed Y1 receptor, blood (Fig. 1). In contrast to the invessels, smooth muscle cells, cerebral hibitory effect of NPY which cortex, hippocampal and cortical appeared to be mediated by the neurons, SK-N-MC neuroblastoma cells Ye subtype, the facilitation of PYY > NPY > NPY13-36 > > [Pro34]NPY* Nerve terminals, renal tubules, Y2 VDCC appeared to be a result of hippocampus, SK-N-BE(2) the activation of a Yt receptor. neuroblastoma cells NPY I> [Pro34]NPY* I> NPY13-36 > > PYY Brain stem (NTS), cardiac membranes, Y3 Indeed, application of agonists adrenal medullary chromaffin cells, selective for Y1 and Ye receptors sympathetic neurons could result in the enhancement * [Leu31Pro34]NPY is a widely-used analogue with similar potency and selectivity to [Pro34]NPY.Abbreviations: and inhibition of VDCCs, respectNPY, neuropeptide Y; NTS, nucleus tractus solitarius; and PPY, peptide YY. Modified, with permission, from ively, in the same neuron 1:; Ref. 3. (Fig. 1). 374
TINS, Vol. 17, No. 9, 1994
Although a functional role for NPY in the spinal cord has not yet been established, there are a number of possible physiological functions that NPY could fulfill. Given that NPY is able to modulate transmitter release and Ca2+ influx in sensory neurons, a role for NPY might be anticipated in the control of nociception by the spinal cord. Indeed, intrathecal injections of NPY have been shown to have antinociceptive effects 14. Thermal nociception was inhibited by Y2receptor agonists (consistent with the inhibition, mediated by Y2 receptors, of Ca~+ currents in the DRG). However, mechanical nociception was inhibited only by C-terminal NPY fragments, and not by full-length analogues or even the intact peptide. This latter receptor's agonist profile is not consistent with any known or proposed NPY-receptor subtype 14. Sympathetic neurons. Neuropeptide Y inhibits the release of noradrenaline from sympathetic nerve terminals and, simultaneously, potentiates the effects of noradrenaline postsynaptically3. As in sensory neurons, NPY had been shown to inhibit Ca2+ currents in sympathetic cells 15. Although a Y2 receptor for NPY has been shown to mediate inhibition of noradrenaline release from sympathetic terminals in intact preparations 3, it appears that the inhibition of VDCCs in acutely isolated superior cervical ganglion (SCG) cells is mediated by a different receptor 1~. The agonist profile of this receptor resembles that of the proposed Y3 recepto r17'18 because it is unresponsive to PYY (Ref. 16), unlike the release of noradrenaline, which is very sensitive to PYY (Ref. 3). The type of VDCC inhibited by NPY appears to be the to-conotoxin GVIA sensitive N-type VDCC, and this NPY receptor is also coupled to the Ca2+ channels by a substrate that is sensitive to pertussis toxin. Interestingly, SCG cells also possess a separate receptor for the related molecule, pancreatic polypeptide (PP). Activation of this receptor by PP results in the inhibition of Ca ~+ influx, even in neurons that are unresponsive to NPY (Ref. 16). Bullfrog sympathetic ganglion 'C' cells that innervate blood vessels, contain NPY, as do the noradrenergic neurons that innervate mammalian blood vessels. Bullfrog 'B' fibers and sympathetic neurons innervating skin in mammals tend not to contain NPY (for example, see Ref. 3). Neuropeptide Y reduces a Ca ~+ current in C cells also 19. In addition, NPY, like noradrenaline, activates an inwardly rectifying K + current in these cells 2°. Currently, this is the only description of NPY activating voltage-gated currents other than Ca2+ currents, although it has been demonstrated that NPY can potentiate, but not activate, the inwardly rectifying K + current activated by o~2-adrenoceptors in LC neurons 21. Physiologically, NPY could be important in regulation (by autoreceptors) of the excitability of the sympathetic neurons that innervate blood vessels. Mechanism of presynaptic action Several potential mechanisms are known whereby NPY might inhibit release of noradrenalfile from sympathetic nerve terminals, including an increase in K ÷ conductance, a decrease in Ca 2+ conductance, or an action on the release mechanism. The majority of studies performed using NPY have examined effects on neuronal cell bodies. The disadvantage in using such preparations is that the techniques used, such as TINS, Vol, 17, No. 9, 1994
+10
(mY) -70
0.25 nA 20 ms
Fig. 1. Neuropeptide Y (NPY) receptors have differential effects on Ca 2÷ currents in single acutely dissociated nodose ganglion neurons. Calcium currents were evoked from V~ = - 7 0 m V by an 8 0 m V voltage step defivered via the patch pipette. Middle traces, control and washout; bottom, in the presence of 100 nM of the Y1-receptor-selective agonist [Pro34]NPY; and top, in the presence of lOOnM of the Y2-receptor-selective agonist NPY13-36. Activation of neither receptor affected sustained, high-threshold Ca2+ currents eficited from Vh = --40mY in these cells. Reproduced, with permission, from
Ref. 13.
