Central neural production of periodic respiratory movements

Trends in NeuroSciences August 1982

during everyday behavior. A major unanswered question is the nature of the control and co-ordination of these pumps by the CNS. Neurophysiological studies 2,1z,v~ have provided detailed inforOver 80 scientists gathered at Lake Bluff, Illinois, U.S.A *., on 14-18 April 1982 to mation regarding the biophysical and discuss the most recent developments covering the neural generation o f periodic synaptic properties of single phrenic and respiratory movements in mammals 5. The past 20 years have seen a marked intercostal motoneurons yet there is still a expansion o f knowledge in this field. Application o f a variety o f techniques including lack of understanding of how these results intracellular recording to study post-synaptic properties of cells; staining o f single cells relate to the overall functioning of the to investigate anatomical properties; cross-correlation analysis, spike-triggered thoraco-abdominal pump system. averaging, and microstimulation to investigate connectivity are now all used routinely Respiratory motor units receive three to investigate questions in this field. The use o f these approaches was quite evident in major neural drives: respiratory, behavioral many o f the presentations at the meeting. and spinal reflex (see Goldman, Ref. 5, p. 11 ). These drives in fact operate on differWhile a considerable body of knowledge apposed surfaces of the lung and a radial ent respiratory muscles in varying degrees. has been built-up concerning the properties pump acting on the rib-cage apposed sur- For example in the normocapnic state of the individual elements (CNS cell types face (see Mead, Ref. 5, p. 5). The two (arterial Pco2 approx. 40 Torr) the drive is and cellular aggregations), which have a pumps interact, so that inspiratory action of primarily inspiratory and onto the diarole in the production of periodic respiratone pump causes the other pump to react phragm and parasternal intercostal muscles, ory movements, a serious deficit has been with an expiratory action. Central neural with small increases in arterial Pco., external the general failure of neurophysiological mechanisms must control for these effects. intercostal (inspiratory) activity becomes workers in the field to consider their work The CNS, in addition to producing periodic evident and at higher levels of CO2 expiratin terms ofthemodus operandi of the syscontraction of the respiratory muscles must ory activity of internal intercostal and tem, i.e. the production of homeostatically also constantly make adjustments as the abdominal muscles is seen. These results regulated movement of the respiratory body is bent, twisted, flexed and extended Continued on page 258 musculature. To redress this point the initial part of the meeting consisted of a series of plenary lectures on respiratory movements and respiratory-related sensory reception. Two important issues were defined that need to be incorporated in neurophysiologStudies on intact flying locusts are revealing the important part played by phasic ical studies. sensory inputs in regulating the flight motor output in insects and require a radical The mechanical action of the mammalian revision o f the concept o f the flight pattern generator. respiratory pump is complex and comprises an axial pump working on the diaphragm Insect flight has become a classic, textbook intact locusts 15,'s. No wing-beat related, example of a centrally generated rhythmic phasic influences were found and the idea * A limitednumberof copies of the Proceedingsof the motor pattern. Just over 20 years ago, Wilhas become established that sensory inputs meeting, including 29 plenary and research lecture son is first showed that the rhythmic alternaare of little importance in the generation of presentationsas well as abstractsof 44 free communi- tion of firing in wing elevator and depressor the flight motor pattern. cations, are available for purchase from: J.L. motor neurones in locusts was still proIn the last 10 years, however, attention Feldman, Ph.D., Department of Physiology, Northhas turned more and more to the role of the western University, 303 East Chicago Avenue, duced correctly in the absence of sensory input from the wings. This was the first sensory inputs in flight. A recent internaChicago. IL 60611, U.S.A. that such tional Symposium* has highlighted the Please enclose a check lbr U.S. $25.00 made pay- conclusive demonstration able to Departmentof Physiology, Northwestern Uni- rhythmic motor patterns could be produced quiet revolution that these studies are causversity, l'hese volumes will be forwarded via surface by a central pattern generator (CPG) within * The Biophysics and Physiology of Insect Flight, held mail. the CNS, rather than by a chain of sensory in Saarbrficken, F.R.G.. on 3-5 March, 1982. The This meeting was supported by NIH grant R13 reflexes. The numerous sensory receptors meetingwas organizedby W. Nachtigalland B. MOhl, HL/TW 27961 and a grant from The Institute for and financed by the Deutsche ForschungsgemcinTobacco Research. Additionalfunds were providedby of the wings and wing hinges appeared to the Department of Anesthesia (Northwestern Univer- have only a tonic excitatory effect, raising schaft. The proceedingswill be publishedas a BIONA Report (Stuttgart)later this year. sity,), Schering Corp. and Smith, Kline, and French the frequency of the CI:'G from its basal rate Laboratories. ( ~mtinued o n p a g e 258 in deafferented preparations to that found in

