ANALYTICAL
BIOCHEMISTRY
1%,69-76
(1991)
In lho Voltammetric Detection of Neuropeptides with Micro Carbon Fiber Biosensors: Possible Selective Detection of Somatostatin Francesco
Crespi’
Department of Physiology and Pharmacology, Nottingham University, Nottingham and Instituto di Ricer&e Farmacologiche M. Negri, via Eritrea 62, 20157 Milano,
Received
May
NG7 2UH, Italy
United
Kingdom,
30, 1990
The electrochemical activity of catecholand indoleamines, measured by differential pulse voltammetry (DPV) with specifically electrically pretreated carbon fiber microelectrodes, has been utilized to develop sensitive assays for amine neurotransmitters and metabolites. So far, four oxidation peaks have been recorded in viva between -200 and +500 mV and are well identified. We now report that by increasing the potential sweep range to +950 mV, a further peak, called Peak 5, was detected at +800 mV in vivo in the striatum of anesthetized rats. Neuropeptides containing tyrosine, tryptophan and/or cysteine appear to be electrochemically active between +600 and +900 mV in vitro in a buffered solution at pH 7.4. The present study investigates the chemical nature of Peak 5 and the possible contribution of electroactive neuropeptides to this in vivo voltammetric signal. Experiments performed in vitro and in vivo with amino acids, neuropeptides, or bacitracin (a potent peptidase inhibitor) support the view that Peak 5 is peptidergic. Furthermore, peripheral administration of cysteamine and intrastriatal injection of specific somatostatin antisera both cause the eventual disappearance of Peak 5, suggesting that somatostatin (which oxidises in vitro at approx +800 mV), or a structurally related peptide, could be the principal component of striatal Peak 5. o iesi Academic POW, I~C.
The electrdchemical activity of catechol- and indoleamines and their metabolites has been utilized to develop sensitive assays for amine neurotransmitters and metabolites (1). Voltammetry is an electrochemical
I Address correspondence Farmacologiche M. Negri,
to the author at Instituto via Eritrea 62, 20157 Milano,
0003-2697191 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
di Ricerche Italy.
technique which has the advantage, compared to classical biochemical methods, of enabling continuous analysis in uiuo and in situ of extracellular electroactive compounds when employed with carbon-based microelectrodes (for a review see Ref. (2)). Differential pulse voltammetry (DPV)’ has been successfully used in vivo to study ascorbic acid and dihydrophenylacetic acid (DOPAC), metabolites of dopamine (DA) (3) or Shydroxyindoleacetic acid (5HIAA), and metabolites of serotonin (5HT) (4) in different brain areas of rat using specifically pretreated carbon fiber electrodes. The development of new electrical pretreatments for carbon fiber microelectrodes (CFE) makes it possible to simultaneously monitor in uiuo extracellular ascorbic acid (AA, Peak 1 at -50 mV), DOPAC (Peak 2 at +70/100 mV), and a mixture of 70% 5HIAA and 30% uric acid (UA) (Peak 3 at +270/310 mV) when used in association with DPV (5,6). Under certain conditions a fourth peak is observed at +400/ 430 mV which is probably due to homovanillic acid (HVA) in rat striatum (5,7) or to 3-methoxytyramine (3MT) in mouse striatum (8). It has been reported that several neuropeptides are electrochemically active at the surface of the untreated carbon paste electrode (CPE) (9), their oxidation appearing to be related to the presence and combination of
* Abbreviations used: DPV, differential pulse voltammetry; DOPAC, dihydrophenylacetic acid; DA, dopamine, BHIAA, 5-hydroxyindoleacetic acid, 5HT, serotonin, CFE, carbon fiber microelectrodes; AA, ascorbic acid; UA, uric acid; HVA, homovanillic acid; 3MT, 3methoxytyramine; CPE, carbon paste electrode; CCK-8, cholecystokinin octapeptide; LH-RH, gonadotropin-releasing hormone; (YMSH, cy-melanocyte-stimulating hormone; AVP, vasopressin, ACTH, adrenocorticotropic hormone; PBS, phosphate-buffered saline; SRIF, somatostatin; CNS, central nervous system. 69
70
FRANCESCO
I
L
10
20
III
30’ ’
CRESPI
I
I
c
I
1
I
40
50
60
70
&I
90
t
I
la,
110
4
120 mln
t SALINE
FIG. 1.
(A) In uitro response of CFE not electrically pretreated in a mixture of AA, 5 mM; DOPAC, 50 pM; 5HIAA, 25 NM; and somatostatin, 1 in PBS, 0.1 M; pH 7.4. (B) The electrical pretreatment for the CFE (details under Methods and Results). (C and D) In vitro response of electrically pretreated CFE in PBS or in the same mixture as in A, respectively. Peak 1, AA at -50 mV, Peak 2, DOPAC at +lOO mV; Peak 3, BHIAA at +300 mV, and Peak 5, SRIF at +800 mV. (E) DPV obtained when the same CFE was placed into the striatum of anesthetized rats. (F) Typical in uiuo DPV illustrating the stability of the peaks recorded in the striatum of one rat after injection of NaCl, 0.9% (2 ml/kg ip). The scans (lasting 2 min each) were performed every 5 min; only one every 10 min is shown here. mM;
specific amino acids (tyrosine, tryptophan, cysteine) in the peptide sequence (10). In the present study, untreated CPE (9) or electrically pretreated CFE, 12 pm in diameter (4,7), have been employed in vitro in order to compare their ability in the selective analysis of different electroactive neuropeptides. In addition, using DPV associated with the electrically pretreated CFE, we have found that increasing the potential sweep range from -200 to +950 mV allows the detection of a further (fifth) peak, called Peak 5, at about +800 mV in the striatum of anesthetized rats. The chemical nature of this newly reported voltammetric signal has then been analyzed using different pharmacological approaches to determine the possibility that carbon fiber microelectrodes can monitor specific electroactive neuropeptides in the striatum of anesthetized rats.