whole-cell voltage-clamp electrophysiology, require adequately sized structures. How tenable, then, is it to extrapolate observations made on the cell soma to nerve terminals, the sites of transmitter release? One way to justify such extrapolations is to correlate results from studies of the cell soma with synaptic events. Unfortunately, in such systems, it is often difficult to establish sites of action unequivocally. However, imaging methods, in combination with electrophysiology, have provided clues to the mechanism of action of NPY. Sympathetic cells grown in culture with atrial myocytes form synaptic connections between axon terminals and target cells. In these cultures, the spontaneous activity of the muscle cells is inhibited by stimulation of the presynaptic neuron (in this case, trains of action potentials are induced by stimulation with a patch pipette). Bath application of NPY blocks the effect of neuronal activation 22. To identify synaptic terminals, the styryl dye, RH414, can be used, which partitions into the outer leaflet of the membrane, and stains the interior of synaptic vesicles upon recyclinge3. By recording from the neuron using a patch pipette fried with the Ca2+-sensitive dye fura-2, intracellular Ca2+ concentrations in proximal, RH414positive, presynaptic terminals can be measured. Trains of action potentials elevate [Ca2+]i in the terminal region, but not in the axon. Application of NPY reversibly reduces the rise in intraterminal [Ca2+] elicited by the action potential (Fig. 2). When ¢o-conotoxin GVIA is applied to block N-type Ca 2+ channels, the stimulated rise in [Ca2+]i is reduced irreversibly by approximately 60%, but subsequent 375
or both, during elevated or prolonged activity. It might be specu600 NPY lated that such effects will result in less noradrenaline release with a concomitant increase in postsynaptic-receptor sensitivity, thus maintaining tone with reduced adrenoceptor activation (and presumably desensitization). A proper c- 400 test of such a hypothesis awaits the availability of effective, sub..ICgTX type-specific, antagonists to NPY NPY receptors. Moreover, it is un0 certain what the Ya-receptormediated inhibition of somatic Ica 200 means for sympathetic physiology. Myenteric plexus neurons. Actions of NPY have also been studied in myenteric plexus neurons in primary cell culture. Similar results to S 10 S S S S S S S S S S S S those obtained in autonomic neur1540 1840 2140 ons were found, that is, NPY 1240 HO 940 inhibits VDCCs in myenteric plexus neurons also. The current inhibited Time (s) by NPY was insensitive to dihydropyridines, and was characterized as Fig. 2. Neuropeptide Y (NPY) inhibits an w-conotoxin GVIA (CgTx)-sensitive Ca 2+ current at identified synapses between sympathetic nerve terminals and atrial myocytes in culture. Nerve an N-type VDCC (Ref. 27). Again terminals were identified both by staining with the styryl dye RH414, and by the response to action the actions of NPY were sensitive potentials elicited in the cell soma by a patch pipette filled with fura-2. Trains of action potentials to pretreatment with pertussis (20 Hz, 2 s), elicited approximately every 70-100s, elicited simultaneous increases in [Ca2+], in toxin. Hirning and colleagues z7 also three separate presynaptic regions (traces shown superimposed). Application of 100n44 NPY demonstrated that NPY did not act reduced the Ca 2+ influx by 38%. Washout of NPY reversed the inhibition. Application of CgTx to via an easily diffusible second the preparation reduced the influx of Ca2+ by 6_6% (the response labeled 10 is to a lOHz, 4 s train messenger, as the activity of single of action potentials). After application of conotoxin, NPY reduced the influx of Ca2+ only by 9% of N-type VDCC recorded in the cellthe first response, and had no effect on the second response. Figure courtesy of V. P. Bindokas, attached mode was unaffected by data reported in Ref. 22. bath application of NPY.
