- Email: [email protected]
Early developmental exposure to benzodiazepine ligands alters brain31P-NMR spectra in young adult rats
Brain Research, 506 (1990) 85-92 Elsevier
85
BRES 15089
Early developmental exposure to benzodiazepine ligands alters brain 31p-NMR spectra in young adult rats Rajesh Miranda ~, Toni Ceckler 2, Ronnie Guillet ~ and Carol Kellogg 1 1Department of Psychology, Meliora Hall, University of Rochester, Rochester, NY 14627 and 2Department of Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 (U. S. A.) (Accepted 6 June 1989) Key words: Diazepam; Clonazepam; PKll195; RO15-1788; Haloperidol; pH; Phosphocreatine utilization; Adenosine triphosphate
Alterations in brain high energy phosphate compounds, using 3~p-NMR (nuclear magnetic resonance) spectroscopy, were measured in vivo in young adult (3-4 months) rats following prenatal exposure to ligands acting specifically at benzodiazepine (BDZ) binding sites. The exposure induced a decrease in intraceUular pH that indicated a predominant interaction of the drugs in utero with central-type BDZ receptor sites. Late gestational exposure to BDZ ligands also induced changes in brain phosphocreatine (PCr) utilization. Exposure to the lowest dose of DZ (1.0 mg/kg) but not the higher dose (2.5 mg/kg) induced a significant change in PCr utilization. Exposure to the central-type BDZ receptor antagonist RO15-1788 alone clearly altered PCr utilization in adult offspring, and DZ (2.5 mg/kg) when administered concurrently was not able to preventthis effect. Though exposure to a peripheral-type ligand (PKIl195) had no effect by itself, it converted the effect of the high dose of DZ to that of the low dose. Together, these results indicate an interaction during development between the central and peripheral-type BDZ binding sites on organization and/or regulation of cellular energy metabolism. Normalized ATP levels were not changed by any prenatal treatment indicating adequate buffering of intracellular ATP by phosphocreatine. The dopaminergic antagonist haloperidol did not alter intracellular pH or any index of phosphate metabolism indicating a selective rceptor mediated role for BDZ ligands in influences on the long term organization of intracellular phosphate metabolism. INTRODUCTION Previous research has demonstrated that prenatal exposure to benzodiazepine ( B D Z ) compounds induces a variety of effects observable in the adult organism 24'53. These effects include long term alterations in cytotoxic aldehydes that are products of cellular oxidative metabolism 3x. Furthermore, the effects on metabolism do not appear to be related to in utero actions at characteristic central-type B D Z binding sites. To date, two major classes of B D Z recognition sites have been identified, The first is the central-type receptor, associated with the 7-aminobutyric acid ( G A B A ) chloride (CL-) channel 5° and considered to mediate the anti-anxiety and anti-convulsant effects of the BDZs. This site is characterized by its affinity for the B D Z clonazepam TM and the antagonist RO15-178818,22. Different subsets of the B D Z - G A B A channel complex may exist, as defined by selectivity for different B D Z ligands 2°. A n o t h e r major B D Z recognition site that has been identified is termed the peripheral-type receptor 3"3s. This receptor population is characterized by its affinity for the ligands Ro5-4864 and PKl11954'29 and is present in glia137"4s as well as neuronal elements 2 in the brain.
Recent evidence has linked the peripheral receptor subtype to mitochondria in brain and other tissues 12'29 and to mitochondrial outer membranes in the adrenal gland 4, implicating that receptor population in regulation of energy metabolism. Acute administration of drugs that act at the G A B A B D Z complex has revealed a differential role in intracellular energy metabolism for the different ligand recognition sites on the B D Z - G A B A - C L channel complex. The B D Z s have been reported to increase brain glucose uptake and transiently increase glycolysis as well 1v'54, while the barbiturates decrease glycolytic flux 3° and the utilization of high energy phosphates by the brain 46. Specific G A B A antagonists, however, increase the utilization of brain high energy phosphates and decrease cellular pH 39'4°'55. To study the impact of drug interaction at B D Z receptors during development on the long term control of cellular energy metabolism, changes in intracellular pH (pHi) and high energy phosphorus compounds were measured in vivo in adult rats following prenatal exposure to BDZs, using 3~p nuclear magnetic resonance (NMR) spectroscopy. Phosphate compounds measured by N M R represent that component of high energy
Correspondence: C. Kellogg, Department of Psychology, Meliora Hall, University of Rochester, Rochester, NY 14627, U.S.A.
86 p h o s p h a t e s that is freely a v a i l a b l e for cellular reactions. In the rat, B D Z and G A B A
r e c o g n i t i o n sites a p p e a r
d u r i n g late g e s t a t i o n 5'7~47. D u r i n g this p e r i o d of n e u r a l d e v e l o p m e n t , the d e v e l o p i n g B D Z - G A B A
system m a y
be sensitive to a l t e r a t i o n s in its c h e m i c a l e n v i r o n m e n t . R e c e n t w o r k has s h o w n that d i a z e p a m ( D Z ) can influence GABA-mediated
c h l o r i d e flux in fetal rat s y n a p t o -
n e u r o s o m a l p r e p a r a t i o n s 25, d e m o n s t r a t i n g an early effector r e s p o n s e to D Z action at r e l e v a n t sites. F u r t h e r m o r e , BDZ
receptors
(the
peripheral-type
especially)
may
r e g u l a t e s e v e r a l aspects o f n e u r a l d e v e l o p m e n t including cell p r o l i f e r a t i o n and n e u r i t e o u t g r o w t h 9'3353. T h e present
experiment
assessed
the
possibility
of e n d u r i n g
c h a n g e s in i n t r a c e l l u l a r m e t a b o l i s m f o l l o w i n g early dev e l o p m e n t a l e x p o s u r e to B D Z c o m p o u n d s such as D Z .
by shimming on the proton signal at 85.56 MHz. ' P signals were acquired at 34.635 MHz, under partially saturated conditions, using a 3 s recycle time and a 20 ~us pulse, producing maximum excitation approximately 5 mm away from the coil. The sampling region mainly included cortical tissue (Fig. l). The spectral bandwidth was +3012.04 Hz, Signals were acquired using quadrature detection and were averaged over 512 acquisitions. The signal was transformed using Fourier analysis following exponential apodization. The contributions to the spectrum from immobilized phosphates in the bone and of the membrane phospholipids were subtracted out using convolution difference techniques (line broadcnings of 10 Hz and 300 Hz). Peak areas were obtained using a program supplied by GE. A sample spectrum (A), the fitted spectrum (B 1), and the individual peaks (132) are shown in Fig. 2. From each treatment group, animals not tested in the magnet were randomly selected for blood gas analysis. Animals were injected with the same combination and dosage of anesthetics as was used during spectral acquisition, and their femoral arteries were catheterized. Samples of heparinized arterial blood were analyzed on an IL system 1302 pH/blood gas analyzer.
T h e r e c e p t o r specificity o f the effect of p r e n a t a l e x p o s u r e to D Z was also e x a m i n e d . In a d d i t i o n , as a c o n t r o l , the effect of p r e n a t a l e x p o s u r e to the b u t y r o p h e n o n e halop e r i d o l , a d o p a m i n e r g i c a n t a g o n i s t and t r a n q u i l i z e r , was also studied.
METHODS
Data analysis Chemical shifts were expressed relative to the phosphocreatine (PCr) peak in ppm according to convention (1 ppm = 34,635 Hz), with the PCr peak at 0 ppm. pH~ was determined from the chemical shift of inorganic phosphate (P~) from P C r 16'~6 according to the following equation 21: (d-3.27) pH i = 6.72 + log,) - -
(5.69-d)
Prenatal paradigm Female rats (Long-Evans Strain, Blue Spruce Farms, Altamount, N.Y.) were bred with in house stock. Pregnant rats were injected intraperitoneally, once daily, with one or a combination of two drugs, on days 14 to 20 of gestation. Day 0 was defined as the day on which the vaginal smear tested sperm positive. On the 13th day of gestation, animals were weighed and assigned to one of the drug exposure groups. Doses were computed based on the dam's weight on day 13 of gestation and held constant thereafter. Within 24 h after birth, all litters were culled to 10-12 pups. Animals were weaned at 28 days of age. Rats from two or more litters were used for each experimental condition and all animals were tested at 3-4 months of age. An approximately equal number of males and females was included in each group. Pregnant females were assigned to one of 4 groups: uninjected, vehicle injected (40% propylene glycol + 10% ethanol), or DZ injected (at 1.0 or 2.5 mg/kg/day). To evaluate the receptor specificity of the effects observed following exposure to DZ, other pregnant females were administered the central-type BDZ receptor antagonist RO15-1788 [ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo4H-imidazo(1,5-a)(1,4)benzodiazepine-3-carboxylate] (10 mg/kg/ day), either alone or in combination with DZ (2.5 mg/kg/day), the central-type BDZ receptor agonist clonazepam (1.0 mg/kg/day), the peripheral-type BDZ receptor ligand PK11195 [1-(2-chlorophenyl)N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide] (5.0 mg/ kg/day), either alone or in combination with DZ (2.5 mg/kg/day), or the dopaminergic antagonist haloperidol (2.5 mg/kg/day). Drug dosages were based on values used in previous experiments. For sample size of each group see the legend to Fig. 3. ~ P-NMR spectral acquisition Animals were anesthetized with ketamine (12.5 mg/kg) and xylazine (15 mg/kg), and placed in a foam restraint. Anesthetics were necessary in order to immobilize the animal for spectral acquisition. A 2 cm diameter, 2 turn, surface coil was taped to the head of the animal. Animals were placed within the bore of a 2.0 Tesla, Oxford Instruments superconducting magnet interfaced to a GE CSI II imaging/spectroscopy system (GE NMR Instruments, Fremont. CA). The homogeneity of the magnetic field was adjusted
(1)
where d is the chemical shift (in ppm) of P~ from PCr, measured from peak to peak. The PCr utilization ration ([Fl~p)4~ was defined as:
[FLp -
PCr
(2)
(PCr + Pt) The total energy phosphate pool [P]t was defined as the percentage of total observable energy phosphates that are a part of the total observable mobile phosphate poolS: [P]t =
Pi + PCr + 7ATP + flATP + aATP
(3)
ix] where IX] = total area under the curve attributable to all the observable mobile phosphates. The fl-phosphate peak of adenosine triphosphate (flATP) was normalized to [P]t, after [P]t was determined to be contant across conditions and the proportion was expressed as ATP'. ATP'-
flATP
[Pit
(4)
In all cases, the ratios are unitless. Data were analyzed using a commercial statistical package (SAS v5.16), Statistical analysis involved a General linear model analysis of variance to control for unbalanced experimental designs, followed by post-hoc t-tests (difference between least square means test using pre-planned comparisons to control for repeated testing). For purposes of statistical analysis, the uninjected animals and the animals prenatally exposed to vehicle were combined for the control group since no significant differences were observed between these groups (P values ranged from 0.35 to 0.80). Data was expressed as mean __+ S.E.M.