METHODS
AND
RESULTS
In Vitro Experiments The electrochemical activity of the following synthetic peptides was tested: caerulein (Farmitalia C. Erba), cholecystokinin octapeptide (CCK-8 Squibb, Inc.), /3-endorphin (ICI Ltd., Pharmaceutical Division), gonadotropin-releasing hormone (LH-RH, Beckman), a-melanocyte-stimulating hormone (a-MSH, Bachem), somatostatin (Peninsula Laboratory, Inc.), neurotensin (Bachem), vasopressin (AVP, Bachem), oxytocin (Bathem), leucine (Leu) and methionine (Met) enkephalin (ICI Ltd., Pharmaceutical Div.), adrenocorticotropic hormone (ACTH, Bachem). The amino acids tyrosine, tryptophan, and cysteine were also tested. Peptides or amino acids were dissolved at concentrations of 0.1 and 1 mrvr, respectively, in phosphate-
IN TABLE
WV0 VOLTAMMETRIC
DETECTION
OF
C
6
1
CCK-8
in Vitro Oxidation Potentials of Electroactive Amino Acids and Neuropeptides at Neutral pH when Carbon Paste or Carbon Fiber Electrodes Were Used The
Substance Tyrosine Tryptophan Cysteine Neurotensin Oxytocin Vasopressin Caerulein Leu-enkephalin Met-enkephalin ACTH,,., fl-Endorphin Somatostatin Cholecystokinin Cholecystokinin LH-RH a-MSH ACTH,.,,
(CCK-4) (CCK-8)
71
NEUROPEPTIDES
Peak oxidation potential at which maximum current generated with
(mV) was
Carbon paste (PI-I 7.4)
fiber 7.4)
680 740 710 640 660 680 780 680 680 680 650 800 770 800 640 800 640
Carbon (PH
720 840 860 670 585 610 670 605 570 700 800 805 730
I .5
.l
AVP
I 10 nA IOnA -',,
.9v
.4
CAERULIN
.8
v
II
CAERULIN P
d!k
.5
.l
OXYTOCIN
.9 v
SOMATOSTATIN
&ENDORPHIN
ic 1
.5 .l
I
J,,
,
.9 v
.a v
.4
810
700 795 650
MET-ENKEPHALIN
MET-ENKEPHALIN
& .5 .7 .9 v buffered saline (PBS) 0.1 M at pH 7.4. Their electroactivity was determined by DPV using a Princeton 174A or a Tacussel PRG5 polarograph with the electrochemical electrodes (auxiliary, reference, and working (CPE or CFE) electrode) suspended in a 500-~1 solution of the peptides or amino acids: saline (NaC10.9%) was the vehicle for all the compounds tested. The CPE were prepared as reported in Ref. (9). The CFE were prepared using a 12pm-diam carbon fiber (Carbon Lorraine, France) as described in Refs. (4,8). The auxiliary electrode was a platinum wire; the reference electrode was a micro silver/silver chloride electrode. Before use, the CFE was electrically treated in PBS (0.1 M, pH 7.4) with a triangular voltage’(from 0 to 3 V, 70 Hz, 8 s; O-2.5 V, 70 Hz, 10 s; O-l.5 V, 70 Hz, 10 s), and then two successive continuous potentials were applied (+1.5 V for 5 s, and -0.9 V for 5 s, see Fig. 1B). This treatment enables the detection of three separate peaks in vitro in a solution of ascorbic acid 5, mM; DOPAC, 50 PM; and 5HIAA, 25 PM, as already demonstrated (5,7). Furthermore, it also allows the in vitro detection of a further oxidation peak when the DPV recordings were made in the same solution with the addition of an electroactive peptide (see Fig. 1D). The scan rate used was 10 mV/s from -250 to +950 mV at a step size of 50 mV. The electrical pretreatment appeared to be essential when the CFE were used as it allowed the selective sepa-
.4
.6
.8
LEU-ENKEPHALIN
V
FIG. 2. Comparison between the in oitro response of CPE (column A) and CFE (columns B and C) to various peptides. Note that the oxidation peaks recorded with CFE are sharper and show clearer separation between the different peptides than those recorded with CPE.
ration of the peptidergic signal from that of other electrochemical compounds in vitro and in vivo when compared to the voltammogram obtained with the untreated CFE. However, the electrical pretreatment
TABLE
2
Effect on Peak 5 of Saline, Bacitracin, or L-Tryptophan Iniected into the Striatum Time Treatment
0
NaCI 0.9% (2ng, n = 9) Bacitracin (10 ng, n = 5) L-Tryptophan CM, n = 5) Note. k SD).
Results
80
120
100 (-c5)
102 (rt.3)
95 (+11)
93 (&16)
100 (-tB)
130 (+13)
159 (-c16)”
153 (+14)’
100 (-+lO)
270 (+62)b
161 (+43)
111(&22)
as percentage
of control
are expressed
o P < 0.05. b P < 0.01, Tukey
40
(mid
test.
values
(mean
12
FRANCESCO
CRESPI
w
I
I
15
30’
,
I
/
I
4s
I
60
Somalostalin
1
1
1
75
90
105
1
120 mln
FIG. 3.
(Top) Effect of local injection into the striatum of SRIF (0,2 pg) or CCK-8 (or P-endorphin or a-MSH) (0,2 pg) compared to the local injection of vehicle (A, NaClO.9%, 2 ~1) on the height of Peak 5 (rz = 7 for each experiment). Results are expressed as percentage of the control values (mean *SD) *P < 0.05, **P < 0.01, Tukey test. Note the largest increase in Peak 5 after SRIF treatment. fl-Endorphin and a-MSH results are omitted for clarity because they were very similar to those observed after CCK-8. (Bottom) DPV observed in one rat after the local injection of SRIF. Note the increase of Peak 5 while Peaks 2 and 3 did not change significantly. The scans were performed every 5 min; only one every 15 min is shown here.
for the CPE was not followed by any significant improvement in sensitivity or selectivity; thus these electrodes were used untreated. The oxidation profiles of all the neuropeptides and amino acids tested were determined with five separate working electrodes, i.e., five CPE or five CFE were suspended, one by one, in the solution containing the substance studied. A peak current value (nA) was determined by constructing a tangent to the shoulders of each respective
peak and measuring the perpendicular height between the tangent and the center of the peak. The five oxidation values obtained from the five different CFE or CPE for each substance studied were plotted to determine the specific oxidation potential of each peptide or amino acid tested. The electrochemical oxidation potentials of these compounds appeared to be distinct and separable from those of the amines DA and 5HT and their metabolites since they oxidized at potentials between +600 and +900 mV with both types of working electrodes (see Ta-
IN
VZVO
VOLTAMMETRIC
DETECTION
B
-VT i ; i : i :
i ! I a f J li 5 It I Iti I ICI I ICI I y; ; 5 I iI I :I I ;i;
3; I 2nA
!: :i ; : i I ~ i; :i j i -LuL .3.5.7.9 v
.3.5.7.9
OF
NEUROPEPTIDES
73
the consistency and the stability of the three respective oxidation potentials recorded, and thus the reliability of the electrically pretreated CFE. The comparison between the respective peak oxidation potentials of the various peptides and amino acids recorded in vitro with DPV using CFE or CPE indicated that the peaks obtained with the first type of electrode were sharper and narrower at the base. Thus, the CFE allows a clearer separation of the oxidation potentials between the different neuropeptides analyzed than the CPE (see Fig. 2 and Table 1). This result, together with the reduced size of the CFE (8-12 pm in diameter) which produces minor histological damage compared to the larger CPE (300 Km in diameter), indicates the advantage of using CFE for in viva studies. In addition, individual neuropeptides appeared to have clearer characteristic oxidation profiles at the CFE when compared to the results obtained with the CPE, thus suggesting the possibility of selective detection of extracellular neuropeptides in uivo. In Vivo Experiments
v
FIG. 4. Zrz uiuo voltammograms illustrating Peak 3 (3) and Peak 5 (5) before (broken line) and 20 min after (solid line) local injection into the striatum of SRIF (A, 2 rg) or L-tryptophan (B, 2 pg). Note that the oxidation potential of Peak 5 was shifted from +800 mV to approx +850 mV by infusion of L-tryptophan, but was unchanged by injection of SRIF.