Central neural production of periodic respiratory movements

The role of sensory inputs in insect flight motor pattern generation

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258 airstream, small rune shifts m the firing ,,t the flight motor neurones relati',c to one another are observed:. These shitt.s re~uh from feedback of phasic intbnnation abota the wing beat to the flight motor neurones. first demonstrated by Wendler '~, who examined the motor output in intact flying locusts when one wing was forcibly moved at a different frequency Io the others. Within one wing beat cycle, the motor neurones oI all lour wings became entrained to the frequency and phase of the imposed signal. Both G. Wendler and B. M6hl have extended this approach by determining the excitatory and inhibitory inputs from the various wing and wing hinge sense organs to the motor ncurones in flying locusts. An electric shock to a sense organ at an appropriate time in the wingbeat cycle results in a time shift of the next muscle potential; excitatory inputs cause firing to come earlier, inhibitory ones delay it. The size of the time shift reflects the magnitude of the potentials. This technique has the great advantage over intracellular recording because it detects those potentials that are of importance in flight and allows the examination of the interaction of inputs from several wings, both of which are dilZ flcult in fixed, dissected preparations. It does however rely on being able to stimulate a single population of receptors. But insect wings do not simply move up and down. Rather, they describe a complex figure-of-eight course, which involves a forward tilt, or pronation, during the downstroke and a backward tilt, supination, on the upstroke ~. The detailed studies of wing kinematics made by W. Zarnack. together with H.-K. Pfau's excellent analysis of the functional morphology of the wing articulation, emphasized how essential it is to know how the periphery works in order to understand the role of the nervous system. They have shown that pronation is caused by two of the downstroke muscles, which were previously thought to be antagonists, acting as synergists. Supination is a passive function of the wing hinge but is controlled by tension on the .joint provided by the previously ignored wing closer muscle. Pfau has also defined exactly the wing-beat parameters which stimulate the wing hinge receptors, showing that, rather than separate signals for upstroke and for pronation, there is an integrated output lYom the sense organ representing the total path of the wing at the top of its stroke. More intriguing even than the phasic feedback from the wing sense organs is J. Bacon and B. M6hl's discovery that the Phasic feedback and wing.beat wind-sensitive head hairs receive phasic regulation The flight motor pattern is stereotyped stimulation from the beating wing,',~, which only in a tethered insect flying straight for- cause rhythmic changes in the airflow ward When flying locusts are turned in an t ontinued ~m o ~ e 259

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do not imply that central expiratory activity is not present in normocapnia since experiments have shown that the presence of phasic inhibition of inspiratory motoneurons during expiration may be sub-threshold for excitation of expiratory alpha motoneurons2,~4. Thus, another important issue that needs further investigation is the mechanisms involved in integration of inputs to respiratory motoneurons. The long-standing question of the location and properties of neural structures responsible for the production of respiratory rhythm (the respiratory oscillator or oscillators), while receiving a good deal of attention at the meeting, still remains unanswered. in the medulla of cat there are two dense bilateral aggregations of respiratory neurons (one the DRG - dorsal respiratory group, located in the region of the ventrolateral solitary nucleus; and the second the VRG - ventral respiratory group, associated with nucleus retroambigualis and nucleus ambiguus). These sites have been thought to be the location of the respiratory pattern generator (see for example Ref. 16). Speck and Eeldman (see Ref. 5, p. 67) reported that extensive bilateral destruction of both DRG and VRG in anesthetized cats produced only small changes in respiratory rhythm with reduction in phrenic nerve amplitude. They concluded from these results that the respiratory oscillator is: ( 1) diffusely distributed throughout the DRG-VRG; (2) located in the DRG or VRG, or both, with redundant oscillators at alternative sites; or (3) lies outside the DRG and VRG ~5. Although considerable attention has been placed by workers in this field on both the DRG and VRG because concentrated respiratory-related neural activity can be easily recorded at these sites, it seems clear from their results that alternative locations, mechanisms, or both, need to be investigated in order to unde~tand respiratory rhythm generation. tt is quite possible that the principles underlying the mammalian respiratory pattern generator are similar to the cellular and network properties of central pattern generators in invertebrates (see Feldman, Ref. 5, p. 49). An intriguing possibility is raised by the crustacean respiratory oscillator which contains non-spiking interneurons having oscillating membrane potentials; these interneurons determine the output period of the motoneurons8. Tonic shifts in membrane potential of these cells produce concommitant adjustments in both intemeuron and ventilation rhytlml. It will be very difficult to determine if such a system operates within the mammalian CNS as