87
la
1t O O
2b
2a
Fig. 1. A proton magnetic resonance image of the rat head and brain showing coil placement over the rat brain in sagittal section (la) and coronal section (2a). White spots above the rat head indicate placement of a water filled tube above the surface coil. A tracing of the rat brain in sagittal (lb) and coronal (2b) section indicates the depth of effective signal acquisition beneath the plane of the coil. Arrow in lb indicates the level of the image in 2b. Abbreviations used: ob, olfactory bulbs: bo, bone; sk, skin; ctx, cortex; cb, cerebellum; sc. spinal cord; s, septum; cp, caudate-putamen; Lv, lateral ventricles; 3v, third ventricle.
RESULTS Alterations in p H i Prenatal exposure to both doses of D Z (1.0 and 2.5 'd'
8
w
o
I I0
,
i
.
6
, , i , j , , . | l | , l
, 0
-10
-20
P~
Fig. 2. Sample 3~P-NMR spectrum (A), with combined fitted peaks (B1) and individual fitted peaks (B2). 1 = Phosphomonoesters (PME), 2 = Inorganic phosphate (P~), 3 = Phosphodiesters (PDE), 4 = Phosphocreatine (PCr), 5 = yATP, 6 = aATE 7 = flATP, 'd' = chemical shift in "ppm' between PCr and Pi (see Eq. 1).
mg/kg) p r o d u c e d a significant decrease in intracellular p H (pHi) (P < 0.0006 and P < 0.0001, respectively) in the brains of young adults, c o m p a r e d to control values (Fig. 3). Exposure to the central-type B D Z rceptor antagonist RO15-1788 alone did not significantly affect pHi, but the antagonist partially p r e v e n t e d the effects of p r e n a t a l exposure to the higher dose of D Z (2.5 mg/kg) when a d m i n i s t e r e d concurrently with D Z . The p H i was higher in this group than in animals e x p o s e d only to D Z at 2.5 mg/kg ( P < 0.01). RO15-1788 was unable to reverse the effect of D Z completely, however, since the pH~ in animals exposed to D Z (2.5 mg/kg) plus RO15-1788 was lower than that in controls ( P < 0.03). Exposure to the central-type B D Z receptor agonist Clonazepam also induced a significant decrease in p H i from control values (P < 0.008), though the magnitude of the effect was less than that produced by either dose of D Z . Exposure to the p e r i p h e r a l - t y p e [igand P K l l 1 9 5 by itself led to a modest though significant decrease in p H i
88
0.88
714"
~/ //..-/
7.10" /~.J~/ 0,83
=..
,/////~
)
//~
H,
7.06-
f//, r//
i
078 t' U+V
DZ1.0
DZ2.5
FO
DZd:IO
CLON
PK
DZ+PK HAt
U+V
DZ1.0
DZ2.5
R~
DZ+RO CLON
PK
D Z + P K HAL
DZ25
FO
702
N
f;;;;
E 100' 90"
6.98 r' U+V
DZ1 0
DZ2.5
FO
DZ.-dql
CLQN
PK
DZ+PK
HAL 80"
PRENATAL DRUG EXPOSURE
Fig. 3. C h a n g e s in pH~ at 3 m o n t h s of age following in utero exposure over gestation days 14-20 to different drugs. U + V = u n i n j e c t e d + v e h i c l e injected controls (n = 8), D Z 1 . 0 = exposure to D Z at 1.0 mg/kg/day (n = 7), D Z 2 . 5 = exposure to D Z at 2.5 mg/kg/day (n = 8), R O = exposure to RO15-1788 at 10 mg/kg/day (n = 6), D Z + R O = exposure to D Z (2.5 mg/kg/day) + RO15-1788 at 10 mg/kg/day (n = 10), C L O N = exposure to clonazepam at 1.0 mg/kg/day (n = 8), PK = exposure to P K l l 1 9 5 at 5.0 mg/kg/day (n = 10), D Z + P K = exposure to D Z at 2.5 mg/kg/day + P K l l 1 9 5 at 5.0 mg/kg/day (n = 10), H A L = exposure to haloperidol at 2.5 mg/kg/day (n = 9). * P < 0.05, ** P < 0.00l, *** P < 0.0001 c o m p a r e d to controls. Standard error bars represent the variance between subjects in a t r e a t m e n t group.
K 70'
60-
50
•
0.3"
T
O0
U+V
DZ1 0
DZ+RO CLON
PK
DZ+PK HAL
P R E N A T A L DRUG EXPOSURE
(P < 0.05). Following combined exposure to PKll195 and the high dose of D Z (2.5 mg/kg), the intracellular pH i was comparable to that observed in animals given D Z (2.5 mg/kg) alone, and this value was decreased significantly from control values (P < 0.001). Prenatal exposure to the dopaminergic antagonist haloperidol did not alter pHi measured in young adult rats. Alterations in PCr utilization [Fiep was most dramatically reduced from control values by prenatal exposure to the central-type antagonist RO15-1788 (P < 0.0001) (Fig. 4A). The high dose of D Z by itself had no effect on [FLp, and concurrent exposure to the high dose of D Z (2.5 mg/kg) did not prevent the effects of RO15-1788. H e n c e [F]cp in animals exposed in utero to this combination of drugs also differed from controls (P < 0.0001). Prenatal exposure to the lower dose of D Z (1.0 mg/kg) induced a significant decrease in [FLp relative to control values (P < 0.002). Neither the central-type agonist clonazepam nor the peripheral-type ligand PKl1195 had any effect on [FIfo measured in young adults. However, concurrent in utero exposure to PKll195 and to D Z (2.5 mg/kg) induced a significant decrease in [Fiep (P < 0.01). Combined exposure to DZ and the peripheral ligand had the apparent effect of
Fig. 4. Changes in A: [Fie p, B: [Plt and C: ATP" at 3 m o n t h s of age following prenatal drug exposure over gestation days 14-20. Exposure groups, levels of significance and sample size are as indicated in Fig. 3. [Flop, [Pit and ATP" are defined in Eqs. 2,3 and 4 respectively.
shifting the response of the that produced by the lower ergic antagonist haloperidol tine utilization from control
higher dose of D Z towards dose of DZ. The dopamindid not alter phosphocreavalues.
