Proc. Nati. Acad. Sci. USA Vol. 82, pp. 3897-3900, June 1985 Neurobiology

N-Acetylaspartylglutamate: Possible role as the neurotransmitter of the lateral olfactory tract (neuropeptide/glutamate/aspartate) J. M. H. FFRENCH-MULLEN*, K. KOLLERt, R. ZACZEKt, J. T. COYLEtt, N. HoRI*, AND D. 0. CARPENTER* *Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201; and tDivision of Child Psychiatry, Departments of Psychiatry and Behavioral Sciences, Pediatrics, Neuroscience and Pharmacology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Communicated by Seymour S. Kety, January 2, 1985

ABSTRACT N-Acetylaspartylglutamate, an endogenous from the horse (14) and subsequently from other mammalian brain peptide that binds with high affinity to a subpopulation species (11). Both Ac-Asp and Ac-Asp-Glu are found only in of glutamate-binding sites in rat brain, is excitatory on rat nervous tissue (15-17). A related tripeptide, fB-hydroxy- piriform cortex pyramidal cells studied in a perfused brain butyrylaspartylaspartylglutamate, has been isolated from the slice. Both the monosynaptic excitation of the pyramidal cells spinal cord (18). While the functional roles of these peptides elicited by stimulation of the lateral olfactory tract and the are yet unknown, preliminary reports have indicated that response to N-acetylaspartylglutamate were blocked by DL-2- Ac-Asp-Glu excites cortical neurons and inhibits thalamic amino-4-phosphonobutyrate but not by other excitatory amino and cerebellar neurons in the rat (19), while f-hydroxybu- acid antagonists. Responses to glutamate and aspartate, previ- tyrylaspartylaspartylglutamate is excitatory on cat spinal ously considered to be candidates as the lateral olfactory tract neurons (18). transmitter, were unaffected by 2-amino-4-phosphonobu- Recently Ac-Asp-Glu was found to exhibit a high affinity tyrate. Three days after unilateral bulbectomy there was a for a subset ofbrain receptor sites labeled by L-[3H]glutamate significant decrease in concentrations of N-acetylas- and to produce potent convulsive actions when injected into partylglutamate as well as aspartate, N-acetylaspartate, and rat hippocampus (20). These observations suggest that Ac- y-aminobutyrate in the pyriform cortex of the side from which Asp-Glu could be a transmitter at a site where the receptors the olfactory bulb had been removed. These results are are similar to those for the excitatory amino acids, such as consistent with the possibility that N-acetylaspartylglutamate 'aspartate and glutamate. Consequently we have investigated is the endogenous transmitter of the lateral olfactory tract. the possible actions and pharmacology of Ac-Asp-Glu on rat piriform neurons. The major excitatory input to the olfactory piriform cortex is from fibers of the lateral olfactory tract (LOT) that project METHODS from the olfactory bulb to terminate on the pyramidal neurons. The neurotransmitter released by the LOT termi- Electrophysiology. Tangential piriform cortex slices (300 to nals has been believed to be either aspartate or glutamate. 