lng in our understanding of the neural control of insect flight, which has come about chiefly through investigations in whole flying insects rather than in dissected preparations. No fewer than 12 out of 23 contributions dealt with sensory inputs to the flight system, several of which reported phasic, wing-beat related inputs with a direct influence on the flight motor output 214. The aecanmlated evidence shows that such phasic sensory inputs play a crucial role in regulating the output of the flight generator and are essential for stable flight in freeflying insects. The pendulum has swung so rapidly that more is known now about the role of the sensory inputs than about the organization of the CPG. which has proved very difficult to investigate. Most of the detailed studies on the neural control of flight have been made on locusts. After comparison with work on a range of other species described at the meeting, it was generally agreed that the locust provides a good model for sustained fliers. Even so, there is always a danger in extrapolating from one species to all, as each has its own specializations. For example, in the cockchafer, where flights are short and rapid, peripheral inputs appear to play very little part, whereas flies, which make complex aerial manoeuvres, are very dependent on sensory feedback. Here 1 will report on the new ideas about the relationship between sensory inputs and the CPG that are emerging from the studies on locusts. Sensory inputs to the flight motor come not only from receptors of the wings and wing hinges t. ~2.~6but also from those on the head ~°."~ and tail", and perhaps from the whole body surface. It has become clear that these receptors have three different functions in flight. Firstly, they maintain flight through the well-documented tonic excitatory input, which keeps the frequency of the CPG well above its intrinsic rate '~''5' ,,;. Secondly, they regulate stability by adjusting the wing beat and interwing co-ordination through phasic, wing-beat related inputs~'~4. Lastly, they control course correction and steering°'°. These functional categories are not mutually exclusive because many of the receptors are involved in more than one of them and the effects on the motor neurones may all be achieved through the same neuronal circuitry. However, the categories are useful because different experimental strategies are required for studying each of them.