Alterations in [P]t [P]t was not altered by prenatal exposure to any drug (Fig. 4B). The flATP peak was therefore normalized to
[P],. Alterations in ATP" The ratio of flATP t o [P]t (i.e. ATP') did not change with prenatal exposure to any of the ligands specific to the BDZ receptor (Fig: 4C). Haloperidol also did not alter ATP" from controls. Blood gas analysis There were no differences in blood pH, paO2, paCO2 or bicarbonate between groups. The arterial blood pH of
89 all groups was in the normal range (7.35-7.45). All groups, including controls, were slightly hypercarbic: a condition attributable to the anesthetic agents used. The paO2s were 70-80 mm Hg, indicating a greater than 90% saturation of haemoglobin at physiologic pH, whilst pCO2s were 40-50 mm Hg. Bicarbonate levels were 22-27 mEq/l. DISCUSSION Exposure to BDZ ligands during the last week of gestation induced significant changes in pHi and PCr utilization measured in young adults. These results provide further evidence that early developmental exposure to these compounds can exert a long-term influence on cellular metabolism. The significant decrease in pH i measured following in utero drug exposure, while not of a magnitude that might endanger cell survival, could nevertheless be indicative of several changes in cell function. Changes in pH i may reflect an alteration in the proton generating capacities of a variety of cellular processes. In line with Mitchell's chemiosmotic hypothesis, the importance of proton gradients in coupling mitochondrial respiration with ATP synthesis has been well established 49. Cellular pH is altered with synaptic activity26"27, as well as by specific GABAergic antagonists 39'4°'s5. In addition, the buffering capacity of the creatine kinase reaction is pH-dependent s . The induction of decreased pH i appeared relatively selective to actions at BDZ binding sites, since exposure to the butyrophenone haloperidol had no effect on phi. Furthermore, the induction of changes in phi by the BDZ ligands appeared to be mediated primarily by in utero action of drugs at the central-type BDZ receptor as indicated by the following evidence. (1) Exposure to both doses of DZ induced a decrease in pH i. (2) Exposure to the central-type agonist clonazepam also led to a decrease in pH i. The less pronounced effect of clonazepam may relate to the lesser efficacy of this drug than of DZ at the BDZ receptor 6. (3) Exposure to the central-type BDZ receptor antagonist (RO15-1788) partially prevented the effects of exposure to DZ even though its half-life of bioavailability is much shorter than that of D Z 32. In contrast, the peripheral-type ligand PKll195 produced a very small (though statistically significant) decrease in pH i indicating that interactions of BDZs at the peripheral-type site may have a less profound influence on pH i. 3~p-NMR also provides an index of 'energy state' or phosphocreatine utilization s'42. Brain creatine (Cr) buffers ATP levels via the creatine kinase [EC 2.7.3.2] reaction. In the brain, under resting conditions, the
reaction catalyzed by creatine kinase is close to equilibrium H'34"42, and is very much faster than ATP utilization 42. Since ATP levels are well buffered by PCr s'42, under steady state energy consumption, alterations in P~ reflect net hydrolysis of PCr and not ATP. Changes in phosphocreatine utilization have been observed with defects in mitochondrial metabolism and with other states of tissue activation ~9"21"3941"43, The availability of the mobile high energy phophates ([Pit) or ATP (ATP') in brains of 3-month-old animals was not altered by prenatal exposure to any drug. This result is consistent with evidence indicating that under most conditions, in the brain, ATP is adequately buffered by PCr. The net steady state loss of P~ is therefore from PCr and not from ATP (since ATP" remains constant). Prenatal exposure to the central antagonist (RO15-1788) alone clearly altered phosphocreatine utilization ([Flop). The effect of RO15-1788 was not attenuated by concurrent prenatal exposure to DZ at 2.5 mg/kg even though the affinities of these drugs for the central-type receptor are virtually similar 22'32, and the half-life of RO15-1788 is much shorter than that of D Z 32. Since ATP availability was not altered, early exposure to RO15-1788 may actually have altered steady state buffering of ATP by PCr, thereby leading to increased utilization of PCr to maintain constant ATP levels. The dramatic consequence of prenatal exposure to the central-type antagonist (RO15-1788) on [Flop may be consistent with the suggestion that this drug is not just an antagonist, but may have intrinsic actions of its o w n 14. An alternative possibility that must be entertained, however, is that RO15-1788 produced its effects by displacing specific endogenous ligands from the centraltype receptor, thereby interfering with cellular function during a critical developmental stage. A variety of putative BDZ receptor-specific ligands have so far been described in the literature T M . However, it is not yet clear what role any of these endogenous ligands might play in brain development, nor is it clear what role the antagonist RO15-1788 might have in the modification of the action of these endogenous ligands. A third possibility to be considered is that the action of RO15-1788 was not mediated via specific receptor action, though one is hard pressed to explain a possible non-receptor mediated mechanism that might account for an action 3 months after administration of the drug, when the drug is no longer present. Since, in the same animal, RO15-1788 prevented the effects of DZ on pH., without having any effect by itself, the action of this drug on PCr utilization may be interpreted to be receptor-mediated. Exposure to the low dose of DZ (1.0 mg/kg) also altered PCr utilization in young adults whereas exposure to the higher dose (2.5 mg/kg) was ineffective when given
90 alone. The peripheral type ligand PKll195, while nol inducing any alterations in PCr utilization when administered alone, did permit changes leading to a decrease in the ratio, when administered concurrently with the high dose of D Z (2.5mg/kg). The peripheral type ligand thus appeared to convert the effect of a higher dose of D Z to that of a lower dose, implicating some sort of antagonistic action between these drugs. The inverse dose-response relationship of D Z observed in this case is consistent with previous research in our laboratory demonstrating inverse dose-response relationships in the alteration of cellular cytotoxic aldehyde content 3J, as well as in behavioral measures 23'24. The effects of the B D Z ligands do not appear to be related to sedative effects of the drugs (during exposure) since exposure to haloperidol, a potent tranquilizer, had no effect on pH~ or IF]q,. The alteration in PCr utilization by in utero exposure to D Z (1.0 mg/kg) was not clearly related to an action of the drugs during early development at one particular class of B D Z recognition sites. Rather, an alteration in the balance between the activity of different sites seems critical. The effect of exposure to the central antagonist alone clearly seems to implicate the central-type site. But the ability of the peripheral-type ligand to convert an ineffective dose of D Z to an effective dose implicates the peripheral receptor also. The fact that exposure to a given B D Z influences pHi and PCr utilization in different ways in the same animal points to separate mechanisms regulating these two measures. In addition, the lack of effect of haloperidol in this study, but the profound effect such exposure had on levels of cytotoxic aldehydes in the brain 3j, further supports the receptor specificity of the regulation of cellular metabolism. The need to anesthetize the rats in order to obtain phosphate spectra could be considered a confounding factor in the interpretation of the results; however, that it was seems unlikely. The anesthetics were selected specifically because they do not alter brain oxygen consumption l°. While ketamine may interact at the N-methyl-D-aspartate receptor 5~, in this study, the dose of ketamine (12.5 mg/kg) used in combination with xylazine was considerably lower than the anesthetic dose generally used (50-80 mg/kg), Any anesthetic may be expected to have some depressant effect on brain metabolism. Our observations suggest, however, that the combination of drugs used to achieve anesthesia had little observable effect as compared, e.g., to the effect of pentobarbital, which markedly altered phosphate metabolism (unpublished observations). Additionally, many volatile anesthetics may act directly at the B D Z - G A B A receptor 35. Furthermore, trying to collect spectra from restrained, unanesthetized rats could have more con-
founding problems than the use of anesthetics, because of interference from stress responses. Another possible confounding factor to interpretation of the results may be drug induced changes in nuclear spin-lattice relaxation mechanisms (Tj). Alterations in Tl may contribute to changes in relative peak areas in NMR spectra acquired under partially saturated conditions (recycle time < 5 * TO is. However, in the present study, alterations in phosphocreatine utilization reflected decreases in PCr paralleled by increases in P, without any changes in ATP'. It is unlikely that T~ times would be altered in opposite directions for PCr and Pi by exactly the same amount, without any change in the T~ for ATE The present study has demonstrated, therefore, using an in vivo measure of energy metabolism, that prenatal exposure to specific BDZs altered intracellular pH as well as phosphocreatine utilization. The specificity and reversibility of the drug actions argues against nonspecific, non-receptor mediated mechanisms. Furthermore, these drugs may compete with one or more of the recently described endogenous ligands, thus modifying the normal developmental role of these endogenous ligands. It has been suggested that G A B A and the BDZs may have a t r o p h i c function during development that is different from their transmitter function in mature tissue 2~. Recent evidence indicates that the BDZs inhibit cell proliferation 53, neurite outgrowth ~3, and stimulate the expression of an incogene product related to the control of cell proliferation and survival ~). It is very likely, therefore, that prenatal exposure to the BDZs may result in enduring changes in cell function. Current research in our laboratory indicates that acute exposure of naive young adult rats to different doses of D Z produces a dose related increase in pHi without changes in any other measure of energy metabolism, indicating that the effects induced by the BDZs during late gestation on adult cellular metabolism may be very different from their acute effects. Thus, the alteration of brain intracellular 31P-NMR spectra following in utero exposure to BDZs suggests that during neural development these drugs affect the organization of cellular metabolism.
Acknowledgements. This work was supported by PHS Grant MH-31850 and by Research Scientist Development Award (MH00651) to C. K., both from the National Institute of Mental Health. Diazepam, RO15-1788 and clonazepam were gifts from Dr. Peter Sorter, Hoffman-LaRoche Inc., Nutley, NJ. PKll195 was a gift from Dr. G. LeFur, Pharmuka Laboratories, Gennevillers, France. Haloperidol was a gift from Dr. John Kleis, McNeil Laboratories Fort Washington, PA. The authors thank Dr. Robert Bryant, Dept. of Biophysics for the use of the NMR spectrometer, Dr. Richard K. Miller, Dept. of Obstetrics and Gynecology for the use of the blood gas analyzer, and Dr. Richard Connett, Dept. of Physiology for his valuable guidance.
91
REFERENCES 1 Alho, H., Costa, E., Ferrero, P., Fujimoto, M. and CosenzaMurphy, D., Diazepam binding inhibitor: a neuropeptide located in selected neuronal populations of rat brain, Science, 229 (1985) 179-182. 2 Anholt, R.R.H., Murphy, K.M.M., Mack, G.E. and Snyder, S.H., Peripheral-type benzodiazepine receptors in the central nervous system: localization to olfactory nerves, J. Neurosci., 4 (1984) 593-603. 3 Anholt, R.R.H., DeSouza, E.B., Oster-Granite, M.L. and Snyder, S.H., Peripheral-type benzodiazepine receptors: autoradiographic localization in whole-body sections of neonatal rats, J. Pharmacol. Exp. Ther., 233 (1985) 517-526. 4 Anholt, R.R.H., Peterson, P.L., DeSouza, E.B. and Snyder, S.H., The peripheral benzodiazepine receptor: localization to mitochondrial membrane, J. Biol. Chem., 261 (1986) 576-583. 5 Braestrup, C. and Nielsen, M., Ontogenetic development of benzodiazepine receptors in the rat brain, Brain Research, 147 (1978) 170-173. 6 Chan, C.Y. and Farb, D.H., Modulation of neurotransmitter action: control of the y-aminobutyric acid response through the benzodiazepine receptor, J. Neuroseience, 5 (1985) 2365-2373. 7 Chisholm, J., Kellogg, C. and Lippa, A., Development of benzodiazepine binding subtypes in three regions of rat brain, Brain Research, 267 (1983) 388-39l. 8 Connett, R.J., Analysis of metabolic control: new insights using a scaled Creatine Kinase model, Am. J. Physiol., 254 (1988) R949-R959. 9 Curran, T. and Morgan, J.l., Superinduction of c-fos by nerve growth factor in the presence of peripherally active benzodiazepines, Science, 229 (1985) 1265-1268. 10 Dawson, B., Michenfelder, J.D. and Theye, R.A., Effects of ketamine on canine cerebral blood flow and metabolism, Anesth. Analg., 50 (1971) 443-447. 11 Degani, H., Alger, J.R., Shulman, R.G., Petroff, O.A.C. and Prichard, J.W., 31p magnetization transfer studies of Creatine Kinase kinetics in living rabbit brain, Magn. Reson. Med., 5 (1987) 1-12. 12 Doble, A., Malgouris, C., Daniel, M., Daniel, N., Imbault, F., Basbaum, A., Uzan, A., Gueremy, C. and LeFur, G., Labelling of peripheral-type benzodiazepine binding sites in human brain with [3H]PKll195: anatomical and subcellular distribution, Brain Res. Bull., 18 (1987) 49-61. 13 Ferrero, P., Guidotti, A., Conti-Tronconi, B. and Costa, E., A brain octadecaneuropeptide generated by tryptic digestion of DBI (diazepam binding inhibitor) functions as a proconflict ligand of benzodiazepine recognition sites, Neuropharmacology, 23 (1984) 1359-1362. 14 File, S.E. and Pellow, S., Intrinsic actions of the benzodiazepine receptor antagonist RO15-1788, Psychopharmacology, 88 (1986) 1-11. 15 Fukushima, E. and Roeder, S.B.W,, Experimental pulse NMR: A Nuts and Bolts Approach, Addison-Wesley Publishing Co. Ma. (1981). 16 Gadian, D.G., Nuclear Magnetic Resonance and its Applications to Living Systems, Clarendon Press, Oxford, U.K., 1982. 17 Gey, K., Effect of benzodiazepines on carbohydrate metabolism in rat brain. In S. Grattini, E. Mussini and L.O. Randall (Eds.), The Benzodiazepines, Raven Press, NY, 1973, pp. 243-256. 18 Haefely, W., The biological basis of benzodiazepine actions, J. Psychoative Drugs, 15 (1983) 19-39. 19 Hayes, D.J., Hilton-Jones, D., Arnold, D.L., Galloway, G., Styles, P., Duncan, J. and Radda, G.K., A mitochondrial encephalomyopathy: a combined 31p magnetic resonance and biochemical investigation, J. Neurol. Sci., 71 (1985) 105-118. 20 Hebebrand, J., Friedl, W. and Propping, P., The concept of isoreceptors: application to the nicotinic acetylcholine receptor and the gamma-aminobutyric acidA/benzodiazepine receptor complex, J, Neural. Transm., 71 (1988) 1-9.