400 um thick) from Harlan Sprague-Dawley albino rats Both amino acids are excitatory on the pyramidal neurons (150-200 g) were cut by hand, incubated for 1 hr in (1). Electrical stimulation of the LOT results in a calcium- Krebs-Ringer solution, and mounted pial side down for dependent release of aspartate in the rat (2) and glutamate in recording in a chamber, where the slice was totally sub- the guinea pig (3, 4). Removal of the olfactory bulb, which merged and perfused as previously described (8, 9). Bipolar causes a degeneration ofLOT fibers (5), significantly reduces stimulating electrodes were positioned on the LOT. In a few the cortical content ofaspartate and glutamate in both the rat studies the population field potential, reflecting the nearly (2, 5) and guinea pig (6, 7). Furthermore, bulbectomy de- synchronous excitation ofpyramidal neurons by LOT stimu- creases the evoked release of aspartate in the rat (2) and of lation, was examined, and agents that might alter the re- both aspartate and glutamate in the guinea pig (3, 4). sponse were applied by bath perfusion (9). Despite the evidence from these release studies, there is In most studies, single units were recorded extracellularly reason to question the identification of aspartate, glutamate, and identified as pyramidal neurons by demonstrating or both as the transmitter of the LOT fibers. Hori et al. (8, 9) monosynaptic excitation on LOT stimulation. In these stud- observed that DL-2-amino-4-phosphonobutyrate (APB), a ies, transmitter candidates were iontophoretically applied structural analog of glutamate, blocks the LOT-evoked with a constant current source, using one- to three-barrel response but not the responses to iontophoretically applied micropipettes positioned independently of the recording aspartate and glutamate. They concluded that the LOT electrode. The barrels contained Ac-Asp-Glu, Ac-Asp, transmitter is neither aspartate nor glutamate and suggested aspartate, glutamate, and N-methyl-DL-aspartate (Me-Asp) the possibility that the measured release of aspartate and at 10 mM and pH 5.5-7.0 and were applied in a time- glutamate could result from degradation of a peptide rich in controlled sequence (30 sec apart) with ejection currents excitatory amino acids. adjusted to produce responses that were submaximal but as Several compounds derived from aspartate or glutamate equal as possible. Ac-Asp (1 mM) and the excitatory amino in was acid antagonists APB, DL-2-amino-5-phosphonovalerate are present brain tissue. N-Acetylaspartate (Ac-Asp) (APV), glutamate diethyl ester, DL-2-amino-7-phosphono- identified in human brain (10) and subsequently in other and mammalian species (11) and is not a neuronal excitant (12, heptanoate, 'y-D-glutamylglycine, cis-2,3-piperidine 13). N-Acetylaspartylglutamate (Ac-Asp-Glu) was isolated Abbreviations: LOT, lateral olfactory tract; APB, DL-2-amino4 phosphonobutyrate; Ac-Asp, N-acetylaspartate; Ac-Asp-Glu, N- The publication costs of this article were defrayed in part by page charge acetylaspartylglutamate; Me-Asp, N-methyl-DL-aspartate; APV, DL- payment. This article must therefore be hereby marked "advertisement" 2-amino-5-phosphonovalerate; GABA, -aminobutyrate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