ble l), with sensitivities similar to the oxidation of catechol- and indoleamines. At the end of each recording, the working electrode was washed in PBS, then used for the following substance tests. Thus for all these substances the same five working electrodes were used, this allows a direct comparison between the different oxidation potentials. Using DPV, we have, for example, observed that with either five untreated CPE (9) or five electrically pretreated CFE (4,8), the three amino acids tyrosine, tryptophan, and cysteine oxidized between +680 and +740 mV or between +720 and +860 mV, respectively, when dissolved at concentrations of 0.1 mM in 0.1 M PBS at pH 7.4 (Table 1). When the five CFE were used, the value of the standard deviation (SD) for the oxidation potential obtained for each compound tested was on the order of f10 mV, but it was *25 mV when the five CPE were employed. This further underlines the greater reliability of the electrically pretreated CFE for the analysis of the characteristic oxidation potential of electroactive compounds. However, before and at the end of each series of recordings, each CFE was placed in a solution containing AA, 5 mM; DOPAC, 50 FM; and 5HIAA, 25 pM, to verify
In vivo differential pulse scans were performed every 5 min in male Sprague-Dawley rats (270-300 g weight), anesthetized with chloral hydrate (500 mg/kg ip) and held in a stereotaxic frame (David Kopf) with a carbon fiber microelectrode implanted in the striatum as previously described (5). It appeared that increasing the potential sweep range from -200 to +950 mV allowed the detection of a fifth peak at approx +800 mV, which we have called Peak 5 (see Fig. 1E). The possibility that this newly reported in uiuo voltammetric signal may correspond to the oxidation of extracellular neuropeptide(s) seems to be supported by the results obtained in the following experiments: (i) Via a Hamilton needle (diameter 100 pm), positioned close to the CFE (maximal distance 1 mm), the intrastriatal administration of bacitracin (10 pg in 1 ~1 saline, n = 5), which is a potent peptidase inhibitor (12), significantly increased the height of Peak 5 (159% of the control value 90 min later, see Table 2), thus supporting the view that Peak 5 is peptidergic. A control group of nine rats received 1~1 saline (NaCI 0.9%) into the striaturn, but Peak 5 was then not modified (Table 2). (ii) Since in vitro experiments demonstrated that only CCK-8, MSH, fl-endorphin, and somatostatin (SRIF) oxidize at approx +800 mV (see Table l), these four peptides were locally injected into the striatum (2 pg in 1 ~1 saline, n = 7 for each peptide) in order to observe the sensitivity of the CFE to these compounds when present in the extracellular fluid. The finding was an increase in the height of Peak 5 with no significant shift in its potential (see Fig. 4A), suggesting that one or more of these neuropeptides may be responsible for the signal in
74
FRANCESCO
CRESPI A-4
control antisera
M
IgG SRIF antisera
20
Time lmin)
FIG. 5.
Effect of cysteamine n = 7) on the height of striatal
(0, 100 mg/kg ip, n = 7), local Peak 5. Results are expressed
injection of control antisera (A, 2 ~1, n = 7), or purified SRIF antisera as percentage of control values (mean -t SD) *P < 0.01, Tukey test.
vim. However, it appeared that SRIF produced the largest increase (450% of controls) compared to the other three peptides (+50%) which lasted longer (90 min) than that following the other peptides (40 min) (Fig. 3). (iii) Peripheral injection of L-tryptophan (100 mg/kg ip, n = 5) did not significantly increase the height of Peak 5; however, following intrastriatal injection of this peptide (2 pg in 1~1 saline, n = 5) Peak 5 was increased to 380% of the preinjection values 20 min after injection (Table 2), but a shift in the oxidation potential of the signal from +800 to +850 mV was also observed (see Fig. 4B). Similar results were obtained with cysteine injected locally (not shown). By contrast, local injection of 2 pg SRIF selectively increased the size of Peak 5 with no shift in its potential (Fig. 4A). (iv) Peripheral administration of cysteamine (100 mg/kg ip) markedly decreased the height of Peak 5 measured in seven rats and the peak was not measurable 30 min after administration (Figs. 5 and 6C). No change of the size of Peak 5 was observed after injection of its vehicle (NaCl 0.9%, 2 ~1, n = 5; see, for example, Fig. 1F). (v) Local injection into the striatum of purified somatostatin antisera (2 ~1, Sanofi, France) also caused the eventual disappearance of Peak 5 within 120 min, but had no significant effect on either the DOPAC or 5HIAA oxidation peak (see Figs. 5 and 6B). Vice versa, injection of control antisera (2 ~1) had no significant effect on any of the peaks recorded in the striatum (Figs. 5 and 6A) (n = 7 for each experiment).
(0,2
pl,
DISCUSSION
We used DPV with carbon fiber electrodes electrically pretreated to record simultaneously AA (Peak l), DOPAC (Peak 2), and 5HIAA (Peak 3) (5,7), and when the potential sweep range was increased to +950 mV the carbon fiber electrode detected the same peptides and amino acids observed previously with carbon paste electrodes at potentials between f600 and +800 mV in vitro (9), but with sharper and more clearly separated oxidation peaks (Fig. 2). A fifth peak, subsequently called Peak 5, was also detected in uiuo at about +800 mV when the carbon fiber electrode was located in the striaturn of anesthetized rats. Since in vitro experiments demonstrated that only CCK-8, a-MSH, /3-endorphin, and somatostatin were oxidized at approx +800 mV, these four peptides were locally injected into the striaturn and shown to increase the height of Peak 5 without alteration of its oxidation potential, indicating that they might contribute to in uiuo Peak 5. However, the largest increase in this signal was observed after the somatostatin administration, suggesting that the carbon fiber electrode is more sensitive in uiuo to this peptide than to the other peptides. It is also possible that a metabolite of somatostatin could participate in Peak 5. This could explain why the increase in Peak 5 following SRIF injection was delayed in onset and lasted longer than that following the other peptides locally injected. Further support that Peak 5 is caused by the oxidation of a peptide comes from the observation that bacitracin, a po-
IN
VZVO
VOLTAMMETRIC
Cantml
IgC Somatostatln
I
1
10
OF
75
NEUROPEPTIDES
Antlsen
lo’
B
DETECTION
I
I
1
I
I
50
70
90
110
130
t
150 mln
Antlrera
/
1
30’
I
50
I
I
I
10
20
3o
I
70
,
I
t
I
90
1
110
I
130
I
I
I
40
50
60
1% mln
I
70 min
Cysteamine
FIG. 6. DPV obtained in the striatum of a single animal to illustrate the effect antisera (2 el), or (C) peripheral administration of cysteamine (100 mg/kg ip).