]
A
v
|
|
|
I I
|
|
•
f
•
I •
"
applications of NPY have no effect on the remainder of the stimulated rise in [Ca2+]i (Fig. 2). Therefore, NPY causes presynaptic inhibition at terminals of sympathetic neurons in culture largely by inhibiting N-type VDCCs at the nerve terminal 2e. The receptor subtype mediating this effect remains to be determined. Inclusion of GTPyS in the patch pipette renders the effects of NPY on Ca 2+ influx at these presynaptic terminals irreversible, suggesting that the receptor mediating this inhibition is coupled by a G protein 22. The coupling might be direct, as N-type VDCCs have recently been demonstrated to bind the a subunit of G,, (Ref. 24). The actions of NPY at the sympathetic neuroeffector junction are twofold: a presynaptic inhibition mediated by the Y2 receptor, and a potentiation of the postsynaptic actions of noradrenaline and other excitatory transmitters via a YFreceptor-mediated mechanism (see Ref. 3). The effect of NPY via the Y1 receptor correlates well with the potentiation of Ltype VDCCs by NPY observed in smooth muscle cells, which results from a shift in the steady-state activation curve of the channel e5. It has been suggested that this potentiation provides for an increase in 'synaptic gain'26, by amplifying postganglionic output. The inhibition of noradrenaline release that is mediated by Y2 receptors might be important in limiting the amplitude of the postsynaptic response or the desensitization of the adrenoceptors, 376
*•
I
Central nervous system Effects of NPY on the electrophysiology of neurons of the CNS have been examined in several brain regions. To date, most observed effects of NPY on central neurons are inhibitory. Although most work has been done in acutely prepared hippocampal slices and hippocampal neurons in culture, other areas of the CNS have also been studied. Hippocampus. Many GABAergic interneurons in hippocampus contain NPY and also somatostatin~; electron microscopy shows immunoreactivity for NPY in varicosities near to putative glutamatergic terminals in areas CA1 and CA3 (Ref. 29). When NPY is applied to a hippocampal slice, the result is a reduction in elicited synaptic excitation of pyramidal cells in areas CA1 and CA3 (Refs 30-32). One possible explanation for this response is that NPY has effects on some of the conventional electrical properties of neurons. However, in vitro, neither the resting membrane potential, conductance, axonal excitability nor the action-potential parameters are affected by NPY (Refs 30 and 33). Furthermore, NPY does not affect Ba 2+dependent action potentials in hippocampal pyramidal cells in slices 3a, or their Ca ~+ currents in primary culture a4, nor does it affect their responses to glutamate aa-aa neither in culture nor in slices (but see Refs 36 and 37). Neuropeptide Y had no effect on synapfically mediated GABAergic inhibition 32. Having excluded all these possibilities, the most parsimonious explanation for the observed reduction in excitability TINS, Vol. 17, No. 9, 1994
A
krnV
B
2s
/
100 ms
Fig. 3. Presynaptic inhibition by neuropeptide Y, but not postsynaptic inhibition by 5-HT, inhibits stimulus-tram-induced bursting (STIB) in rat hlppocampal area CA3 neurons in vitro. Extracellular chart recording from the pyramidal ceil layer in area CA3B of a rat hippocampal slice. A stimulus train (600 ms, 60Hz) is applied to stratum radiatum of CA3 (Ref. 40), resulting in the prolonged afterdischarge in CA3. (A) STIB response in control (left) and during application of 1i~44 NPY (right). Remaimng response represents discharge of a single (unidentified) extraceflular unit. (B) STIB response in the same slice in control (left) and during application of 3 l~M 5-carboxamidotryptamine (right), which acts at 5-HT1A receptors to activate an inwardly rectifying K ÷ current in the postsynaptic pyramidal cells. Expansions, below, show digital oscilloscope traces of representative regions of the