21 Hope, P.L., Costello, A.M. DeL., Cady, E.B., Delpy, D.T., Torts, P.S., Chu, A., Hamilton, P.A., Reynolds, E.O.R. and Wilkie, D.R., Cerebral energy metabolism studied with phophorus NMR spectroscopy in normal and birth-asphyxiated infants, Lancet, ii (1984) 366-370. 22 Hunkeler, W., Mohler, H., Pieri, L., Pole, P., Bonetti, E.P., Cumin, R., Schaffner, R. and Haefely, W., Selective antagonists of the benzodiazepines, Nature (Lond.), 290 (1981) 514-516. 23 Kellogg, C., Ison, J.R. and Miller, R.K., Prenatal diazepam exposure: effects on auditory temporal resolution in rats, Psychopharmacology, 79 (1983) 332-339. 24 Kellogg, C.K., Benzodiazepines: influence on the developing brain, Prog. Brain Res., 73 (1988) 207-228. 25 Kellogg, C.K. and Pleger, G., GABA-stimulated chloride uptake and enchancement by diazepam in synaptoneurosomes from rat brain during prenatal and postnatal development, Dev. Brain Res., 79 (1989) 87-95. 26 Kraig, P.R., Ferreira-Filho, C.R. and Nicholson, C., Alkaline and acid transients in cerebellar microenvironment, J. Neurophysiol.. 49 (1983) 831-850. 27 Krishtal, O.A., Osipchuk, Y.V., Shelest, T.N. and Smirnoff, S.V., Rapid extracellular pH transients related to synaptic transmission in rat hippocampal slices, Brain Research, 436 (1987) 352-356. 28 Madtes, P., Ontogeny of the G A B A receptor complex. In D.A. Redburn and A. Schousboe (Eds.), Neurotrophic Activity of G A B A during Development, Neurology and Neurobiology, Vol. 32, Alan R. Liss Inc., NY, 1987, pp. 161-187. 29 Marangos, P.J., Patel, J., Boulenger, J. and Clark-Rosenberg, R., Characterization of peripheral-type benzodiazepine binding sites in brain using [3H]RO5-4864, Mol. Pharmacol., 22 (1982) 26-32. 30 Miller, L.P., Mayer, S., Braun, L.D., Geigcr, P. and Oldendorf, W.H., The effect of pretreatment with pentobarbital on the extent of 14C incorporation from [U-14C]glucose into various rat brain glycolytic intermediates: relevance to regulation at hexokinase and phosphofructokinase, Neurochem. Res., 13 (1988) 377-382. 31 Miranda, R. and Kellogg, C.K., Alterations in brain cellular metabolism of rats following in utero exposure to diazepam, Neurosci. Abstr., 13 (1987) 958. 32 Mohler, H., Burkard, W.P., Keller, H.H., Richards,, J.G. and Haefely, W., Benzodiazepine antagonist RO15-1788 binding characteristics and interaction with drug induced changes in dopamine turnover in cerebellar cGMP levels, J. Neurochem., 37 (1981) 714-722. 33 Morgan, J.l., Johnson, M.D., Wang, J.K.T., Sonnenfeld, K.H. and Spector, S., Peripheral-type benzodiazepines influence ornithine decarboxylase levels and neurite outgrowth in pcl2 cells, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 5223-5226. 34 Morris, P.G., Feeney, J., Cox, D.W.G. and Bachelard, H.S., 3~P-saturation-transfer nuclear-magnetic-resonance measurements of phosphocreatine turnover in guinea-pig brain slices, Biochem. J., 227 (1985) 777-782. 35 Moody, E,S., Serzdak, P.D. and Skolnik, P., Modulation of the benzodiazepine/y-aminobutyric acid receptor chloride channel complex by inhalation anesthetics, J. Neurochem., 51 (1988) 1386-1393. 36 Nunnally, R.L., In vivo monitoring of metabolism with nuclear magnetic resonance spectroscopy, Semin. Nucl. Med., 8 (1983) 377-382. 37 Owen, F., Poulter, M., Waddington, J.L., Mashal, R.D. and Crow, T.J., [3H]RO5-4864 benzodiazepine binding in the kainate lesioned striatum and in temporal cortex of brains from patients with senile dementia of the Alzheimer type, Brain Research, 278 (1983) 373-375. 38 Pazos, A., Cymerman, U., Probst, A. and Palacios, J.M., "Peripheral' benzodiazepine binding sites in human brain and kidney: autoradiographic studies, Neurosci. Lett.. 66 (1986) 147-152.
92 39 Petroff, O.A.C., Prichard, J.W., Ogino, "1., Avison, M., Alger, J.R. and Shulman, R.G., Combined ~H and ~tp Nuclear Magnetic Resonance spectroscopic studies of bicuculline-induced seizures in vivo, Ann. Neurol., 20 (1986) 185-193. 40 Petroff, O.A.C., Prichard, J.W., Behar, K.L., Alger, J.R. and Shulman, R.G., In vivo phosphorus nuclear magnetic resonance spectroscopy in status epilepticus, Ann. Neurol., 16 (1984) 169-177. 41 Radda, G.K., Bore, P.J., Gadian, D.G., Ross, B.D., Styles, P.. Taylor, D.J. and Morgan-Hughes, J., 3~p NMR examination of two patients with NADH-CoQ reductase deficiency, Science, 295 (1982) 608-609. 42 Radda, G.K., Control of bioenergetics: from cells to man by phosphorus nuclear-magnetic-resonance spectroscopy, Biochem. Soc. Trans., 14 (1986) 517-525. 43 Radda, G.K. and Chien, S., Cellular biochemistry in animals and man observed by 31p NMR spectroscopy. In S. Chien and H. Chien (Eds.), N M R in Biology and Medicine., Raven Press, NY, 1986, pp. 217-240. 44 Sangameswaran, L. and DeBlass, A., Demonstration of benzodiazepine-like molecules in the mammalian brain with monoclonal antibodies to benzodiazepines, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 5560-5564. 45 Sangameswaran, L., Fales, H.M., Friedrich, P. and DeBlass, A.L., Purification of benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 9236-9240. 46 Sauter, A. and Rudin, M., Effects of calcium antagonists on high-energy phosphates in ischemic rat brain measured by 31p
NMR spectroscopy, Magn. Reson. Me~i.. 4 ([9~7) 1-~. 47 Schlumpf, M., Richards, J.G., Lichtensteiger, W. and Mohler, H., An autoradiographical study of the prenatal development of benzodiazepine binding sites in rat brain..I Neurosei., 3 (1983j 1478-1487. 48 Schoemaker, H., Morelli, M., Deshmukh, P. and Yamamura, H.I., [3H]RO5-4864 benzodiazepine binding in the kainatelesioned rat striatum and Huntington's diseased basal ganglia, Brain Research, 248 (1982) 396-401. 49 Stryer, L., Biochemistrv, 3rd edn., W.H. Freeman and Co., NY, (1988), pp. 410. 50 Tallman, J.E and Gallager, D.W., The GABA-ergic system: a locus of benzodiazepine action, Ann. Rev. Neuros'ci.. 8 (1985) 21-44. 51 Thomson, A.M., West, D.C. and Lodge, D., An N-methylaspartate receptor-mediated synapse in rat cerebral cortex: a site of action of ketamine?, Nature (Lond.), 313 (1985) 479-481. 52 Tucker, J.C., Benzodiazepines in the developing rat: a critical review, Neurosci. Biobehav. Rev., 9 (1985) 101-111. 53 Wang, J.K.T., Morgan, J.I. and Spector, S., Benzodiazepines that bind at peripheral sites inhibit cell proliferation. Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 753-756. 54 Young, R.L,, Albano, R.E, Charnecki, A.M. and Demesak, G., Effect of diazepam on regional levels of glucose and malate in the central nervous system, Fed. Proc., 28 (1969) 444. 55 Young, R.S.K., Osbakken, M.D., Briggs, R.W., Yagel, S.K., Rice, D.W. and Goldberg, S., 31p NMR study of cerebral metabolism during prolonged seizures in the neonatal dog, Anal. Neurol.. 18 (1985) 14-20.