tent peptidase inhibitor (12), increased the height of the peak. Somatostatin is a peptide widely distributed within the CNS and relatively high levels are found in the striatum (13,14). Cysteamine is a thiol reagent reported to selectively affect brain somatostatin and prolactin
of local
injection
of (A) control
antisera
(2 rl),
(B) purified
(X,16,17). Another ex uivo study (18) demonstrated that cysteamine given systemically selectively depletes somatostatin in the rat central nervous system while Beal and Martin (19) found that local cysteamine injection decreased the somatostatin content of striatal slices. Somatostatin (which is electroactive) and prolac-
76
FRANCESCO
tin (not electroactive) are the only neuropeptides studied thus far that have been shown to be significantly affected by cysteamine while LH-RH, VIP, CCK-8, AVP, substance P, TRH, fl-endorphin, and neuropeptide Y (all electroactive except substance P) are unaltered (17,18,20,21). The mechanism whereby cysteamine depletes somatostatin is unclear, though there are indications that the disulfide bridge in the somatostatin molecule is chemically modified by cysteamine, thus altering the biological properties of the peptide. In the present study peripheral injection of cysteamine decreased Peak 5 supporting the view that this peak is due to (a) a peptide and (b) possibly somatostatin. Local injection into the striatum of purified somatostatin antisera, which would be expected to combine with the somatostatin in the extracellular space and therefore prevent its oxidation at the surface of the working electrode, also caused the eventual disappearance of Peak 5 within 120 min. However, local injection of control antisera had no significant effect on the size of Peak 5, thus providing further evidence implicating oxidation of somatostatin in Peak 5. L-Tryptophan and cysteine are the principal electroactive amino acids present in the somatostatin sequence and our data indicate that the CFE is sensitive to L-tryptophan and cysteine in Go. However, they suggest that oxidation of either L-tryptophan or cysteine alone in extracellular fluid is not solely responsible for Peak 5 since following their local injection the oxidation position of the peak was significantly altered. Similar observations have been made concerning the involvement of UA (which oxidizes at +260/280 mV in uitro) in the indole signal (Peak 3 at +300 mV) (4) which increases when UA is infused close to the CFE in the striatum of rat, but again with a shift of the oxidation potential of Peak 3 from +300 to +270 mV (6). The concomitant presence of L-tryptophan and cysteine within the chemical structure of somatostatin may determine the value of the oxidation potential of this peptide at about +800 mV in vitro. Thus, while injected alone each one of these two amino acids altered the oxidation potential of in uiuo Peak 5, they did not alter this oxidation potential when injected as part of the whole somatostatin molecule. In conclusion, these data suggest that the CFE seems to be more sensitive to the oxidation of somatostatin than that of other neuropeptides in the extracellular fluid, thus SRIF could be the major component of Peak 5 in uiuo. Obviously, further work is required to confirm this view, and the importance of such studies (in pro-
CRESPI
gress) is increased by the interest in somatostatin in relation to its possible involvement in Alzheimer’s senile dementia (22) and other brain disorders (23,24). ACKNOWLEDGMENT We thank port.
the Medical
Research
Council
(UK)
for financial
sup-
REFERENCES 1. Adams, R. H., and Marsden, C. A. (1982) Handb. Psychopharmacol. 15, l-74. 2. Marsden, C. A., Joseph, M. H., Kruk, Z., Maidment, N., O’Neill, R., Schenk, J., and Stamford, J. (1988) Neuroscience 25,389-400. 3. Gonon, F., Buda, M., Cespuglio, R., Jouvet, M., and Pujol, J. F. (1980) Nature (London) 286,902-904. 4. Crespi, F., Cespuglio, R., and Jouvet, M. (1982) Brain Res. 270, 45-54. 5. Crespi, F., Sharp, T., Keane, P., and Morre, M. (1984) Neurosci. Lett. 52, 159-164. 6. Crespi, F., Sharp, T., Maidment, N., and Marsden, C. A. (1983) Neurosci. L&t. 43, 207-211. 7. Crespi, F. (1986) Neurosci. Lett. 66, l-6. K., Heal, D., Marsden, C. A., Buckett, 8. Crespi, F., Martin, Sanghera, M. (1989) Brain Res. 500, 241-246. G. W., Brazell, M. P., and Marsden, C. A. (1981) 9. Bennett, 29,1001-1007. 10. Brabec, V. (1980) Bioelectrochem. Bioenerg. 7,69-82. F., Fombarlet, C. M., Buda, M., and Pujol, 11. Gonon, Anal. Chem. 53, 1386-1389.
W., and Life Sci.
J. F. (1981)
12. Hokfelt, T., Lundberg, J. M., Schulzberg, M., Johansson, O., Skirboll, L., Anggard, A., Fredholm, B., Hamberger, B., Pernow, B., Rehfeld, J., and Goldstein, M. (1980) Proc. R. Sot. London B 210,63-77. 13. Beal, M. F. (1983) Brain Res. 278,103-108. 14. Finley, J. L. W., Madernt, J. L., Rogers, L. T., and Petrusz, (1981) Neuroscience 6, 2173-2192. 15. Bakhit, C., Benoit, R., and Bloom, F. E. (1983) Regul. Pept. 169-177.
P. 6,
16. Palkovits, M., Browstein, M. J., Eiden, L. E., Beinfeld, M. C., Russel, J., Arimura, A., and Szabo, S. (1982) Brain Res. 240, 178-180. S. (1981) Endocrinology 109,2255-2257. 17. Szabo, S., and Reichlin, 18. Sagar, S. M., Laudry, D., Millard, W. J., Badger, T. M., Arnold, M. A., and Martin, J. B. (1982) J. Neuroscience 2,225-231. 19. Beal, M. F., and Martin, J. B. (1984) Brain Res. 308,319-324. 20. Geetinder, K. C., and Beal, M. F. (1987) Brain Res. 401,359-364. 21. Millard, W. J., Sagar, S. M., Badger, T. M., Carr, D. B., Arnold, M. A., Spindel, E., Kasting, N. W., and Martin, J. B. (1983) Endocrinology 112,51&525. 22. Davies, P., Katzman, R., and Terry, R. D. (1980) Nature (Londonl 288,279-280. 23. Cooper, P. E., Aronin, H., Bird, E. D., Leeman, S. E., and Martin, J. B. (1981) Neurology 31,64. 24. Epelbaum, J., Ruberg, M., Moyse, E., Javoy-Agid, F., Dubois, B., and Agid, Y. (1983) Brain Res. 278.376-379.