afterdischarge on a faster timescal¢. Note that the morphology of the afterdischarge components is altered by 5-carboxamidotryptamine, although the afterdischarge is as prolonged as in control. (Ref. 40 and unpublished data.) would be an action of NPY at a presynaptic site, most likely the nerve terminal, to selectively inhibit excitatory synaptic transmission. As with the studies performed on peripheral neurons, it seems that the Y2 receptor mediates this inhibitory action of NPY, since NPY and PYY are equipotent agonists and C-terminal fragments as short as NPY16-36 are agonists with lower potency (although efficacy was not assessed34'38). In a few hippocampal neurons in cell culture, excitatory responses were produced by NPY via a Y1 receptor ~4, a situation reminiscent of excitatory effects seen in nodose ganglion neurons (see above). The selective suppression of excitatory transmission by NPY in the hippocampus suggests that its physiological rote in hippocampus might relate to the control of excitability. The prime example of uncontrolled excitability in hippocampus is epileptic seizure. Indeed, there are models of epileptiform activity induced in in vitro slices in which such a hypothesis can be tested. If hippocampal slices are exposed to saline containing nominally zero Mg ~+, epileptiform activity results, often resembling interictal discharges 3"~. Neuropeptide Y potently inhibits the frequency of these discharges, without affecting their waveform 4°. In another such model, stimulus trains applied to area CA3 result in afterdischarges that closely resemble electrographic seizures recorded in vivo41. When applied to such a preparation, NPY causes cessation of the elicited responses 4° TINS, Vol. 17, No. 9, 1994
(Fig. 3). Interestingly, activation of dendritic K + currents by an agonist acting at the 5-HT1A receptors did not suppress the afterdischarges 4° (Fig. 3). In cultures of hippocampal pyramidal neurons, networks of excitatory synaptic connections are formed which cause resonating synaptic potentials resembling epileptiform discharges. Neuropeptide Y, by activating a Y2 receptor, potently inhibits these resonating synaptic potentials 34. Finally, there are several key experimental observations from studies in vivo that point to NPY having effects in models of epilepsy. For example, the release of NPY is enhanced following kindling 42, and the expression of NPY is increased 43 or even following several following convulsions', repetitions of relatively brief periods of intense activity 44. N P Y and the sigma (a) receptor site. Several reports have suggested that NPY might be a natural ligand for the putative 0 receptor site in the hippocampus. Experiments have shown that NPY was able to displace ligands for this receptor, and the iontophoretic application of NPY and related peptides onto CA3 pyramidal cells of rat dorsal hippocampus in vivo potentiated the extracellularly recorded responses of these neurons to iontophoretic pulses of N-methyl-Daspartate (NMDA)'36"'"37.45 ' . In the same cells, NPY did not potentiate either quisqualate [acting at D,L-O~amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors] or kainate responses 36'37. However, this response is difficult to fit to any of the
377
known binding profiles for NPY receptors, and other workers have been unable to reproduce the competitive displacement of NPY binding by ligands for the 0 receptor 46'47. Furthermore, responses of rat dorsal hippocampal CA3 neurons in brain slices to iontophoretic applications of NMDA were insensitive to NPY, although the presynaptic actions on mossyfiber responses described above were observed 35. Interestingly, binding studies in the mouse in vivo show that a certain percentage of binding to 0 receptors is displaced by NPY and related peptides in a pattern consistent with a Y~ receptor 4s. The failure of the results obtained in vivo to be reproduced in vitro is puzzling, and suggests that