85
BRES 15089
Early developmental exposure to benzodiazepine ligands alters brain 31p-NMR spectra in young adult rats Rajesh Miranda ~, Toni Ceckler 2, Ronnie Guillet ~ and Carol Kellogg 1 1Department of Psychology, Meliora Hall, University of Rochester, Rochester, NY 14627 and 2Department of Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642 (U. S. A.) (Accepted 6 June 1989) Key words: Diazepam; Clonazepam; PKll195; RO15-1788; Haloperidol; pH; Phosphocreatine utilization; Adenosine triphosphate
Alterations in brain high energy phosphate compounds, using 3~p-NMR (nuclear magnetic resonance) spectroscopy, were measured in vivo in young adult (3-4 months) rats following prenatal exposure to ligands acting specifically at benzodiazepine (BDZ) binding sites. The exposure induced a decrease in intraceUular pH that indicated a predominant interaction of the drugs in utero with central-type BDZ receptor sites. Late gestational exposure to BDZ ligands also induced changes in brain phosphocreatine (PCr) utilization. Exposure to the lowest dose of DZ (1.0 mg/kg) but not the higher dose (2.5 mg/kg) induced a significant change in PCr utilization. Exposure to the central-type BDZ receptor antagonist RO15-1788 alone clearly altered PCr utilization in adult offspring, and DZ (2.5 mg/kg) when administered concurrently was not able to preventthis effect. Though exposure to a peripheral-type ligand (PKIl195) had no effect by itself, it converted the effect of the high dose of DZ to that of the low dose. Together, these results indicate an interaction during development between the central and peripheral-type BDZ binding sites on organization and/or regulation of cellular energy metabolism. Normalized ATP levels were not changed by any prenatal treatment indicating adequate buffering of intracellular ATP by phosphocreatine. The dopaminergic antagonist haloperidol did not alter intracellular pH or any index of phosphate metabolism indicating a selective rceptor mediated role for BDZ ligands in influences on the long term organization of intracellular phosphate metabolism. INTRODUCTION Previous research has demonstrated that prenatal exposure to benzodiazepine ( B D Z ) compounds induces a variety of effects observable in the adult organism 24'53. These effects include long term alterations in cytotoxic aldehydes that are products of cellular oxidative metabolism 3x. Furthermore, the effects on metabolism do not appear to be related to in utero actions at characteristic central-type B D Z binding sites. To date, two major classes of B D Z recognition sites have been identified, The first is the central-type receptor, associated with the 7-aminobutyric acid ( G A B A ) chloride (CL-) channel 5° and considered to mediate the anti-anxiety and anti-convulsant effects of the BDZs. This site is characterized by its affinity for the B D Z clonazepam TM and the antagonist RO15-178818,22. Different subsets of the B D Z - G A B A channel complex may exist, as defined by selectivity for different B D Z ligands 2°. A n o t h e r major B D Z recognition site that has been identified is termed the peripheral-type receptor 3"3s. This receptor population is characterized by its affinity for the ligands Ro5-4864 and PKl11954'29 and is present in glia137"4s as well as neuronal elements 2 in the brain.
Recent evidence has linked the peripheral receptor subtype to mitochondria in brain and other tissues 12'29 and to mitochondrial outer membranes in the adrenal gland 4, implicating that receptor population in regulation of energy metabolism. Acute administration of drugs that act at the G A B A B D Z complex has revealed a differential role in intracellular energy metabolism for the different ligand recognition sites on the B D Z - G A B A - C L channel complex. The B D Z s have been reported to increase brain glucose uptake and transiently increase glycolysis as well 1v'54, while the barbiturates decrease glycolytic flux 3° and the utilization of high energy phosphates by the brain 46. Specific G A B A antagonists, however, increase the utilization of brain high energy phosphates and decrease cellular pH 39'4°'55. To study the impact of drug interaction at B D Z receptors during development on the long term control of cellular energy metabolism, changes in intracellular pH (pHi) and high energy phosphorus compounds were measured in vivo in adult rats following prenatal exposure to BDZs, using 3~p nuclear magnetic resonance (NMR) spectroscopy. Phosphate compounds measured by N M R represent that component of high energy
Correspondence: C. Kellogg, Department of Psychology, Meliora Hall, University of Rochester, Rochester, NY 14627, U.S.A.
86 p h o s p h a t e s that is freely a v a i l a b l e for cellular reactions. In the rat, B D Z and G A B A
r e c o g n i t i o n sites a p p e a r
d u r i n g late g e s t a t i o n 5'7~47. D u r i n g this p e r i o d of n e u r a l d e v e l o p m e n t , the d e v e l o p i n g B D Z - G A B A
system m a y
be sensitive to a l t e r a t i o n s in its c h e m i c a l e n v i r o n m e n t . R e c e n t w o r k has s h o w n that d i a z e p a m ( D Z ) can influence GABA-mediated
c h l o r i d e flux in fetal rat s y n a p t o -
n e u r o s o m a l p r e p a r a t i o n s 25, d e m o n s t r a t i n g an early effector r e s p o n s e to D Z action at r e l e v a n t sites. F u r t h e r m o r e , BDZ
receptors
(the
peripheral-type
especially)
may
r e g u l a t e s e v e r a l aspects o f n e u r a l d e v e l o p m e n t including cell p r o l i f e r a t i o n and n e u r i t e o u t g r o w t h 9'3353. T h e present
experiment
assessed
the
possibility
of e n d u r i n g
c h a n g e s in i n t r a c e l l u l a r m e t a b o l i s m f o l l o w i n g early dev e l o p m e n t a l e x p o s u r e to B D Z c o m p o u n d s such as D Z .
by shimming on the proton signal at 85.56 MHz. ' P signals were acquired at 34.635 MHz, under partially saturated conditions, using a 3 s recycle time and a 20 ~us pulse, producing maximum excitation approximately 5 mm away from the coil. The sampling region mainly included cortical tissue (Fig. l). The spectral bandwidth was +3012.04 Hz, Signals were acquired using quadrature detection and were averaged over 512 acquisitions. The signal was transformed using Fourier analysis following exponential apodization. The contributions to the spectrum from immobilized phosphates in the bone and of the membrane phospholipids were subtracted out using convolution difference techniques (line broadcnings of 10 Hz and 300 Hz). Peak areas were obtained using a program supplied by GE. A sample spectrum (A), the fitted spectrum (B 1), and the individual peaks (132) are shown in Fig. 2. From each treatment group, animals not tested in the magnet were randomly selected for blood gas analysis. Animals were injected with the same combination and dosage of anesthetics as was used during spectral acquisition, and their femoral arteries were catheterized. Samples of heparinized arterial blood were analyzed on an IL system 1302 pH/blood gas analyzer.
T h e r e c e p t o r specificity o f the effect of p r e n a t a l e x p o s u r e to D Z was also e x a m i n e d . In a d d i t i o n , as a c o n t r o l , the effect of p r e n a t a l e x p o s u r e to the b u t y r o p h e n o n e halop e r i d o l , a d o p a m i n e r g i c a n t a g o n i s t and t r a n q u i l i z e r , was also studied.
METHODS
Data analysis Chemical shifts were expressed relative to the phosphocreatine (PCr) peak in ppm according to convention (1 ppm = 34,635 Hz), with the PCr peak at 0 ppm. pH~ was determined from the chemical shift of inorganic phosphate (P~) from P C r 16'~6 according to the following equation 21: (d-3.27) pH i = 6.72 + log,) - -
(5.69-d)
Prenatal paradigm Female rats (Long-Evans Strain, Blue Spruce Farms, Altamount, N.Y.) were bred with in house stock. Pregnant rats were injected intraperitoneally, once daily, with one or a combination of two drugs, on days 14 to 20 of gestation. Day 0 was defined as the day on which the vaginal smear tested sperm positive. On the 13th day of gestation, animals were weighed and assigned to one of the drug exposure groups. Doses were computed based on the dam's weight on day 13 of gestation and held constant thereafter. Within 24 h after birth, all litters were culled to 10-12 pups. Animals were weaned at 28 days of age. Rats from two or more litters were used for each experimental condition and all animals were tested at 3-4 months of age. An approximately equal number of males and females was included in each group. Pregnant females were assigned to one of 4 groups: uninjected, vehicle injected (40% propylene glycol + 10% ethanol), or DZ injected (at 1.0 or 2.5 mg/kg/day). To evaluate the receptor specificity of the effects observed following exposure to DZ, other pregnant females were administered the central-type BDZ receptor antagonist RO15-1788 [ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo4H-imidazo(1,5-a)(1,4)benzodiazepine-3-carboxylate] (10 mg/kg/ day), either alone or in combination with DZ (2.5 mg/kg/day), the central-type BDZ receptor agonist clonazepam (1.0 mg/kg/day), the peripheral-type BDZ receptor ligand PK11195 [1-(2-chlorophenyl)N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide] (5.0 mg/ kg/day), either alone or in combination with DZ (2.5 mg/kg/day), or the dopaminergic antagonist haloperidol (2.5 mg/kg/day). Drug dosages were based on values used in previous experiments. For sample size of each group see the legend to Fig. 3. ~ P-NMR spectral acquisition Animals were anesthetized with ketamine (12.5 mg/kg) and xylazine (15 mg/kg), and placed in a foam restraint. Anesthetics were necessary in order to immobilize the animal for spectral acquisition. A 2 cm diameter, 2 turn, surface coil was taped to the head of the animal. Animals were placed within the bore of a 2.0 Tesla, Oxford Instruments superconducting magnet interfaced to a GE CSI II imaging/spectroscopy system (GE NMR Instruments, Fremont. CA). The homogeneity of the magnetic field was adjusted
(1)
where d is the chemical shift (in ppm) of P~ from PCr, measured from peak to peak. The PCr utilization ration ([Fl~p)4~ was defined as:
[FLp -
PCr
(2)
(PCr + Pt) The total energy phosphate pool [P]t was defined as the percentage of total observable energy phosphates that are a part of the total observable mobile phosphate poolS: [P]t =
Pi + PCr + 7ATP + flATP + aATP
(3)
ix] where IX] = total area under the curve attributable to all the observable mobile phosphates. The fl-phosphate peak of adenosine triphosphate (flATP) was normalized to [P]t, after [P]t was determined to be contant across conditions and the proportion was expressed as ATP'. ATP'-
flATP
[Pit
(4)
In all cases, the ratios are unitless. Data were analyzed using a commercial statistical package (SAS v5.16), Statistical analysis involved a General linear model analysis of variance to control for unbalanced experimental designs, followed by post-hoc t-tests (difference between least square means test using pre-planned comparisons to control for repeated testing). For purposes of statistical analysis, the uninjected animals and the animals prenatally exposed to vehicle were combined for the control group since no significant differences were observed between these groups (P values ranged from 0.35 to 0.80). Data was expressed as mean __+ S.E.M.