BIOCHEMISTRY
1%,69-76
(1991)
In lho Voltammetric Detection of Neuropeptides with Micro Carbon Fiber Biosensors: Possible Selective Detection of Somatostatin Francesco
Crespi’
Department of Physiology and Pharmacology, Nottingham University, Nottingham and Instituto di Ricer&e Farmacologiche M. Negri, via Eritrea 62, 20157 Milano,
Received
May
NG7 2UH, Italy
United
Kingdom,
30, 1990
The electrochemical activity of catecholand indoleamines, measured by differential pulse voltammetry (DPV) with specifically electrically pretreated carbon fiber microelectrodes, has been utilized to develop sensitive assays for amine neurotransmitters and metabolites. So far, four oxidation peaks have been recorded in viva between -200 and +500 mV and are well identified. We now report that by increasing the potential sweep range to +950 mV, a further peak, called Peak 5, was detected at +800 mV in vivo in the striatum of anesthetized rats. Neuropeptides containing tyrosine, tryptophan and/or cysteine appear to be electrochemically active between +600 and +900 mV in vitro in a buffered solution at pH 7.4. The present study investigates the chemical nature of Peak 5 and the possible contribution of electroactive neuropeptides to this in vivo voltammetric signal. Experiments performed in vitro and in vivo with amino acids, neuropeptides, or bacitracin (a potent peptidase inhibitor) support the view that Peak 5 is peptidergic. Furthermore, peripheral administration of cysteamine and intrastriatal injection of specific somatostatin antisera both cause the eventual disappearance of Peak 5, suggesting that somatostatin (which oxidises in vitro at approx +800 mV), or a structurally related peptide, could be the principal component of striatal Peak 5. o iesi Academic POW, I~C.
The electrdchemical activity of catechol- and indoleamines and their metabolites has been utilized to develop sensitive assays for amine neurotransmitters and metabolites (1). Voltammetry is an electrochemical
I Address correspondence Farmacologiche M. Negri,
to the author at Instituto via Eritrea 62, 20157 Milano,
0003-2697191 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
di Ricerche Italy.
technique which has the advantage, compared to classical biochemical methods, of enabling continuous analysis in uiuo and in situ of extracellular electroactive compounds when employed with carbon-based microelectrodes (for a review see Ref. (2)). Differential pulse voltammetry (DPV)’ has been successfully used in vivo to study ascorbic acid and dihydrophenylacetic acid (DOPAC), metabolites of dopamine (DA) (3) or Shydroxyindoleacetic acid (5HIAA), and metabolites of serotonin (5HT) (4) in different brain areas of rat using specifically pretreated carbon fiber electrodes. The development of new electrical pretreatments for carbon fiber microelectrodes (CFE) makes it possible to simultaneously monitor in uiuo extracellular ascorbic acid (AA, Peak 1 at -50 mV), DOPAC (Peak 2 at +70/100 mV), and a mixture of 70% 5HIAA and 30% uric acid (UA) (Peak 3 at +270/310 mV) when used in association with DPV (5,6). Under certain conditions a fourth peak is observed at +400/ 430 mV which is probably due to homovanillic acid (HVA) in rat striatum (5,7) or to 3-methoxytyramine (3MT) in mouse striatum (8). It has been reported that several neuropeptides are electrochemically active at the surface of the untreated carbon paste electrode (CPE) (9), their oxidation appearing to be related to the presence and combination of
* Abbreviations used: DPV, differential pulse voltammetry; DOPAC, dihydrophenylacetic acid; DA, dopamine, BHIAA, 5-hydroxyindoleacetic acid, 5HT, serotonin, CFE, carbon fiber microelectrodes; AA, ascorbic acid; UA, uric acid; HVA, homovanillic acid; 3MT, 3methoxytyramine; CPE, carbon paste electrode; CCK-8, cholecystokinin octapeptide; LH-RH, gonadotropin-releasing hormone; (YMSH, cy-melanocyte-stimulating hormone; AVP, vasopressin, ACTH, adrenocorticotropic hormone; PBS, phosphate-buffered saline; SRIF, somatostatin; CNS, central nervous system. 69
70
FRANCESCO
I
L
10
20
III
30’ ’
CRESPI
I
I
c
I
1
I
40
50
60
70
&I
90
t
I
la,
110
4
120 mln
t SALINE
FIG. 1.
(A) In uitro response of CFE not electrically pretreated in a mixture of AA, 5 mM; DOPAC, 50 pM; 5HIAA, 25 NM; and somatostatin, 1 in PBS, 0.1 M; pH 7.4. (B) The electrical pretreatment for the CFE (details under Methods and Results). (C and D) In vitro response of electrically pretreated CFE in PBS or in the same mixture as in A, respectively. Peak 1, AA at -50 mV, Peak 2, DOPAC at +lOO mV; Peak 3, BHIAA at +300 mV, and Peak 5, SRIF at +800 mV. (E) DPV obtained when the same CFE was placed into the striatum of anesthetized rats. (F) Typical in uiuo DPV illustrating the stability of the peaks recorded in the striatum of one rat after injection of NaCl, 0.9% (2 ml/kg ip). The scans (lasting 2 min each) were performed every 5 min; only one every 10 min is shown here. mM;
specific amino acids (tyrosine, tryptophan, cysteine) in the peptide sequence (10). In the present study, untreated CPE (9) or electrically pretreated CFE, 12 pm in diameter (4,7), have been employed in vitro in order to compare their ability in the selective analysis of different electroactive neuropeptides. In addition, using DPV associated with the electrically pretreated CFE, we have found that increasing the potential sweep range from -200 to +950 mV allows the detection of a further (fifth) peak, called Peak 5, at about +800 mV in the striatum of anesthetized rats. The chemical nature of this newly reported voltammetric signal has then been analyzed using different pharmacological approaches to determine the possibility that carbon fiber microelectrodes can monitor specific electroactive neuropeptides in the striatum of anesthetized rats.