further work is needed to confirm the possibility of interactions between NPY receptors and o ligands. Dorsal raphd nucleus. The dorsal raph6 nucleus of the brainstem comprises the largest group of 5-HTcontaining neurons in the CNS. The 5-HT that these neurons release has been associated with the control of many behaviors, including mood, aggression, food intake and reproductive behavior, the last two of which have been associated with NPY also. Stimulation of the synaptic inputs to the 5-HT-containing neurons in the dorsal raph6 nuclei reveals complex responses, comprising sequentially, rapid, excitatory (AMPA/NMDA) and inhibitory (GABAA) receptormediated potentials, a 5-HTaa-receptor-mediated IPSP, and an oq-adrenoceptor-mediated slow EPSP (Ref. 49). When a Y2 receptor is activated, there is a selective reduction of the 5-HT- and adrenoceptormediated responses, without any change in the potentials mediated by amino acids 5°. As with the hippocampal cells, there was no evidence for an effect of NPY on postsynaptic-membrane properties, nor did it affect the conductance changes induced in the dorsal raph6 cells by activation of either 5-HT~A or oqadrenoceptors, suggesting a selective, presynaptic site of action to inhibit release of noradrenaline and 5-HT (Ref. 50). The actions of NPY at first appear paradoxical in that both excitation and inhibition are reduced. The tonic firing activity observed in dorsal raph6 neurons in vivo results from tonic noradrenergic input, while the IPSP results from activation of an autoreceptor by 5-HT. Therefore, release of NPY in the dorsal raph6 nucleus would probably reduce the tonic activity of dorsal raph~ neurons by suppressing the release of noradrenaline. At the same time, it would enable the dorsal raph6 neurons to fire for longer periods of time in response to the activation of their excitatory amino acid receptors, since it will also reduce the autoinhibition, by reducing the local release of 5-HT. A similar change of neuronal state, termed 'quiet readiness '51, is induced by the actions of noradrenaline on hippocampal pyramidal neurons, albeit by a postsynaptic mechanism52. Locus coeruleus. Many noradrenergic fibers of the brain arise from neurons of the brainstem nucleus LC. Innervation from this group of cells extends to the hippocampus and the surface of the cerebral cortex. Many LC cells that contain noradrenaline express NPY also. Neuropepfide Y hyperpolarizes these neurons by increasing a K + conductance. However, it only does so when o~2-adrenoceptors are activated. Thus, NPY potentiates the K+-channel response to o~2-adrenoceptor agonists'~1. This is specific to the o~2378
adrenoceptor-effector pathway, since NPY has no further effect when the same K + channel is activated via the ~ opioid receptor instead21. In several smooth muscle preparations, NPY potentiates contractile responses mediated by oq-adrenoceptors in smooth muscle via a Y1 receptora; indeed all reported receptor-receptor interactions have been via the Y1 receptor. Surprisingly, therefore, the NPY effect in the LC was consistent with the involvement of a Y2 receptor 53. The release of NPY in the LC could possibly contribute to activity dependent inhibition of the neurons; if NPY is released locally from LC cells it could help potentiate the inhibitory o~2-autoreceptor response 54, and thereby help limit the firing of those LC cells that are strongly stimulated, in another form of the synaptic gain theory26. Concluding remarks Neuropeptide Y has been shown to have numerous actions on the electrophysiology of neurons and their connections in both the peripheral and CNSs. In some cases, we can speculate as to the task this extensively distributed neuromodulator might play in a given part of the nervous system. However, a vast amount of work remains to be carried out. For example, there is, at present, no documentation of the nature or mechanism of action of the Y1 receptor on neurons