87
la
1t O O
2b
2a
Fig. 1. A proton magnetic resonance image of the rat head and brain showing coil placement over the rat brain in sagittal section (la) and coronal section (2a). White spots above the rat head indicate placement of a water filled tube above the surface coil. A tracing of the rat brain in sagittal (lb) and coronal (2b) section indicates the depth of effective signal acquisition beneath the plane of the coil. Arrow in lb indicates the level of the image in 2b. Abbreviations used: ob, olfactory bulbs: bo, bone; sk, skin; ctx, cortex; cb, cerebellum; sc. spinal cord; s, septum; cp, caudate-putamen; Lv, lateral ventricles; 3v, third ventricle.
RESULTS Alterations in p H i Prenatal exposure to both doses of D Z (1.0 and 2.5 'd'
8
w
o
I I0
,
i
.
6
, , i , j , , . | l | , l
, 0
-10
-20
P~
Fig. 2. Sample 3~P-NMR spectrum (A), with combined fitted peaks (B1) and individual fitted peaks (B2). 1 = Phosphomonoesters (PME), 2 = Inorganic phosphate (P~), 3 = Phosphodiesters (PDE), 4 = Phosphocreatine (PCr), 5 = yATP, 6 = aATE 7 = flATP, 'd' = chemical shift in "ppm' between PCr and Pi (see Eq. 1).
mg/kg) p r o d u c e d a significant decrease in intracellular p H (pHi) (P < 0.0006 and P < 0.0001, respectively) in the brains of young adults, c o m p a r e d to control values (Fig. 3). Exposure to the central-type B D Z rceptor antagonist RO15-1788 alone did not significantly affect pHi, but the antagonist partially p r e v e n t e d the effects of p r e n a t a l exposure to the higher dose of D Z (2.5 mg/kg) when a d m i n i s t e r e d concurrently with D Z . The p H i was higher in this group than in animals e x p o s e d only to D Z at 2.5 mg/kg ( P < 0.01). RO15-1788 was unable to reverse the effect of D Z completely, however, since the pH~ in animals exposed to D Z (2.5 mg/kg) plus RO15-1788 was lower than that in controls ( P < 0.03). Exposure to the central-type B D Z receptor agonist Clonazepam also induced a significant decrease in p H i from control values (P < 0.008), though the magnitude of the effect was less than that produced by either dose of D Z . Exposure to the p e r i p h e r a l - t y p e [igand P K l l 1 9 5 by itself led to a modest though significant decrease in p H i
88
0.88
714"
~/ //..-/
7.10" /~.J~/ 0,83
=..
,/////~
)
//~
H,
7.06-
f//, r//
i
078 t' U+V
DZ1.0
DZ2.5
FO
DZd:IO
CLON
PK
DZ+PK HAt
U+V
DZ1.0
DZ2.5
R~
DZ+RO CLON
PK
D Z + P K HAL
DZ25
FO
702
N
f;;;;
E 100' 90"
6.98 r' U+V
DZ1 0
DZ2.5
FO
DZ.-dql
CLQN
PK
DZ+PK
HAL 80"
PRENATAL DRUG EXPOSURE
Fig. 3. C h a n g e s in pH~ at 3 m o n t h s of age following in utero exposure over gestation days 14-20 to different drugs. U + V = u n i n j e c t e d + v e h i c l e injected controls (n = 8), D Z 1 . 0 = exposure to D Z at 1.0 mg/kg/day (n = 7), D Z 2 . 5 = exposure to D Z at 2.5 mg/kg/day (n = 8), R O = exposure to RO15-1788 at 10 mg/kg/day (n = 6), D Z + R O = exposure to D Z (2.5 mg/kg/day) + RO15-1788 at 10 mg/kg/day (n = 10), C L O N = exposure to clonazepam at 1.0 mg/kg/day (n = 8), PK = exposure to P K l l 1 9 5 at 5.0 mg/kg/day (n = 10), D Z + P K = exposure to D Z at 2.5 mg/kg/day + P K l l 1 9 5 at 5.0 mg/kg/day (n = 10), H A L = exposure to haloperidol at 2.5 mg/kg/day (n = 9). * P < 0.05, ** P < 0.00l, *** P < 0.0001 c o m p a r e d to controls. Standard error bars represent the variance between subjects in a t r e a t m e n t group.
K 70'
60-
50
•
0.3"
T
O0
U+V
DZ1 0
DZ+RO CLON
PK
DZ+PK HAL
P R E N A T A L DRUG EXPOSURE
(P < 0.05). Following combined exposure to PKll195 and the high dose of D Z (2.5 mg/kg), the intracellular pH i was comparable to that observed in animals given D Z (2.5 mg/kg) alone, and this value was decreased significantly from control values (P < 0.001). Prenatal exposure to the dopaminergic antagonist haloperidol did not alter pHi measured in young adult rats. Alterations in PCr utilization [Fiep was most dramatically reduced from control values by prenatal exposure to the central-type antagonist RO15-1788 (P < 0.0001) (Fig. 4A). The high dose of D Z by itself had no effect on [FLp, and concurrent exposure to the high dose of D Z (2.5 mg/kg) did not prevent the effects of RO15-1788. H e n c e [F]cp in animals exposed in utero to this combination of drugs also differed from controls (P < 0.0001). Prenatal exposure to the lower dose of D Z (1.0 mg/kg) induced a significant decrease in [FLp relative to control values (P < 0.002). Neither the central-type agonist clonazepam nor the peripheral-type ligand PKl1195 had any effect on [FIfo measured in young adults. However, concurrent in utero exposure to PKll195 and to D Z (2.5 mg/kg) induced a significant decrease in [Fiep (P < 0.01). Combined exposure to DZ and the peripheral ligand had the apparent effect of
Fig. 4. Changes in A: [Fie p, B: [Plt and C: ATP" at 3 m o n t h s of age following prenatal drug exposure over gestation days 14-20. Exposure groups, levels of significance and sample size are as indicated in Fig. 3. [Flop, [Pit and ATP" are defined in Eqs. 2,3 and 4 respectively.
shifting the response of the that produced by the lower ergic antagonist haloperidol tine utilization from control
higher dose of D Z towards dose of DZ. The dopamindid not alter phosphocreavalues.