METHODS
AND
RESULTS
In Vitro Experiments The electrochemical activity of the following synthetic peptides was tested: caerulein (Farmitalia C. Erba), cholecystokinin octapeptide (CCK-8 Squibb, Inc.), /3-endorphin (ICI Ltd., Pharmaceutical Division), gonadotropin-releasing hormone (LH-RH, Beckman), a-melanocyte-stimulating hormone (a-MSH, Bachem), somatostatin (Peninsula Laboratory, Inc.), neurotensin (Bachem), vasopressin (AVP, Bachem), oxytocin (Bathem), leucine (Leu) and methionine (Met) enkephalin (ICI Ltd., Pharmaceutical Div.), adrenocorticotropic hormone (ACTH, Bachem). The amino acids tyrosine, tryptophan, and cysteine were also tested. Peptides or amino acids were dissolved at concentrations of 0.1 and 1 mrvr, respectively, in phosphate-
IN TABLE
WV0 VOLTAMMETRIC
DETECTION
OF
C
6
1
CCK-8
in Vitro Oxidation Potentials of Electroactive Amino Acids and Neuropeptides at Neutral pH when Carbon Paste or Carbon Fiber Electrodes Were Used The
Substance Tyrosine Tryptophan Cysteine Neurotensin Oxytocin Vasopressin Caerulein Leu-enkephalin Met-enkephalin ACTH,,., fl-Endorphin Somatostatin Cholecystokinin Cholecystokinin LH-RH a-MSH ACTH,.,,
(CCK-4) (CCK-8)
71
NEUROPEPTIDES
Peak oxidation potential at which maximum current generated with
(mV) was
Carbon paste (PI-I 7.4)
fiber 7.4)
680 740 710 640 660 680 780 680 680 680 650 800 770 800 640 800 640
Carbon (PH
720 840 860 670 585 610 670 605 570 700 800 805 730
I .5
.l
AVP
I 10 nA IOnA -',,
.9v
.4
CAERULIN
.8
v
II
CAERULIN P
d!k
.5
.l
OXYTOCIN
.9 v
SOMATOSTATIN
&ENDORPHIN
ic 1
.5 .l
I
J,,
,
.9 v
.a v
.4
810
700 795 650
MET-ENKEPHALIN
MET-ENKEPHALIN
& .5 .7 .9 v buffered saline (PBS) 0.1 M at pH 7.4. Their electroactivity was determined by DPV using a Princeton 174A or a Tacussel PRG5 polarograph with the electrochemical electrodes (auxiliary, reference, and working (CPE or CFE) electrode) suspended in a 500-~1 solution of the peptides or amino acids: saline (NaC10.9%) was the vehicle for all the compounds tested. The CPE were prepared as reported in Ref. (9). The CFE were prepared using a 12pm-diam carbon fiber (Carbon Lorraine, France) as described in Refs. (4,8). The auxiliary electrode was a platinum wire; the reference electrode was a micro silver/silver chloride electrode. Before use, the CFE was electrically treated in PBS (0.1 M, pH 7.4) with a triangular voltage’(from 0 to 3 V, 70 Hz, 8 s; O-2.5 V, 70 Hz, 10 s; O-l.5 V, 70 Hz, 10 s), and then two successive continuous potentials were applied (+1.5 V for 5 s, and -0.9 V for 5 s, see Fig. 1B). This treatment enables the detection of three separate peaks in vitro in a solution of ascorbic acid 5, mM; DOPAC, 50 PM; and 5HIAA, 25 PM, as already demonstrated (5,7). Furthermore, it also allows the in vitro detection of a further oxidation peak when the DPV recordings were made in the same solution with the addition of an electroactive peptide (see Fig. 1D). The scan rate used was 10 mV/s from -250 to +950 mV at a step size of 50 mV. The electrical pretreatment appeared to be essential when the CFE were used as it allowed the selective sepa-
.4
.6
.8
LEU-ENKEPHALIN
V
FIG. 2. Comparison between the in oitro response of CPE (column A) and CFE (columns B and C) to various peptides. Note that the oxidation peaks recorded with CFE are sharper and show clearer separation between the different peptides than those recorded with CPE.
ration of the peptidergic signal from that of other electrochemical compounds in vitro and in vivo when compared to the voltammogram obtained with the untreated CFE. However, the electrical pretreatment
TABLE
2
Effect on Peak 5 of Saline, Bacitracin, or L-Tryptophan Iniected into the Striatum Time Treatment
0
NaCI 0.9% (2ng, n = 9) Bacitracin (10 ng, n = 5) L-Tryptophan CM, n = 5) Note. k SD).
Results
80
120
100 (-c5)
102 (rt.3)
95 (+11)
93 (&16)
100 (-tB)
130 (+13)
159 (-c16)”
153 (+14)’
100 (-+lO)
270 (+62)b
161 (+43)
111(&22)
as percentage
of control
are expressed
o P < 0.05. b P < 0.01, Tukey
40
(mid
test.
values
(mean
12
FRANCESCO
CRESPI
w
I
I
15
30’
,
I
/
I
4s
I
60
Somalostalin
1
1
1
75
90
105
1
120 mln
FIG. 3.
(Top) Effect of local injection into the striatum of SRIF (0,2 pg) or CCK-8 (or P-endorphin or a-MSH) (0,2 pg) compared to the local injection of vehicle (A, NaClO.9%, 2 ~1) on the height of Peak 5 (rz = 7 for each experiment). Results are expressed as percentage of the control values (mean *SD) *P < 0.05, **P < 0.01, Tukey test. Note the largest increase in Peak 5 after SRIF treatment. fl-Endorphin and a-MSH results are omitted for clarity because they were very similar to those observed after CCK-8. (Bottom) DPV observed in one rat after the local injection of SRIF. Note the increase of Peak 5 while Peaks 2 and 3 did not change significantly. The scans were performed every 5 min; only one every 15 min is shown here.
for the CPE was not followed by any significant improvement in sensitivity or selectivity; thus these electrodes were used untreated. The oxidation profiles of all the neuropeptides and amino acids tested were determined with five separate working electrodes, i.e., five CPE or five CFE were suspended, one by one, in the solution containing the substance studied. A peak current value (nA) was determined by constructing a tangent to the shoulders of each respective
peak and measuring the perpendicular height between the tangent and the center of the peak. The five oxidation values obtained from the five different CFE or CPE for each substance studied were plotted to determine the specific oxidation potential of each peptide or amino acid tested. The electrochemical oxidation potentials of these compounds appeared to be distinct and separable from those of the amines DA and 5HT and their metabolites since they oxidized at potentials between +600 and +900 mV with both types of working electrodes (see Ta-
IN
VZVO
VOLTAMMETRIC
DETECTION
B
-VT i ; i : i :
i ! I a f J li 5 It I Iti I ICI I ICI I y; ; 5 I iI I :I I ;i;
3; I 2nA
!: :i ; : i I ~ i; :i j i -LuL .3.5.7.9 v
.3.5.7.9
OF
NEUROPEPTIDES
73
the consistency and the stability of the three respective oxidation potentials recorded, and thus the reliability of the electrically pretreated CFE. The comparison between the respective peak oxidation potentials of the various peptides and amino acids recorded in vitro with DPV using CFE or CPE indicated that the peaks obtained with the first type of electrode were sharper and narrower at the base. Thus, the CFE allows a clearer separation of the oxidation potentials between the different neuropeptides analyzed than the CPE (see Fig. 2 and Table 1). This result, together with the reduced size of the CFE (8-12 pm in diameter) which produces minor histological damage compared to the larger CPE (300 Km in diameter), indicates the advantage of using CFE for in viva studies. In addition, individual neuropeptides appeared to have clearer characteristic oxidation profiles at the CFE when compared to the results obtained with the CPE, thus suggesting the possibility of selective detection of extracellular neuropeptides in uivo. In Vivo Experiments
v
FIG. 4. Zrz uiuo voltammograms illustrating Peak 3 (3) and Peak 5 (5) before (broken line) and 20 min after (solid line) local injection into the striatum of SRIF (A, 2 rg) or L-tryptophan (B, 2 pg). Note that the oxidation potential of Peak 5 was shifted from +800 mV to approx +850 mV by infusion of L-tryptophan, but was unchanged by injection of SRIF.