of the neocortex or other forebrain areas, yet this is potentially of great importance to the understanding of mechanisms of anxiety4. The apparent differential distribution of NPY receptors on sympathetic neurons (Y3 receptors at the soma, Y2 receptors at the terminal) is also at present unexplained. The site and mechanism of action of NPY at the atypical Y1 receptor, which mediates the feeding response, is also not yet known but will also be central to the understanding of, and potential therapeutic intervention at, feeding mechanisms that are mediated centrally. Finally, many questions will remain open until appropriate antagonists become available, although the use of antisense oligonucleotides directed against either the receptors' or the peptide's mRNA might assist in the absence of the appropriate pharmacological tools 3'4. In any case, the pace of the field is likely to remain as brisk as it has been in the decade or so since the discovery of NPY.
Selected references 1 Hendry, S, H. C. (1993)in The Biology of Neuropeptide Yand Related Peptides (Colmers, W. and Wahlestedt, C., eds), pp. 65-156, Humana Press 2 Sundler, F., B6ttcher, G,, Ekblad, E. and H,~kanson, R. (1993) in The Biology of Neuropeptide Y and Related Peptides (Colmers, W. and Wahlestedt, C., eds), pp. 157-196, Humana Press 3 Wahlestedt, C. and Reis, D. J. (1993)Annu. Rev. PharmacoL ToxicoL 32, 309-352 4 Wahlestedt, C., Merlo Pich, E., Koob, G. F., Yee, F. and Heilig, M. (1993) Science 259, 528-531 5 Heilig, M., Koob, G. F., Ekman, R. and Britton, K. T. (1994) Trends Neurosci. 17, 80-85 6 Rimland, J. R. etal. (1991) Mol. Pharmacol. 40, 869-875 7 Jazin, E. E. et aL (1993) Regul. Peptides 47, 247-258 8 Walker, M. W., Ewald, D. A., Perney, T. M. and Miller, R. J. (1988) J. NeuroscL 8, 2438-2446 9 Ewald, D. A., Pang, I-H., Sternweis, P. C. and Miller, R. J. (1989) Neuron 2, 1185-1193 10 Bean, B. P. (1989) Nature 340, 153-156 11 Beech, D. J., Bernheim, L., Mathie, A. and Hille, B. (1991) Proc. Natl Acad. 5ci. USA 88, 652-656 12 81eakman, D., Colmers, W. F., Fournier, A. and Miller, R. J. (1991) Br. J. Pharmacol. 103, 1781-1789
TINS, Vo/. 17, No. 9, 1994
13 Wiley, J. W., Gross, R. A. and Macdonald, R. L. (1993) J. Neurophysiol. 70, 324-330 14 Hua, X-Y. et al. (1991) J. Pharmacol. Exp. Ther. 258, 243 -248 15 Plummer, M. R., Rittenhouse, A., Kanevsky, M. and Hess, P. (1991) J. Neurosci. 1 I, 2239-2248 16 Foucart, S., Bleakman, D., Bindokas, V. P. and Miller, R. J. (1993) J. Pharmacol. Exp. Ther. 265, 903-909 17 Balasubramaniam, A., Sheriff, S., Rigel, D. F. and Fischer, J. E. (1990) Peptides 1 I, 545-550 18 Grundemar, L., Wahlestedt, C. and Reis, D. J. (1991) J. Pharmacol. Exp. Ther. 258, 633-638 19 Schofield, G. G. and Ikeda, S. R. (1988) Eur. J. Pharmacol. 151,131-134 20 Zidichouski, J. A., Chen, H. and Smith, P. A. (1990) Neurosci. Left. 117, 123-128 21 liles, P. and Regenold, J. T. (1990) Nature 344, 62-63 22 Toth, P. T., Bindokas, V., Bleakman, D., Colmers, W. F. and Miller R. J. (1993) Nature 364, 635-639 23 Betz, W. and Bewick, G. S. (1992) Science 255, 200-203 24 McEnerny, M. W., Snowman, A. M. and Snyder, S. H. (1994) J. Biol. Chem. 269, 5 - 8 25 Xiong, Z., Bolzon, B. and Cheung, D. W. (1993) Pfl6gers Arch. 423,504-510 26 Horn, J. P. (1992) Can. J. Physiol. Pharmacol. (Suppl.) 70, 19-26 27 Hirning, L. D., Fox, A. P. and Miller, R. J. (1990) Brain Res. 532, 120-130 28 Kohler, C., Eriksson, L., Davies, S. and Chan-Palay, V. (1987) Neurosci. LetL 78, 1 - 6 29 Milner, T. A. and Veznedaroglu, E. (1992) Hippocampus 2, 107-126 30 Colmers, W. F., Lukowiak, K. D. and Pittman, Q. J. (1987) J. PhysioL 383,285-299 31 Haas, H. L., Hermann, A., Greene, R. W. and Chan-Palay, V. (1987) J. Comp. Neurol. 257, 208-215 32 Klapstein, G. J. and Colmers, W. F. (1993) Hippocampus 3, 103-112 33 Colmers, W. F., Lukowiak, K. D. and Pittman, Q. J. (1988) J. Neurosci. 8, 3827-3837 34 Bleakman, D., Harrison, N. L., Colmers, W. F. and Miller, R. J.