( o~ltinued on page 259

259

T I N S - A u g u s t 1982 Central neural cont, from page 258

Semory inputs cont. from page 258

the basis for respiratory rhythm around the head (Horsmann, Heinzel and generatio#. Wendler, unpublished observations). PreThe importance of tonic chemoreceptor viously, the head receptors had been inputs in control of respiratory motor output thought to provide only a tonic excitatory was emphasized by T. A. Sears (see Ref. input to the flight motor 13. That the phasic 5, p. 35). Expiratory intercostal motor units information feeds directly back to the flight are tonically excited by graded increases in motor neurones has been shown by two alveolar CO2 in the absence of respiratory ingenious experiments. The wing beat can rhythm (rhythm being abolished by be coupled to imposed rhythmic disturhypocapnia - low CO2)L Sears and col- bances in the airstream (Horsmann et al., leagues proposed that respiratory rhythm unpublished observations), and single elecseen in expiratory motor units arose from tric shocks to the hair fields are sufficient to periodic inhibition of the CO2-dependent alter the phase relationships between the tonic excitation of expiratory bulbospinal elevator and depressor motor neurones in neurons. Recently, Sears, Berger and the next wing beat cycle (Bacon and M6hl, Phillipson have extended this concept by unpublished observations). Phasic input investigating the effects of hypoxia on in- from the head hairs thus reinforces that spiratory and expiratory motoneuron dis- from the wings and wing hinges; the two charge. Hypoxia, through peripheral subsystems are mutually reinforcing and chemoreceptor activation, specifically interact to ensure regular and stable wing excites inspiratory motoneurons even in the beating. absence of rhythm. This excitation pro- Course correction duces a reciprocal tonic inhibition of Stability in free flight also requires conexpiratory motoneurons. The results tinual correction of deviations in the roll, obtained from these two studies led them to pitch and yaw planes. As insects have no conclude that, in the absence of rhythm (for specialized gravity sensors, these deviaexample, in hypocapnic apnea), respiratory tions are detected entirely as asymmetries in chemical stimuli are fully expressed as the visual environment and in the pattern of tonic activation of inspiratory and expirat- air flow on the head. The wind-sensitive ory motoneurons. Furthermore, they pro- head hairs respond to yaw and pitch moveposed that respiratory rhythm arises from ments, while roll and pitch stimulate the phasic inhibition of these tonic activities. ocelli, the small, simple eyes which detect This is in contrast to other models of the position of the horizon. Both sense respiratory pattern generation, which pro- organs are connected to the motor circuits pose phasic excitation as a basis for pattern by fast, direct pathways involving large, generation~,4. identifiable descending interneurones Richter and colleagues have studied, via (DINs) 3.~°. In the locust, a serendipitous intracellular recordings, spontaneous anatomical quirk makes one pair of windneuronal activity as a means of uncovering sensitive DINs, the tritocerebral commisthe interactions between neurons. Their sure giant neurones (TCG), easily accessstudies have revealed inhibitory interac- ible for single unit recording and stimulations between inspiratory and expiratory tion in flying animalsL Each TCG receives medullary neurons ~z. They reported (see inputs from hairs on one side of the head Ref. 5, p. 56) that many medullary only and makes excitatory connections with inspiratory cells receive short-lasting, both elevator and depressor motor neurones chloride-sensitive, IPSPs late in inspira- in the opposite half of the thorax H. In tion. This input may constitute part of the straight flight it fires rhythmically in phase with the wing beat 2 but the firing pattern inspiratory terminating 'offswitch' alters when the locust pitches or yaws mechanism:'.~. The classic concept that the respiratory (M6hl and Bacon, unpublished observacycle consists of two phases, inspiration tions). The left and right neurones are and expiration, was challenged by Richter antagonistic in yaw, and J. Bacon described (see Ref. 5, p. 56). He contended that there elegant experiments in which yaw was is a phase inbetween inspiration and expira- simulated by electrical stimulation of one tion, termed the post-inspiratory phase, that TCG in a locust flying straight forward. is very important. This period constitutes a This caused depressor motor neurones on time when irreversible inhibition of inspira- the opposite side to fire earlier, resulting in tion can occur with irreversible blockade of a stronger wing beat on that side, to counreactivation of inspiration. He even sug- teract the apparent turn. The two TCGs thus gested that this period may be more impor- function as comparators in the feedback tant than expiration for rhythm generation, circuit: wing beat since, during this period, there is a general- flight motor - ' I ~head hairs ized inhibition of all medullary respiratory neurones.qL..._...._~TCGs ~ l ~ Continued on page 260

in which the optimal state is an identical pattern of fwing in the two TCGs. Presumably, the ocellar interneurones act in similar antagonistic pairs in roll. How do these sensory inputs influence the f'wing of the motor neurones? C. H. F. Rowell offered us some insights with his description of the inputs from the ocellar DINs to the flight motor neurones ~°. Each DIN makes three sets of synapses, one directly with the motor neurones, and the others with two classes of spiking local intemeurones, one of which has subthreshold excitatory, the other inhibitory, inputs to the motor neurones. The network is organized to excite one set of motor neurones while inhibiting its antagonists, so producing course correcting wing movements in response to particular patterns of ocellar stimulation. Most interesting was Rowell's explanation for the duplication of inputs to the motor neurones. The direct input, though shorter latency than the indirect, is a small labile epsp, while the indirect epsp is stable and powerful. The indirect input is however gated as the local interneurones fire only in response to ocellar stimulation when the flight motor is active, so that the input from the ocelli is effective only when it is behaviourally relevant. The direct input, which is active whenever the locust's position with respect to the horizon changes, whether it is flying or not, is insufficient to affect the motor neurone firing on its own but acts as a primer for the longer latency potentials that may follow. Even raising the membrane potential by less than 2 mV at a critical moment boosts a subsequent potential so that the motor neurone approaches threshold more rapidly. We must assume that it is the summing of many such subtle effects from a large number of input channels which controls the timing and strength of the motor neurones' firing in the flying insect. The relationship between sensory inputs and the C P G