ble l), with sensitivities similar to the oxidation of catechol- and indoleamines. At the end of each recording, the working electrode was washed in PBS, then used for the following substance tests. Thus for all these substances the same five working electrodes were used, this allows a direct comparison between the different oxidation potentials. Using DPV, we have, for example, observed that with either five untreated CPE (9) or five electrically pretreated CFE (4,8), the three amino acids tyrosine, tryptophan, and cysteine oxidized between +680 and +740 mV or between +720 and +860 mV, respectively, when dissolved at concentrations of 0.1 mM in 0.1 M PBS at pH 7.4 (Table 1). When the five CFE were used, the value of the standard deviation (SD) for the oxidation potential obtained for each compound tested was on the order of f10 mV, but it was *25 mV when the five CPE were employed. This further underlines the greater reliability of the electrically pretreated CFE for the analysis of the characteristic oxidation potential of electroactive compounds. However, before and at the end of each series of recordings, each CFE was placed in a solution containing AA, 5 mM; DOPAC, 50 FM; and 5HIAA, 25 pM, to verify
In vivo differential pulse scans were performed every 5 min in male Sprague-Dawley rats (270-300 g weight), anesthetized with chloral hydrate (500 mg/kg ip) and held in a stereotaxic frame (David Kopf) with a carbon fiber microelectrode implanted in the striatum as previously described (5). It appeared that increasing the potential sweep range from -200 to +950 mV allowed the detection of a fifth peak at approx +800 mV, which we have called Peak 5 (see Fig. 1E). The possibility that this newly reported in uiuo voltammetric signal may correspond to the oxidation of extracellular neuropeptide(s) seems to be supported by the results obtained in the following experiments: (i) Via a Hamilton needle (diameter 100 pm), positioned close to the CFE (maximal distance 1 mm), the intrastriatal administration of bacitracin (10 pg in 1 ~1 saline, n = 5), which is a potent peptidase inhibitor (12), significantly increased the height of Peak 5 (159% of the control value 90 min later, see Table 2), thus supporting the view that Peak 5 is peptidergic. A control group of nine rats received 1~1 saline (NaCI 0.9%) into the striaturn, but Peak 5 was then not modified (Table 2). (ii) Since in vitro experiments demonstrated that only CCK-8, MSH, fl-endorphin, and somatostatin (SRIF) oxidize at approx +800 mV (see Table l), these four peptides were locally injected into the striatum (2 pg in 1 ~1 saline, n = 7 for each peptide) in order to observe the sensitivity of the CFE to these compounds when present in the extracellular fluid. The finding was an increase in the height of Peak 5 with no significant shift in its potential (see Fig. 4A), suggesting that one or more of these neuropeptides may be responsible for the signal in
74
FRANCESCO
CRESPI A-4
control antisera
M
IgG SRIF antisera
20
Time lmin)
FIG. 5.
Effect of cysteamine n = 7) on the height of striatal
(0, 100 mg/kg ip, n = 7), local Peak 5. Results are expressed
injection of control antisera (A, 2 ~1, n = 7), or purified SRIF antisera as percentage of control values (mean -t SD) *P < 0.01, Tukey test.
vim. However, it appeared that SRIF produced the largest increase (450% of controls) compared to the other three peptides (+50%) which lasted longer (90 min) than that following the other peptides (40 min) (Fig. 3). (iii) Peripheral injection of L-tryptophan (100 mg/kg ip, n = 5) did not significantly increase the height of Peak 5; however, following intrastriatal injection of this peptide (2 pg in 1~1 saline, n = 5) Peak 5 was increased to 380% of the preinjection values 20 min after injection (Table 2), but a shift in the oxidation potential of the signal from +800 to +850 mV was also observed (see Fig. 4B). Similar results were obtained with cysteine injected locally (not shown). By contrast, local injection of 2 pg SRIF selectively increased the size of Peak 5 with no shift in its potential (Fig. 4A). (iv) Peripheral administration of cysteamine (100 mg/kg ip) markedly decreased the height of Peak 5 measured in seven rats and the peak was not measurable 30 min after administration (Figs. 5 and 6C). No change of the size of Peak 5 was observed after injection of its vehicle (NaCl 0.9%, 2 ~1, n = 5; see, for example, Fig. 1F). (v) Local injection into the striatum of purified somatostatin antisera (2 ~1, Sanofi, France) also caused the eventual disappearance of Peak 5 within 120 min, but had no significant effect on either the DOPAC or 5HIAA oxidation peak (see Figs. 5 and 6B). Vice versa, injection of control antisera (2 ~1) had no significant effect on any of the peaks recorded in the striatum (Figs. 5 and 6A) (n = 7 for each experiment).
(0,2
pl,
DISCUSSION
We used DPV with carbon fiber electrodes electrically pretreated to record simultaneously AA (Peak l), DOPAC (Peak 2), and 5HIAA (Peak 3) (5,7), and when the potential sweep range was increased to +950 mV the carbon fiber electrode detected the same peptides and amino acids observed previously with carbon paste electrodes at potentials between f600 and +800 mV in vitro (9), but with sharper and more clearly separated oxidation peaks (Fig. 2). A fifth peak, subsequently called Peak 5, was also detected in uiuo at about +800 mV when the carbon fiber electrode was located in the striaturn of anesthetized rats. Since in vitro experiments demonstrated that only CCK-8, a-MSH, /3-endorphin, and somatostatin were oxidized at approx +800 mV, these four peptides were locally injected into the striaturn and shown to increase the height of Peak 5 without alteration of its oxidation potential, indicating that they might contribute to in uiuo Peak 5. However, the largest increase in this signal was observed after the somatostatin administration, suggesting that the carbon fiber electrode is more sensitive in uiuo to this peptide than to the other peptides. It is also possible that a metabolite of somatostatin could participate in Peak 5. This could explain why the increase in Peak 5 following SRIF injection was delayed in onset and lasted longer than that following the other peptides locally injected. Further support that Peak 5 is caused by the oxidation of a peptide comes from the observation that bacitracin, a po-
IN
VZVO
VOLTAMMETRIC
Cantml
IgC Somatostatln
I
1
10
OF
75
NEUROPEPTIDES
Antlsen
lo’
B
DETECTION
I
I
1
I
I
50
70
90
110
130
t
150 mln
Antlrera
/
1
30’
I
50
I
I
I
10
20
3o
I
70
,
I
t
I
90
1
110
I
130
I
I
I
40
50
60
1% mln
I
70 min
Cysteamine
FIG. 6. DPV obtained in the striatum of a single animal to illustrate the effect antisera (2 el), or (C) peripheral administration of cysteamine (100 mg/kg ip).