3897 Downloaded by guest on September 29, 2021 3898 Neurobiology: ffrench-Mullen et al. Proc. Natl. Acad. Sci. USA 82 (1985) dicarboxylate, each at 0.1 mM, were bath-perfused for up to Table 1. Effects of antagonists of excitatory amino acids on 20 min, followed by a wash period with control Krebs-Ringer responses to iontophoretically applied Ac-Asp-Glu, solution. Stable and consistent responses were obtained for glutamate, aspartate, and Me-Asp and to a minimum of 10 min; then spikes in five cycles of responses electrically stimulated LOT responses to each agonist were counted and averaged before an an- Depression by antagonist* tagonist was applied. Responses during antagonist perfusions and wash periods were similarly counted and averaged. Stimulation APB GDEE APV APH yDGG PDA Results were expressed as percentages of the control re- LOT 101/105 0/35 0/44 0/15 0/33 0/20 sponse. Ac-Asp-Glu 12/14t 0/5 0/5 0/5 0/5 0/3 Bulbectomy. Male Sprague-Dawley albino rats (150-200 g) Glutamate 0/42t 0/20 4/12 0/9 8/13 0/12 were unilaterally bulbectomized by aspiration of the left Aspartate 0/33t 0/15 9/9§ 4/4§ 2/5 0/5 olfactory bulb while the animals were under chloropenta- Me-Asp 10/44 0/15 11/11§ 4/4§ 3/3§ 0/8 The animals were sacrificed 3 barbital anesthesia (6). days GDEE, glutamate diethyl ester; APH, DL-2-amino-7-phosphono- after surgery, and the brains were removed. Both ipsilateral heptanoate; yDGG, y-D-glutamylglycine; PDA, cis-2-3-piperidine and contralateral piriform cortices were dissected. dicarboxylate. Ac-Asp-Glu, Ac-Asp, and Amino Acid Determinations. *Number of cells depressed by >50%o of the control response/ Dissected tissue was immediately sonicated in 10 vol of number of cells tested. Antagonists were present at 0.1 mM. ice-cold MeOH/H20 (9:1, vol/vol) and centrifuged to pellet tNo significant difference (x2) between Ac-Asp-Glu and LOT APB the precipitated protein. An aliquot of the supernatant was blockade. dried and applied to a Dowex AG 50 column. The lyophilized *p <0.001. eluate was analyzed for Ac-Asp-Glu and Ac-Asp by anion- §Total blockade of cells. exchange high-pressure liquid chromatography as described by Koller et al. (17). An additional aliquot of the deprotein- ated supernatant was allowed to react with o-phthalaldehyde Pharmacology of the Ac-Asp-Glu Response. Previous work in the presence of ethanethiol and analyzed for amino acids has demonstrated that the natural synaptic response of these by reversed-phase high-pressure liquid chromatography and neurons elicited by stimulation ofthe LOT is blocked by APB fluorometric detection (21). (0.1 mM) but not by other antagonists of excitatory amino acids (8, 9). The responses of a pyramidal neuron to Ac-Asp- RESULTS Glu, glutamate, and LOT stimulation are illustrated in Fig. 2. Stimulation of the LOT resulted in either one or two spikes Effects of Ac-Asp-Glu on Piriform Neurons. Ac-Asp-Glu, per stimulus, whereas iontophoretic application of the amino purified to homogeneity from rat brain, was excitatory on all acids and agonists elicited multispiked responses. In this cell 50 neurons examined. The response (Fig. 1) was of short APB (0.1 mM) perfusion for 8.5 min totally blocked both the latency, often starting before the iontophoretic pulse ended. Long iontophoretic ejection times (1-3 sec) with ejection Ac-Asp-Glu and LOT responses, while the glutamate-in- currents up to 100 nA often resulted in spontaneous discharge duced response was unaffected. After a 12-min wash with and loss of spike amplitude, indicative of an excitotoxic control Krebs-Ringer solution the LOT response returned to action. The time course of the response and the discharge control and the Ac-Asp-Glu-induced response to 87.5% ofthe pattern to Ac-Asp-Glu were similar to those for Me-Asp (Fig. control response. 1). A systematic iontophoretic study showed that Ac-Asp- The sensitivity and time course of action of APB (0.1 mM) Glu was more potent than Me-Asp (all of 7 cells), glutamate on the LOT and Ac-Asp-Glu responses were compared in 14 (all of 19 cells) (Fig. 1), and aspartate (all of 3 cells, not neurons (Table 1). In 12 neurons the average time to blockade shown). of the LOT response was 11.5 min, and the average time for Ac-Asp was found to be electrophysiologically inactive, recovery to control after washing was 12.1 min. At the time confirming previous reports (12, 13). Bath perfusion of of total LOT blockade in these 12 neurons the multiple spike Ac-Asp (1 mM) had no effect on the amplitude ofthe synaptic responses to Ac-Asp-Glu were depressed to an average of component of the field potential. In contrast, perfusion of 1 18.8% ofthe control response; in three cells the blockade was mM glutamate or aspartate will depress the field potential (9). total (Fig. 2). On washing, the Ac-Asp-Glu response had To further verify the lack of effect of Ac-Asp, aspartate and returned to 91.9% of control when the LOT response had Ac-Asp were iontophoretically applied to five cells from a recovered to control values. In one ofthe two remaining cells double-barreled electrode. These neurons were unresponsive both the LOT and Ac-Asp-Glu responses were unaffected; in to Ac-Asp but excited by aspartate. the other the LOT response was reduced to 45% of control,