Still very little is known about the generation of the intrinsic flight motor pattern. Wilson ~,1~ postulated that there are multiple pacemakers and suggested that they could be the motor neurones. Another much favoured model proposes interneuronal oscillators which provide the motor neurones with a ready-made patterned output, Recent anatomicaP 12 and physiologicals,7,11 evidence indicated that the final integration of inputs is in the motor neurones and not in a pre-motor driver network. Coupling between motor neurones has been demonstrated4, hut it is open to doubt whether this is through an inter(qmtintled on Falge 2~0

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neurons (except the post-inspiratory neurons which themselves may be responsible for this phase). Controversy has surrounded the source of the expiratory phase inhibition of medullary inspiratory neurons 12, as well as spinal inspiratory motoneurons 2.14. The major difficulty stemmed from Merrill's conclusion, based upon his antidromic mapping studies, that the caudal population of medullary expiratory neurons (nucleus retroambigualis) did not appear to have medullary axon collateralsl°. Therefore, it would be unlikely that they would be the source of the inhibition seen in medullary inspiratory neurons. In the rostral medulla near the retrofacial nucleus is another site of medullary expiratory activity (called by some the B6tzinger group)L Lipski and MerrilP have demonstrated that these rostral expiratory neurons do project to the DRG. At the meeting, Merrill, Lipski and Kubin presented evidence from intracellular spike triggered averaging studies showing that these rostral expiratory neurons produce IPSPs in DRG inspiratory cells. Merrill also described more recent results showing that these expiratory neurons also have direct inhibitory projections onto phrenic motoneurons. This latter result is of general neurophysiological interest because Semo~yinputscont.from~ 259 neuronal delay circuit4 or proprioceptive feedback from wing hinge sense organs*. Local intemeurones, which spike rhythmically in phase with either elevator or depressor motor neurones, have now been discovered but their roles are still not clear~. They are only active when the flight motor neurones are firing and most excite or inhibit particular motor neurones. An exciting finding is a type involved in resetting the flight rhythm, which slows down when the intemeurone is depolarized. So far, there is too little information to allow us to say for certain that these interneurones are part of the CPG, particularly as the experiments were done in almost totally deafferented preparations ~, which makes it difficult to evaluate the roles of the interneurones correctly. For example, the resetting type could normally mediate the rapid phase shifts produced by stimulating the wing or head sense organs 2' 1,, In order to identify the CPG it is necessary to have a search image, a clear idea of what we are looking for. In the new picture of the flight pattern generator, the importance of the phasic sensory inputs relegates the CPG to a much more minor role than it had in earlier models. It is now perhaps best considered as only one of a number of oscillators all converging on the huge arboriza-

the rostral expiratory neuron to phrenic motoneuron interconnection constitutes a long direct inhibitory pathway; this type of pathway is relatively rare within the CNS (see also Ref. t l ) . Thus, this rostral aggregation or expiratory neurons may constitute the long sought after, widely divergent, inspiratory inhibitory population. Several themes ,seemed predominant at the meeting. (1) The simple concept of a respiratory center is no longer tenable, New concepts regarding the basis for respiratory rhythm generation are probably necessary. (2) Interactions between respiratory neurons are complex; they involve an abundance of excitatory and inhibitory connections, only a few of which are known. (3) Greater efforts will be necessary to correlate the results generated from neurophysiological studies to the behaviorly relevant production of respiratory movements and ultimately to the overall goal of this system - the maintenance of blood-gas and probably brain acid-base homeostasis.