tent peptidase inhibitor (12), increased the height of the peak. Somatostatin is a peptide widely distributed within the CNS and relatively high levels are found in the striatum (13,14). Cysteamine is a thiol reagent reported to selectively affect brain somatostatin and prolactin
of local
injection
of (A) control
antisera
(2 rl),
(B) purified
(X,16,17). Another ex uivo study (18) demonstrated that cysteamine given systemically selectively depletes somatostatin in the rat central nervous system while Beal and Martin (19) found that local cysteamine injection decreased the somatostatin content of striatal slices. Somatostatin (which is electroactive) and prolac-
76
FRANCESCO
tin (not electroactive) are the only neuropeptides studied thus far that have been shown to be significantly affected by cysteamine while LH-RH, VIP, CCK-8, AVP, substance P, TRH, fl-endorphin, and neuropeptide Y (all electroactive except substance P) are unaltered (17,18,20,21). The mechanism whereby cysteamine depletes somatostatin is unclear, though there are indications that the disulfide bridge in the somatostatin molecule is chemically modified by cysteamine, thus altering the biological properties of the peptide. In the present study peripheral injection of cysteamine decreased Peak 5 supporting the view that this peak is due to (a) a peptide and (b) possibly somatostatin. Local injection into the striatum of purified somatostatin antisera, which would be expected to combine with the somatostatin in the extracellular space and therefore prevent its oxidation at the surface of the working electrode, also caused the eventual disappearance of Peak 5 within 120 min. However, local injection of control antisera had no significant effect on the size of Peak 5, thus providing further evidence implicating oxidation of somatostatin in Peak 5. L-Tryptophan and cysteine are the principal electroactive amino acids present in the somatostatin sequence and our data indicate that the CFE is sensitive to L-tryptophan and cysteine in Go. However, they suggest that oxidation of either L-tryptophan or cysteine alone in extracellular fluid is not solely responsible for Peak 5 since following their local injection the oxidation position of the peak was significantly altered. Similar observations have been made concerning the involvement of UA (which oxidizes at +260/280 mV in uitro) in the indole signal (Peak 3 at +300 mV) (4) which increases when UA is infused close to the CFE in the striatum of rat, but again with a shift of the oxidation potential of Peak 3 from +300 to +270 mV (6). The concomitant presence of L-tryptophan and cysteine within the chemical structure of somatostatin may determine the value of the oxidation potential of this peptide at about +800 mV in vitro. Thus, while injected alone each one of these two amino acids altered the oxidation potential of in uiuo Peak 5, they did not alter this oxidation potential when injected as part of the whole somatostatin molecule. In conclusion, these data suggest that the CFE seems to be more sensitive to the oxidation of somatostatin than that of other neuropeptides in the extracellular fluid, thus SRIF could be the major component of Peak 5 in uiuo. Obviously, further work is required to confirm this view, and the importance of such studies (in pro-
CRESPI
gress) is increased by the interest in somatostatin in relation to its possible involvement in Alzheimer’s senile dementia (22) and other brain disorders (23,24). ACKNOWLEDGMENT We thank port.
the Medical
Research
Council
(UK)
for financial
sup-
REFERENCES 1. Adams, R. H., and Marsden, C. A. (1982) Handb. Psychopharmacol. 15, l-74. 2. Marsden, C. A., Joseph, M. H., Kruk, Z., Maidment, N., O’Neill, R., Schenk, J., and Stamford, J. (1988) Neuroscience 25,389-400. 3. Gonon, F., Buda, M., Cespuglio, R., Jouvet, M., and Pujol, J. F. (1980) Nature (London) 286,902-904. 4. Crespi, F., Cespuglio, R., and Jouvet, M. (1982) Brain Res. 270, 45-54. 5. Crespi, F., Sharp, T., Keane, P., and Morre, M. (1984) Neurosci. Lett. 52, 159-164. 6. Crespi, F., Sharp, T., Maidment, N., and Marsden, C. A. (1983) Neurosci. L&t. 43, 207-211. 7. Crespi, F. (1986) Neurosci. Lett. 66, l-6. K., Heal, D., Marsden, C. A., Buckett, 8. Crespi, F., Martin, Sanghera, M. (1989) Brain Res. 500, 241-246. G. W., Brazell, M. P., and Marsden, C. A. (1981) 9. Bennett, 29,1001-1007. 10. Brabec, V. (1980) Bioelectrochem. Bioenerg. 7,69-82. F., Fombarlet, C. M., Buda, M., and Pujol, 11. Gonon, Anal. Chem. 53, 1386-1389.
W., and Life Sci.
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12. Hokfelt, T., Lundberg, J. M., Schulzberg, M., Johansson, O., Skirboll, L., Anggard, A., Fredholm, B., Hamberger, B., Pernow, B., Rehfeld, J., and Goldstein, M. (1980) Proc. R. Sot. London B 210,63-77. 13. Beal, M. F. (1983) Brain Res. 278,103-108. 14. Finley, J. L. W., Madernt, J. L., Rogers, L. T., and Petrusz, (1981) Neuroscience 6, 2173-2192. 15. Bakhit, C., Benoit, R., and Bloom, F. E. (1983) Regul. Pept. 169-177.
P. 6,
16. Palkovits, M., Browstein, M. J., Eiden, L. E., Beinfeld, M. C., Russel, J., Arimura, A., and Szabo, S. (1982) Brain Res. 240, 178-180. S. (1981) Endocrinology 109,2255-2257. 17. Szabo, S., and Reichlin, 18. Sagar, S. M., Laudry, D., Millard, W. J., Badger, T. M., Arnold, M. A., and Martin, J. B. (1982) J. Neuroscience 2,225-231. 19. Beal, M. F., and Martin, J. B. (1984) Brain Res. 308,319-324. 20. Geetinder, K. C., and Beal, M. F. (1987) Brain Res. 401,359-364. 21. Millard, W. J., Sagar, S. M., Badger, T. M., Carr, D. B., Arnold, M. A., Spindel, E., Kasting, N. W., and Martin, J. B. (1983) Endocrinology 112,51&525. 22. Davies, P., Katzman, R., and Terry, R. D. (1980) Nature (Londonl 288,279-280. 23. Cooper, P. E., Aronin, H., Bird, E. D., Leeman, S. E., and Martin, J. B. (1981) Neurology 31,64. 24. Epelbaum, J., Ruberg, M., Moyse, E., Javoy-Agid, F., Dubois, B., and Agid, Y. (1983) Brain Res. 278.376-379.