Alterations in [P]t [P]t was not altered by prenatal exposure to any drug (Fig. 4B). The flATP peak was therefore normalized to
[P],. Alterations in ATP" The ratio of flATP t o [P]t (i.e. ATP') did not change with prenatal exposure to any of the ligands specific to the BDZ receptor (Fig: 4C). Haloperidol also did not alter ATP" from controls. Blood gas analysis There were no differences in blood pH, paO2, paCO2 or bicarbonate between groups. The arterial blood pH of
89 all groups was in the normal range (7.35-7.45). All groups, including controls, were slightly hypercarbic: a condition attributable to the anesthetic agents used. The paO2s were 70-80 mm Hg, indicating a greater than 90% saturation of haemoglobin at physiologic pH, whilst pCO2s were 40-50 mm Hg. Bicarbonate levels were 22-27 mEq/l. DISCUSSION Exposure to BDZ ligands during the last week of gestation induced significant changes in pHi and PCr utilization measured in young adults. These results provide further evidence that early developmental exposure to these compounds can exert a long-term influence on cellular metabolism. The significant decrease in pH i measured following in utero drug exposure, while not of a magnitude that might endanger cell survival, could nevertheless be indicative of several changes in cell function. Changes in pH i may reflect an alteration in the proton generating capacities of a variety of cellular processes. In line with Mitchell's chemiosmotic hypothesis, the importance of proton gradients in coupling mitochondrial respiration with ATP synthesis has been well established 49. Cellular pH is altered with synaptic activity26"27, as well as by specific GABAergic antagonists 39'4°'s5. In addition, the buffering capacity of the creatine kinase reaction is pH-dependent s . The induction of decreased pH i appeared relatively selective to actions at BDZ binding sites, since exposure to the butyrophenone haloperidol had no effect on phi. Furthermore, the induction of changes in phi by the BDZ ligands appeared to be mediated primarily by in utero action of drugs at the central-type BDZ receptor as indicated by the following evidence. (1) Exposure to both doses of DZ induced a decrease in pH i. (2) Exposure to the central-type agonist clonazepam also led to a decrease in pH i. The less pronounced effect of clonazepam may relate to the lesser efficacy of this drug than of DZ at the BDZ receptor 6. (3) Exposure to the central-type BDZ receptor antagonist (RO15-1788) partially prevented the effects of exposure to DZ even though its half-life of bioavailability is much shorter than that of D Z 32. In contrast, the peripheral-type ligand PKll195 produced a very small (though statistically significant) decrease in pH i indicating that interactions of BDZs at the peripheral-type site may have a less profound influence on pH i. 3~p-NMR also provides an index of 'energy state' or phosphocreatine utilization s'42. Brain creatine (Cr) buffers ATP levels via the creatine kinase [EC 2.7.3.2] reaction. In the brain, under resting conditions, the
reaction catalyzed by creatine kinase is close to equilibrium H'34"42, and is very much faster than ATP utilization 42. Since ATP levels are well buffered by PCr s'42, under steady state energy consumption, alterations in P~ reflect net hydrolysis of PCr and not ATP. Changes in phosphocreatine utilization have been observed with defects in mitochondrial metabolism and with other states of tissue activation ~9"21"3941"43, The availability of the mobile high energy phophates ([Pit) or ATP (ATP') in brains of 3-month-old animals was not altered by prenatal exposure to any drug. This result is consistent with evidence indicating that under most conditions, in the brain, ATP is adequately buffered by PCr. The net steady state loss of P~ is therefore from PCr and not from ATP (since ATP" remains constant). Prenatal exposure to the central antagonist (RO15-1788) alone clearly altered phosphocreatine utilization ([Flop). The effect of RO15-1788 was not attenuated by concurrent prenatal exposure to DZ at 2.5 mg/kg even though the affinities of these drugs for the central-type receptor are virtually similar 22'32, and the half-life of RO15-1788 is much shorter than that of D Z 32. Since ATP availability was not altered, early exposure to RO15-1788 may actually have altered steady state buffering of ATP by PCr, thereby leading to increased utilization of PCr to maintain constant ATP levels. The dramatic consequence of prenatal exposure to the central-type antagonist (RO15-1788) on [Flop may be consistent with the suggestion that this drug is not just an antagonist, but may have intrinsic actions of its o w n 14. An alternative possibility that must be entertained, however, is that RO15-1788 produced its effects by displacing specific endogenous ligands from the centraltype receptor, thereby interfering with cellular function during a critical developmental stage. A variety of putative BDZ receptor-specific ligands have so far been described in the literature T M . However, it is not yet clear what role any of these endogenous ligands might play in brain development, nor is it clear what role the antagonist RO15-1788 might have in the modification of the action of these endogenous ligands. A third possibility to be considered is that the action of RO15-1788 was not mediated via specific receptor action, though one is hard pressed to explain a possible non-receptor mediated mechanism that might account for an action 3 months after administration of the drug, when the drug is no longer present. Since, in the same animal, RO15-1788 prevented the effects of DZ on pH., without having any effect by itself, the action of this drug on PCr utilization may be interpreted to be receptor-mediated. Exposure to the low dose of DZ (1.0 mg/kg) also altered PCr utilization in young adults whereas exposure to the higher dose (2.5 mg/kg) was ineffective when given
90 alone. The peripheral type ligand PKll195, while nol inducing any alterations in PCr utilization when administered alone, did permit changes leading to a decrease in the ratio, when administered concurrently with the high dose of D Z (2.5mg/kg). The peripheral type ligand thus appeared to convert the effect of a higher dose of D Z to that of a lower dose, implicating some sort of antagonistic action between these drugs. The inverse dose-response relationship of D Z observed in this case is consistent with previous research in our laboratory demonstrating inverse dose-response relationships in the alteration of cellular cytotoxic aldehyde content 3J, as well as in behavioral measures 23'24. The effects of the B D Z ligands do not appear to be related to sedative effects of the drugs (during exposure) since exposure to haloperidol, a potent tranquilizer, had no effect on pH~ or IF]q,. The alteration in PCr utilization by in utero exposure to D Z (1.0 mg/kg) was not clearly related to an action of the drugs during early development at one particular class of B D Z recognition sites. Rather, an alteration in the balance between the activity of different sites seems critical. The effect of exposure to the central antagonist alone clearly seems to implicate the central-type site. But the ability of the peripheral-type ligand to convert an ineffective dose of D Z to an effective dose implicates the peripheral receptor also. The fact that exposure to a given B D Z influences pHi and PCr utilization in different ways in the same animal points to separate mechanisms regulating these two measures. In addition, the lack of effect of haloperidol in this study, but the profound effect such exposure had on levels of cytotoxic aldehydes in the brain 3j, further supports the receptor specificity of the regulation of cellular metabolism. The need to anesthetize the rats in order to obtain phosphate spectra could be considered a confounding factor in the interpretation of the results; however, that it was seems unlikely. The anesthetics were selected specifically because they do not alter brain oxygen consumption l°. While ketamine may interact at the N-methyl-D-aspartate receptor 5~, in this study, the dose of ketamine (12.5 mg/kg) used in combination with xylazine was considerably lower than the anesthetic dose generally used (50-80 mg/kg), Any anesthetic may be expected to have some depressant effect on brain metabolism. Our observations suggest, however, that the combination of drugs used to achieve anesthesia had little observable effect as compared, e.g., to the effect of pentobarbital, which markedly altered phosphate metabolism (unpublished observations). Additionally, many volatile anesthetics may act directly at the B D Z - G A B A receptor 35. Furthermore, trying to collect spectra from restrained, unanesthetized rats could have more con-
founding problems than the use of anesthetics, because of interference from stress responses. Another possible confounding factor to interpretation of the results may be drug induced changes in nuclear spin-lattice relaxation mechanisms (Tj). Alterations in Tl may contribute to changes in relative peak areas in NMR spectra acquired under partially saturated conditions (recycle time < 5 * TO is. However, in the present study, alterations in phosphocreatine utilization reflected decreases in PCr paralleled by increases in P, without any changes in ATP'. It is unlikely that T~ times would be altered in opposite directions for PCr and Pi by exactly the same amount, without any change in the T~ for ATE The present study has demonstrated, therefore, using an in vivo measure of energy metabolism, that prenatal exposure to specific BDZs altered intracellular pH as well as phosphocreatine utilization. The specificity and reversibility of the drug actions argues against nonspecific, non-receptor mediated mechanisms. Furthermore, these drugs may compete with one or more of the recently described endogenous ligands, thus modifying the normal developmental role of these endogenous ligands. It has been suggested that G A B A and the BDZs may have a t r o p h i c function during development that is different from their transmitter function in mature tissue 2~. Recent evidence indicates that the BDZs inhibit cell proliferation 53, neurite outgrowth ~3, and stimulate the expression of an incogene product related to the control of cell proliferation and survival ~). It is very likely, therefore, that prenatal exposure to the BDZs may result in enduring changes in cell function. Current research in our laboratory indicates that acute exposure of naive young adult rats to different doses of D Z produces a dose related increase in pHi without changes in any other measure of energy metabolism, indicating that the effects induced by the BDZs during late gestation on adult cellular metabolism may be very different from their acute effects. Thus, the alteration of brain intracellular 31P-NMR spectra following in utero exposure to BDZs suggests that during neural development these drugs affect the organization of cellular metabolism.
Acknowledgements. This work was supported by PHS Grant MH-31850 and by Research Scientist Development Award (MH00651) to C. K., both from the National Institute of Mental Health. Diazepam, RO15-1788 and clonazepam were gifts from Dr. Peter Sorter, Hoffman-LaRoche Inc., Nutley, NJ. PKll195 was a gift from Dr. G. LeFur, Pharmuka Laboratories, Gennevillers, France. Haloperidol was a gift from Dr. John Kleis, McNeil Laboratories Fort Washington, PA. The authors thank Dr. Robert Bryant, Dept. of Biophysics for the use of the NMR spectrometer, Dr. Richard K. Miller, Dept. of Obstetrics and Gynecology for the use of the blood gas analyzer, and Dr. Richard Connett, Dept. of Physiology for his valuable guidance.
91
REFERENCES 1 Alho, H., Costa, E., Ferrero, P., Fujimoto, M. and CosenzaMurphy, D., Diazepam binding inhibitor: a neuropeptide located in selected neuronal populations of rat brain, Science, 229 (1985) 179-182. 2 Anholt, R.R.H., Murphy, K.M.M., Mack, G.E. and Snyder, S.H., Peripheral-type benzodiazepine receptors in the central nervous system: localization to olfactory nerves, J. Neurosci., 4 (1984) 593-603. 3 Anholt, R.R.H., DeSouza, E.B., Oster-Granite, M.L. and Snyder, S.H., Peripheral-type benzodiazepine receptors: autoradiographic localization in whole-body sections of neonatal rats, J. Pharmacol. Exp. Ther., 233 (1985) 517-526. 4 Anholt, R.R.H., Peterson, P.L., DeSouza, E.B. and Snyder, S.H., The peripheral benzodiazepine receptor: localization to mitochondrial membrane, J. Biol. Chem., 261 (1986) 576-583. 5 Braestrup, C. and Nielsen, M., Ontogenetic development of benzodiazepine receptors in the rat brain, Brain Research, 147 (1978) 170-173. 6 Chan, C.Y. and Farb, D.H., Modulation of neurotransmitter action: control of the y-aminobutyric acid response through the benzodiazepine receptor, J. Neuroseience, 5 (1985) 2365-2373. 7 Chisholm, J., Kellogg, C. and Lippa, A., Development of benzodiazepine binding subtypes in three regions of rat brain, Brain Research, 267 (1983) 388-39l. 8 Connett, R.J., Analysis of metabolic control: new insights using a scaled Creatine Kinase model, Am. J. Physiol., 254 (1988) R949-R959. 9 Curran, T. and Morgan, J.l., Superinduction of c-fos by nerve growth factor in the presence of peripherally active benzodiazepines, Science, 229 (1985) 1265-1268. 10 Dawson, B., Michenfelder, J.D. and Theye, R.A., Effects of ketamine on canine cerebral blood flow and metabolism, Anesth. Analg., 50 (1971) 443-447. 11 Degani, H., Alger, J.R., Shulman, R.G., Petroff, O.A.C. and Prichard, J.W., 31p magnetization transfer studies of Creatine Kinase kinetics in living rabbit brain, Magn. Reson. Med., 5 (1987) 1-12. 12 Doble, A., Malgouris, C., Daniel, M., Daniel, N., Imbault, F., Basbaum, A., Uzan, A., Gueremy, C. and LeFur, G., Labelling of peripheral-type benzodiazepine binding sites in human brain with [3H]PKll195: anatomical and subcellular distribution, Brain Res. Bull., 18 (1987) 49-61. 13 Ferrero, P., Guidotti, A., Conti-Tronconi, B. and Costa, E., A brain octadecaneuropeptide generated by tryptic digestion of DBI (diazepam binding inhibitor) functions as a proconflict ligand of benzodiazepine recognition sites, Neuropharmacology, 23 (1984) 1359-1362. 14 File, S.E. and Pellow, S., Intrinsic actions of the benzodiazepine receptor antagonist RO15-1788, Psychopharmacology, 88 (1986) 1-11. 15 Fukushima, E. and Roeder, S.B.W,, Experimental pulse NMR: A Nuts and Bolts Approach, Addison-Wesley Publishing Co. Ma. (1981). 16 Gadian, D.G., Nuclear Magnetic Resonance and its Applications to Living Systems, Clarendon Press, Oxford, U.K., 1982. 17 Gey, K., Effect of benzodiazepines on carbohydrate metabolism in rat brain. In S. Grattini, E. Mussini and L.O. Randall (Eds.), The Benzodiazepines, Raven Press, NY, 1973, pp. 243-256. 18 Haefely, W., The biological basis of benzodiazepine actions, J. Psychoative Drugs, 15 (1983) 19-39. 19 Hayes, D.J., Hilton-Jones, D., Arnold, D.L., Galloway, G., Styles, P., Duncan, J. and Radda, G.K., A mitochondrial encephalomyopathy: a combined 31p magnetic resonance and biochemical investigation, J. Neurol. Sci., 71 (1985) 105-118. 20 Hebebrand, J., Friedl, W. and Propping, P., The concept of isoreceptors: application to the nicotinic acetylcholine receptor and the gamma-aminobutyric acidA/benzodiazepine receptor complex, J, Neural. Transm., 71 (1988) 1-9.