A B C D Ac-Asp-Glu Me-Asp Glu Glu

100 A.V 1 sec FIG. 1. Responses to iontophoretically applied Ac-Asp-Glu, Me-Asp, and L-glutamate on a pyramidal neuron. lontophoretic ejection currents and durations were as follows: (A) Ac-Asp-Glu, 160 nA, 500 msec; (B) Me-Asp, 160 nA, 600 msec; (C) glutamate, 160 nA, 500 msec; (D) glutamate, 180 nA, 500 msec. Each drug barrel had a 5-nA retaining current and contained 0.01 M agonist in 100 mM sodium chloride. Each agonist was ejected in a controlled sequence at 30-sec intervals. Even with a pulse duration greater by 20%o, the Me-Asp response was less vigorous than that to Ac-Asp-Glu. At the same current and duration as Ac-Agp-Glu, glutamate elicited no response (160 nA; C) while with a higher ejection current (180 nA; D) glutamate elicited a brief high-frequency discharge. The deflections in C are the shock artifacts from iontophoresis, which are also present in all other traces. Downloaded by guest on September 29, 2021 Neurobiology: ffrench-Muffen et aL Proc. Natl. Acad. Sci. USA 82 (1985) 3899

Control APB Wash

Ac-Asp-Glu

Glu NI I

100 JAV

1 sec

LOT

100 .V 10 msec

FIG. 2. Reversible blockade of responses to iontophoretically applied Ac-Asp-Glu (100 nA) and stimulation of the LOT by APB (0.1 mM), with no effect on the response to L-glutamate (130 nA). lontophoretic current pulses were of 1 sec duration and each barrel had a 2.5-nA retaining current. After APB perfusion for 8.5 min the Ac-Asp-Glu and LOT responses were totally blocked, while the glutamate response was essentially unaffected. After a 12-min wash the Ac-Asp-Glu and LOT responses had returned to 87.5% and 100% of control responses, respectively. In the LOT trace an upward shock artifact precedes the extracellular spike. In the presence ofAPB only the LOT shock artifact and the Ac-Asp-Glu iontophoretic current artifact remain.

and the Ac-Asp-Glu response was virtually unaffected (90% than 50%o of control. yD-Glutamylglycine, an Me-Asp and of control). kainate antagonist (23), had no effect on the Ac-Asp-Gln and The glutamate and aspartate responses were unaffected by LOT responses but totally blocked the Me-Asp responses; APB in all neurons examined (Table 1). The Me-Asp re- the aspartate and glutamate responses were depressed in sponses showed a variable and partial depression by APB, some but not all neurons. Piperidine dicarboxylate, an an- which has been attributed to the D isomer (22). In 10 of 44 tagonist reported to block glutamate, aspartate, and Me-Asp neurons the Me-Asp responses were depressed to less than receptors (23), had no effect on any response. Aminophos- 50% of the control (average 46%) after 15 min of APB phonoheptanoate, an Me-Asp antagonist (23), had no effect perfusion. The responses in the remaining neurons were on the LOT, Ac-Asp-Glu, and glutamate responses but 50-100%o of control. totally blocked the aspartate and Me-Asp responses in all The sensitivity of the LOT, Ac-Asp-Glu, glutamate, neurons tested. Thus the LOT and Ac-Asp-Glu responses aspartate, and Me-Asp responses to other antagonists is also show similar pharmacologic sensitivities, which differ from shown in Table 1. Glutamate diethyl ester, an antagonist of those of the other agonists tested. the glutamate-preferring receptor (23), had no effect on any Ac-Asp-Glu Content in Piriform Cortex. Since Ac-Asp-Glu response. APV, reported to be the most selective and potent is excitatory and exhibits a pharmacologic profile similar to Me-Asp antagonist (23), had no effect on the Ac-Asp-Glu or that of the endogenous LOT transmitter, we conducted exper- LOT responses. However, APV totally blocked the Me-Asp iments to determine the concentration of Ac-Asp-Glu in piri- and aspartate responses in all neurons tested, and it de- form cortex. Ac-Asp-Glu, glutamate, aspartate, and t-amino- pressed the glutamate responses in 4 of 12 neurons to less butyrate (GABA) are present in piriform cortex (Table 2).