Reaaing Ust

R~)~,B II~USJlhot'.()l~et~tct. ; i~', llt~ 4 Euler, (" vo. tl980) I'rend~ "41-ur,,.~l i 27%277 5 Fcldman.J. 1_ and Berger. A. J ( 1u~2H)ro~eed ings of lnwrnational Symposium: tPnttal Neural Producoon o/ Perit)di~ Respiratory Movernent~, p 2hi. Chicago Feldman, J L and Cleland. C L. i 1982) ¢ elluhtr Pacemakers (Carpenter. D. O., ed / Vol. 2. pp. 101-119, Wiley. New York 7 Lipski. J. and Merrill. t.. G. (19801 Brain Rc~. 197.521-524 8 Mendelson. M (1971)Se/ence 171, 1171~1173 9 Menill, E. G. (1970)Brain Res. 24. 11-28 10 Merrill. E. G 11974) Essay's On the Nervous System (Beilairs. R. and Gray. E G. eds). pp. 45:L.486. ClarendonPress. Oxford I I Rapo[x-,rt. S.. Susswein, A. Uchino. Y. and Wilson. V. J (1977)J. Physiol. (London) 272. 367-382 12 Richter, D. W., Camerer, H.. Meesmann,M. and R6hrig, N. (1979) Pfli~gersA rch, 380.24.5--257 13 Sears, T. A. 11964),/. Physiol. ¢London) 175, 386-.4O3 14 Sears. T. A (~964)J. PhysioL (L¢md.n) t75. 404-424 15 Speck. D. F. and Fcldman. J. L. d N, urosci. (in press) 16 Wyman. R. J. 11'977)Annu. Rev. Physiol. 3% 417-448 ,\ J. BERGER* J I, FELDMAN*

1 Bainton, C.R.. Kirkwood, P. A. and Sears, T A 11978) J. Physiol. (London) 280, 249-272 2 Berger. A. J. (1979)J. NeurophysioL 42, 76--90 3 Bradley. G. W.. Ealer. ('. yon. Marttila, 1. and

*Department o] Physiology and Biophysics, School of Medicine SJ-40, Universio' of Washington, Seattle. WA 98195, U.S.A. t Department of Physiology, Northwestern University, ~03F Chicago Ave, Chicago, IL 6061 t. U.S..t.

(ions of the flight motor neuronestL The CPG provides a basic common input to the motor neurones, which summates with the phasic sensory inputs to produce a stable yet flexible motor output, Possibly there is no discrete entity that can be labelled the CPG: the basic rhythm may be an emergent property of a network of interneurones and motor neurones which become coupled together only when the inputs which trigger flight are present. This possibility reinforces the feeling that emerged very strongly from the Symposium, that it is essential to consider the flying insect as a whole in order to understand the functions of the various parts. The recent conceptual advances are due not so much to more sophisticated techniques as to the realization that the nervous system must be studied in the context of the flying insect. An early British aeroplane was once described as 100,000 nuts and bolts flying in close formation. Perhaps we should think of a flying locust as a large number of components, mechanical, sensory and central neurones, all oscillating in resonance at flight frequency. In the last analysis, the whole flying locust is the flight motor pattern generator,

NeuroL 172. 409-430 2 Bacon,J. and M6hl, B. (1979)Nature (London) 278,638~40 3 Bacon. J. and Tyrer. M (197;g) I, (ornlL Physiol. 126, 317-325 4 Burrows, M. 11973)J. ('omp. Physiol. 83, 135-164 5 Burrows. M. (1975)J. Exp, Biol. 62, 189-219 6 Fraser, P. J. (19771 Nature (London) 268, 523-524 7 Kien, J. and Airman, J. S. (1979)J. Comp. Physiol. 133, 2'-)9-310 8 M6hl, B. and Nachtigull, W. (1978) J. Comp. PhysioL t28, 57--65 '9 Robertson, R M. and Pearson, K. G. J, Comp. Physiol. (in press) 10 Rowe/l,C. H. F. and "Pearson,K. G.J. Exp. Biol. (in press) 11 Tyrer, N. M. (1981) Adv. Physiol. SeL (SId~a~ki, J., ed.), Vol. 23, pp. 557-571, AkademiaiKiado, Budapest 12 Tyrer, N, M. and Allman, J. S. (1974)J. Ciwnp. Neurol. •57, t17-138 13 Weis-Fogh,T. (1956) Phi/. Trans. R, Soc. London, Set. B 239, 553-584 14 Wendler, G. fl974)J, Comp_ Physiol, 88, 173-21~ 15 Wilson, D. M. (1961)J. Exp. Biol. 38,471-490 16 Wilson, D. M, 11968) Adv. insect Physiol. 5,

1 Altman, J S. and Tyrer, N. M. (1977)J. Comp.

189-338

17 Zamack, W. and M61,,.I, B. 11977) J. Corap. PhysioL 118, 215-233 JENNIFER ALTMAN Freelance Research Consultant in Neurobiology, 7, Ho[ebouom, Todmorden, Lancashire O L t 4 8DD, U.K