21 Hope, P.L., Costello, A.M. DeL., Cady, E.B., Delpy, D.T., Torts, P.S., Chu, A., Hamilton, P.A., Reynolds, E.O.R. and Wilkie, D.R., Cerebral energy metabolism studied with phophorus NMR spectroscopy in normal and birth-asphyxiated infants, Lancet, ii (1984) 366-370. 22 Hunkeler, W., Mohler, H., Pieri, L., Pole, P., Bonetti, E.P., Cumin, R., Schaffner, R. and Haefely, W., Selective antagonists of the benzodiazepines, Nature (Lond.), 290 (1981) 514-516. 23 Kellogg, C., Ison, J.R. and Miller, R.K., Prenatal diazepam exposure: effects on auditory temporal resolution in rats, Psychopharmacology, 79 (1983) 332-339. 24 Kellogg, C.K., Benzodiazepines: influence on the developing brain, Prog. Brain Res., 73 (1988) 207-228. 25 Kellogg, C.K. and Pleger, G., GABA-stimulated chloride uptake and enchancement by diazepam in synaptoneurosomes from rat brain during prenatal and postnatal development, Dev. Brain Res., 79 (1989) 87-95. 26 Kraig, P.R., Ferreira-Filho, C.R. and Nicholson, C., Alkaline and acid transients in cerebellar microenvironment, J. Neurophysiol.. 49 (1983) 831-850. 27 Krishtal, O.A., Osipchuk, Y.V., Shelest, T.N. and Smirnoff, S.V., Rapid extracellular pH transients related to synaptic transmission in rat hippocampal slices, Brain Research, 436 (1987) 352-356. 28 Madtes, P., Ontogeny of the G A B A receptor complex. In D.A. Redburn and A. Schousboe (Eds.), Neurotrophic Activity of G A B A during Development, Neurology and Neurobiology, Vol. 32, Alan R. Liss Inc., NY, 1987, pp. 161-187. 29 Marangos, P.J., Patel, J., Boulenger, J. and Clark-Rosenberg, R., Characterization of peripheral-type benzodiazepine binding sites in brain using [3H]RO5-4864, Mol. Pharmacol., 22 (1982) 26-32. 30 Miller, L.P., Mayer, S., Braun, L.D., Geigcr, P. and Oldendorf, W.H., The effect of pretreatment with pentobarbital on the extent of 14C incorporation from [U-14C]glucose into various rat brain glycolytic intermediates: relevance to regulation at hexokinase and phosphofructokinase, Neurochem. Res., 13 (1988) 377-382. 31 Miranda, R. and Kellogg, C.K., Alterations in brain cellular metabolism of rats following in utero exposure to diazepam, Neurosci. Abstr., 13 (1987) 958. 32 Mohler, H., Burkard, W.P., Keller, H.H., Richards,, J.G. and Haefely, W., Benzodiazepine antagonist RO15-1788 binding characteristics and interaction with drug induced changes in dopamine turnover in cerebellar cGMP levels, J. Neurochem., 37 (1981) 714-722. 33 Morgan, J.l., Johnson, M.D., Wang, J.K.T., Sonnenfeld, K.H. and Spector, S., Peripheral-type benzodiazepines influence ornithine decarboxylase levels and neurite outgrowth in pcl2 cells, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 5223-5226. 34 Morris, P.G., Feeney, J., Cox, D.W.G. and Bachelard, H.S., 3~P-saturation-transfer nuclear-magnetic-resonance measurements of phosphocreatine turnover in guinea-pig brain slices, Biochem. J., 227 (1985) 777-782. 35 Moody, E,S., Serzdak, P.D. and Skolnik, P., Modulation of the benzodiazepine/y-aminobutyric acid receptor chloride channel complex by inhalation anesthetics, J. Neurochem., 51 (1988) 1386-1393. 36 Nunnally, R.L., In vivo monitoring of metabolism with nuclear magnetic resonance spectroscopy, Semin. Nucl. Med., 8 (1983) 377-382. 37 Owen, F., Poulter, M., Waddington, J.L., Mashal, R.D. and Crow, T.J., [3H]RO5-4864 benzodiazepine binding in the kainate lesioned striatum and in temporal cortex of brains from patients with senile dementia of the Alzheimer type, Brain Research, 278 (1983) 373-375. 38 Pazos, A., Cymerman, U., Probst, A. and Palacios, J.M., "Peripheral' benzodiazepine binding sites in human brain and kidney: autoradiographic studies, Neurosci. Lett.. 66 (1986) 147-152.
92 39 Petroff, O.A.C., Prichard, J.W., Ogino, "1., Avison, M., Alger, J.R. and Shulman, R.G., Combined ~H and ~tp Nuclear Magnetic Resonance spectroscopic studies of bicuculline-induced seizures in vivo, Ann. Neurol., 20 (1986) 185-193. 40 Petroff, O.A.C., Prichard, J.W., Behar, K.L., Alger, J.R. and Shulman, R.G., In vivo phosphorus nuclear magnetic resonance spectroscopy in status epilepticus, Ann. Neurol., 16 (1984) 169-177. 41 Radda, G.K., Bore, P.J., Gadian, D.G., Ross, B.D., Styles, P.. Taylor, D.J. and Morgan-Hughes, J., 3~p NMR examination of two patients with NADH-CoQ reductase deficiency, Science, 295 (1982) 608-609. 42 Radda, G.K., Control of bioenergetics: from cells to man by phosphorus nuclear-magnetic-resonance spectroscopy, Biochem. Soc. Trans., 14 (1986) 517-525. 43 Radda, G.K. and Chien, S., Cellular biochemistry in animals and man observed by 31p NMR spectroscopy. In S. Chien and H. Chien (Eds.), N M R in Biology and Medicine., Raven Press, NY, 1986, pp. 217-240. 44 Sangameswaran, L. and DeBlass, A., Demonstration of benzodiazepine-like molecules in the mammalian brain with monoclonal antibodies to benzodiazepines, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 5560-5564. 45 Sangameswaran, L., Fales, H.M., Friedrich, P. and DeBlass, A.L., Purification of benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 9236-9240. 46 Sauter, A. and Rudin, M., Effects of calcium antagonists on high-energy phosphates in ischemic rat brain measured by 31p
NMR spectroscopy, Magn. Reson. Me~i.. 4 ([9~7) 1-~. 47 Schlumpf, M., Richards, J.G., Lichtensteiger, W. and Mohler, H., An autoradiographical study of the prenatal development of benzodiazepine binding sites in rat brain..I Neurosei., 3 (1983j 1478-1487. 48 Schoemaker, H., Morelli, M., Deshmukh, P. and Yamamura, H.I., [3H]RO5-4864 benzodiazepine binding in the kainatelesioned rat striatum and Huntington's diseased basal ganglia, Brain Research, 248 (1982) 396-401. 49 Stryer, L., Biochemistrv, 3rd edn., W.H. Freeman and Co., NY, (1988), pp. 410. 50 Tallman, J.E and Gallager, D.W., The GABA-ergic system: a locus of benzodiazepine action, Ann. Rev. Neuros'ci.. 8 (1985) 21-44. 51 Thomson, A.M., West, D.C. and Lodge, D., An N-methylaspartate receptor-mediated synapse in rat cerebral cortex: a site of action of ketamine?, Nature (Lond.), 313 (1985) 479-481. 52 Tucker, J.C., Benzodiazepines in the developing rat: a critical review, Neurosci. Biobehav. Rev., 9 (1985) 101-111. 53 Wang, J.K.T., Morgan, J.I. and Spector, S., Benzodiazepines that bind at peripheral sites inhibit cell proliferation. Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 753-756. 54 Young, R.L,, Albano, R.E, Charnecki, A.M. and Demesak, G., Effect of diazepam on regional levels of glucose and malate in the central nervous system, Fed. Proc., 28 (1969) 444. 55 Young, R.S.K., Osbakken, M.D., Briggs, R.W., Yagel, S.K., Rice, D.W. and Goldberg, S., 31p NMR study of cerebral metabolism during prolonged seizures in the neonatal dog, Anal. Neurol.. 18 (1985) 14-20.