Table 2. Effect of unilateral bulbectomy on concentrations of Ac-Asp-Glu, Ac-Asp, aspartate, glutamate, and GABA in olfactory piriform cortex Concentration on day 3, nmol/mg of protein Rat group Ac-Asp-Glu Ac-Asp Aspartate GABA Glutamate Control 1.25 ± 0.02 57.82 ± 1.19 27.28 ± 0.97 19.50 ± 1.07 127.18 ± 5.80 Bulbectomized Ipsilaterally 0.98 ± 0.05 45.89 + 1.88 21.80 + 1.50 14.84 + 0.36 113.33 ± 4.55 Change from control -22%* -21%* -20%6* -24%* -11% Contralaterally 1.19 ± 0.06 54.15 ± 2.44 25.64 ± 1.01 14.84 + 0.79 120.05 + 3.34 Change from control -5% -6% -6% -24%* -6% Results are mean + SEM; n = 6. *P < 0.025 (nonpaired Student's t test). Downloaded by guest on September 29, 2021 3900 Neurobiology: ffrench-Mullen et al. Proc. Natl. Acad Sci. USA 82 (1985) Unilateral bulbectomy, which causes degeneration of LOT tions after bulbectomy, and the previously reported release of fibers, resulted in a significant decrease in Ac-Asp-Glu aspartate and glutamate upon stimulation of the LOT (2) are concentration in ipsilateral cortex 3 days after surgery. No all consistent with a transmitter function for Ac-Asp-Glu. significant decrease of Ac-Asp-Glu content was observed These substances should be both the building blocks for contralaterally. Aspartate and GABA also decreased signifi- synthesis and the products of degradation of Ac-Asp-Glu. cantly in the ipsilateral cortex; the decrease of glutamate was While aspartate and glutamate are released on LOT stimula- not statistically significant. Notably, only GABA decreased tion and are physiologically active on these neurons, the significantly contralaterally. pharmacologic differences suggest that neither is the trans- mitter. That the physiologically active substances aspartate and glutamate may be degradation products has not been DISCUSSION previously proposed. Such a possibility is, however, parallel Three types of excitatory amino acid receptors have been to that at cholinergic synapses, where the degradation prod- distinguished in a variety of systems, based principally on uct, choline, is a weak agonist at acetylcholine receptors (28). physiologic and pharmacologic criteria (23). These receptors These considerations emphasize the importance of using are activated specifically by the selective agonists Me-Asp, multiple criteria for identification of a transmitter at a quisqualate, and kainate, respectively, none of which is particular synapse, in that use of only release and binding endogenous. Aspartate and glutamate are mixed agonists, data may point to a degradation product rather than the capable of activating all three receptors but with differing endogenous transmitter. efficacies (23). It is assumed that aspartate is the transmitter Our results indicate that there are more than three types of that has physiologic actions at Me-Asp receptors and gluta- excitatory amino acid receptors and suggest that small mate the endogenous substance acting at quisqualate recep- aspartate- and glutamate-rich peptides may function as tors. Kainate is thought to act at a distinct receptor (23), endogenous transmitters at at least some receptors known to where no endogenous agonist has been definitively identified. bind glutamate or aspartate. Drugs such as APB may be Of a variety of antagonists that are relatively specific for useful tools for the study of these receptors. these receptor types, APV, glutamate diethyl ester, and 1. Constanti, A., Connor, J. D., Galvan, M. & Nistri, A. (1980) Brain y-D-glutamylglycine have proven most useful for distinguish- Res. 195, 403-420. ing Me-Asp, quisqualate, and kainate receptors, respectively 2. Collins, C. G. S. (1979) J. Physiol. (London) 291, 51-60. (23). 3. Bradford, H. F. & Richards, C. D. (1976) Brain Res. 105, 168-172. While neurons in the rat piriform cortex respond to 4. Yamamoto, C. & Matsui, S. (1976) J. Neurochem. 26, 487-491. and on 5. Godfrey, D. A., Ross, C. D., Carter, J. A., Lowry, 0. H. & glutamate aspartate (1, 8, 9) and aspartate is released Matchinsky, F. M. (1980) J. Histochem. Cytochem. 28, 1157-1169. stimulation of the LOT (2), the pharmacologic sensitivities of 6. Harvey, J. A., Scholfield, C. N., Graham, L. T. & Aprison, M. H. the responses to aspartate and the endogenous transmitter (1975) J. Neurochem. 