(1992) Br. J. PharmacoL 107, 334-340 35 McQuiston, A. R. and Colmers, W. F. (1992) Neurosci. Lett. 138, 261-264 36 Monnet, F. P., Debonnel, G. and de Montigny, C. (1990) Eur. J. Pharmacol. 182, 207-208 37 Monnet, F. P., Fournier, A., Debonnel, G. and De Montigny, C. (1992)J. PharmacoL Exp. Ther. 263, 1212-1218 38 Colmers, W. F., Klapstein, G. J., Fournier, A., St-Pierr e, S. and Treherne, K. A. (1991) Br, J. Pharmacol. 102, 41-44 39 Coan, E. J. and Collingridge, G. L. (1985) Neurosci. Lett. 53, 21-26 40 Klapstein, G. J. and Colmers, W. F. (1993) Soc. Neurosci. Abstr. 19, 1869 41 Stasheff, S. A., Bragdon, A. and Wilson, W. A. (1985) Brain Res. 344, 296-302 42 Rizzi, M., Monno, A., Samanin, R., Sperk, G. and Vezzani, A. (1993) Eur. J. Neurosci. 5, 1534-1538 43 Wahlestedt, C. et aL (1990) Brain Res. 507, 65-68 44 Goodman, J. H. and Sloviter, R. S. (1993) Brain Res. 616', 263-272 45 Roman, F. J. etaL (1989) Eur. J. PharmacoL 174, 301-302 46 Tam, S, M. and Mitchell, K. N. (1991) Eur. J. Pharmacol. 193, 121-122 47 Quirion, R., Mount, H., Chaudieu, I., Dumont, Y. and Boksa, P. (1991)in NMDA Receptor Related Agents: Biochemistry, Pharmacology and Behavior (Kamayama, T., Nabeshima, T. and Domino, E. F., eds), pp. 203-210, NPP 48 Bouchard, P., Dumont, Y., Fournier, A., St-Pierre, S. and Quirion, R. (1993) J. Neurosci. 13, 3926-3931 49 Williams, J. T., Colmers, W. F. and Pan, Z. Z. (1988) J. Neurosci. 8, 3499-3506 50 Kombian, S. B. and Colmers, W. F. (1992) J. Neurosci. 12, 1086-1093 51 Livingstone, M. S. and Hubel, D. H. (1981) Nature 291, 554-561 52 Madison, D. V. and Nicoll, R. A. (1982) Nature 299, 636-638 53 Illes, P., Finta, E. and Nieber, K. (1993) Naunyn-Schmied. Arch. PharmacoL 348, 546-548 54 Williams, J. T., Henderson, G. and North, R. A. (1985) Neuroscience 14, 95-101
Acknowledgements We would like to thank our co-authors on our individualand collaborativestudies on neuropeptide Y, mostnotably R.J.Mi//er, V. P. Bindokas, P. Toth, N. Harrison, £ B. Kombian, A. R.McQuiston and G.3. Klapstein,many of whomgenerously supp/iedunpublished data orfigures. Researchin W F.C's laboratory was supportedby the MedicalResearch Councilof Canada. W.E C isa Seholarof the AIberta Heritage Foundation for MedicalResearch.
Columnarorganizationin the midbrainperiaqueductalgray: modulesfor emotionalexpression? R i c h a r d B a n d l e r a n d M i c h a e l T. S h i p l e y
Independent discoveries in several laboratories suggest that the midbrain periaqueductal gray (PAG), the celldense region surrounding the midbrain aqueduct, contains a previously unsuspected degree of anatomical and functional organization. This organization takes the form of longitudinal columns of afferent inputs, output neurons and intrinsic interneurons. Recent evidence suggests: that the important functions that are classically associated with the PAG - defensive reactions, analgesia and autonomic regulation - are integrated by overlapping longitudinal columns of neurons; and that different classes of threatening or nociceptive stimuli trigger distinct co-ordinated patterns of skeletal, autonomic and antinociceptive adjustments by selectively targeting specific PAG columnar circuits. These findings call for a fundamental revision in our concept of the organization of the PAG, and a recognition of the special rolesplayed by different longitudinal PAG columns in co-ordinating distinct strategies for coping with different types of stress, threat and pain.
havioral responses to threatening or stressful stimuli. Traditionally, there was little interchange between these research areas. However, a meeting held during 1990 brought together scientists working on the PAG from a variety of perspectives. A number of important points of consensus emerged from this meeting and a subsequent symposium at the Annual Meeting of the Society for Neuroscience in 1992. First, that the nocicepfive inhibition and behavioral responses elicited from the PAG were best conceptualized as components of co-ordinated reactions important for survival. Second, that functional specificity within the PAG was represented in the form of distinct, longitudinal neuronal columns extending, for varying distances, along the rostrocaudal axis of the PAG 1-3. In this article, the evidence for longitudinal columnar organization within the PAG is reviewed, and the mechanisms by which PAG columns coordinate distinct patterns of behavioral and physiological reactions critical for survival are considered.
RichardBandleris at the Dept of Anatomy and Histology, Universityof Sydney, Sydney, New South Wales2006, Australia, and Michael T. Shipleyis at the Oept of Anatomy, University of Maryland, 655 WestBaltimore Street, Baltimore,MD 21201-1559, USA.
During the past two decades, two largely independent Historical perspective themes have dominated research on the PAG, namely Early studies reported that defensive or 'aversive' inhibition of nociception, and the integration of be- reactions, hypertension and, later, antinociception, TINS, Vol. 17, No. 9, 1994
© 1994, Elsevier ScienceLtd
379