24, 445-449. are different. The aspartate-preferring receptor antagonists, 7. Scholfield, C. N., Moroni, F., Corradetti, R. & Pepeu, G. (1983) J. APV and aminophosphonoheptanoate, while totally blocking Neurochem. 41, 135-138. both the aspartate and Me-Asp responses, had no effect on 8. Hori, N., Auker, C. R., Braitman, D. J. & Carpenter, D. 0. (1981) Cell. Mol. Neurobiol. 1, 115-120. the LOT-evoked responses. The lack of effect of glutamate 9. Hori, N., Auker, C. R., Braitman, D. J. & Carpenter, D. 0. (1982) diethyl ester and y-D-glutamylglycine on the LOT responses J. Neurophysiol. 48, 1289-1301. suggests that the endogenous receptors are not of the 10. Tallan, H. H., Moore, S. & Stein, W. H. (1956) J. Biol. Chem. 219, quisqualate or kainate type. In contrast, APB blocks the 257-264. endogenous transmitter response with no effect on the 11. Reichelt, K. L. & Fonnum, F. (1969) J. Neurochem. 16, 1409-1416. responses to aspartate or glutamate. These observations 12. Jacobson, K. B. (1959) J. Gen. Physiol. 43, 323-333. 13. Curtis, D. R. & Watkins, J. C. (1960) J. Neurochem. 6, 117-141. suggest that the LOT transmitter activates a distinct amino 14. Curatolo, A., D'Arcengelo, P., Lino, A. & Brancatti, A. (1965) J. acid-type receptor that is blocked by APB. Neurochem. 12, 339-342. APB was originally used in invertebrates as a glutamate 15. Nadler, J. V. & Cooper, J. R. (1972) J. Neurochem. 19, 313-319. receptor antagonist, on the basis of its structural similarity to 16. Miyake, M., Kakimoto, Y. & Sorimachi, M. (1981) J. Neurochem. glutamate (24). It is a poor inhibitor of excitatory amino acid 36, 804-810. in system (9, 25). 17. Koller, K. J., Zaczek, R. & Coyle, J. T. (1984) J. Neurochem. 43, responses the mammalian central nervous 1136-1142. Consistent with the present results and those of Hori et al. 18. Kanawaza, I., Sutoo, D. & Munekata, E. (1981) Proc. Jpn. Acad. (9), four classes of binding sites have been demonstrated in 57, 346-349. hippocampus. Three ofthese sites correspond to the Me-Asp, 19. Avoli, M., Barra, P., Brancatti, A., Cecchi, L. & Deodati, M. quisqualate, and kainate receptors, respectively (26); the (1976) Boll. Soc. Ital. Biol. Sper. 52, 1525-1530. fourth is distinguished by binding of APB (27). 20. Zaczek, R., Koller, K. J., Cotter, R., Heller, D. & Coyle, J. T. (1983) Proc. Natl. Acad. Sci. USA 80, 1116-1119. A transmitter function of Ac-Asp-Glu is suggested by the 21. Hill, D., Burnworth, L., Shea, W. & Pfeifer, R. (1982) J. Liq. observation that Ac-Asp-Glu is excitatory on all pyramidal Chromatogr. 5, 2369-2393. neurons in piriform cortex and the similarity in pharmaco- 22. Evans, R. H., Francis, A. A., Jones, A. W., Smith, D. A. S. & logic sensitivities ofthe Ac-Asp-Glu and LOT responses. The Watkins, J. C. (1982) Br. J. Pharmacol. 75, 65-76. quisqualate, Me-Asp, and kainate receptor antagonists had 23. Watkins, J. C. & Evans, R. H. (1981) Annu. Rev. Pharmacol. no effect on the Ac-Asp-Glu and LOT-evoked responses, Toxicol. 21, 165-204. 24. Usherwood, P. N. R. (1976) in Electrobiology of Nerve, Synapse while both were blocked by APB with a similar time course. and Muscle, eds. Reuben, J. P., Purpura, D. P., Bennett, M. U. L. The presence of Ac-Asp-Glu in piriform cortex and the & Kandel, E. R. (Raven, New York), pp. 81-92. decline in Ac-Asp-Glu concentrations after bulbectomy are 25. Evans, R. H., Francis, A. A., Hunt, K., Oakes, D. J. & Watkins, strong evidence that the endogenous transmitter at this J. C. (1979) Br. J. Pharmacol. 67, 591-603. synapse is indeed Ac-Asp-Glu, although there remains a 26. Monaghan, D. T., Holets, V. R. & Cotman, C. W. (1983) Nature that Ac-Asp-Glu is one of a class of small, (London) 306, 176-179. possibility 27. Whittemore, S. R., Mena, E. E., Monaghan, D. T. & Cotman, excitatory, amino acid-rich peptides that act as transmitters. C. W. (1983) Brain Res. 277, 99-107. The presence of considerable aspartate, Ac-Asp, and 28. del Castillo, J. & Katz, B. (1957) Proc. R. Soc. London Ser. B 146, glutamate in piriform cortex, the change in their concentra- 367-381. Downloaded by guest on September 29, 2021