Post-mortem studies of brain phosphatidylinositol

Post-mortem studies of brain phosphatidylinositol hydrolysis in depression and the effect of antidepressant treatment

A RT I C LE

International Journal of Neuropsychopharmacology (2000), 3, 109–115. Copyright # 2000 CINP

Moyra A. Coull1, Sandra Lowther1, Cornelius L. E. Katona2 and Roger W. Horton1 " Department of Pharmacology and Clinical Pharmacology, St. George’s Hospital Medical School, London, UK # Department of Psychiatry, University College London Medical School, London, UK

Abstract Basal, neurotransmitter and G protein-mediated [$H]PI hydrolysis was measured in the frontal cortex, temporal cortex, hippocampus and thalamus from suicides, with a firm retrospective diagnosis of depression, and individually matched controls. Suicides were divided into those who had been free of antidepressant drugs for at least 3 months and those in whom prescription of antidepressants was clearly documented. There were no significant differences in basal, GTPγS-, 5-HT- or ACPD-stimulated [$H]PI hydrolysis in either antidepressantfree or antidepressant-treated suicides and their respective controls in the four brain regions studied. This was also the case when suicides were divided into those dying by violent or non-violent means. The two main conclusions from this study are : (i) neurotransmitter- (5-HT and ACPD) stimulated [$H]PI hydrolysis was unaltered in depression, and (ii) antidepressant treatment did not modify PI-mediated signal transduction. Received 1 December 1999 ; Reviewed 26 January 2000 ; Revised 2 March 2000 ; Accepted 6 March 2000 Key words : Phosphatidylinositol hydrolysis, signal transduction, suicide, depression, post-mortem human brain, antidepressant drugs.

Introduction Research into the pathophysiology of affective disorders and the mechanism of action of antidepressant drugs has primarily focused on altered monoamine neurotransmission. However extensive studies have failed to prove conclusively abnormalities in neurotransmitter or postsynaptic receptor function. This has led to an examination of post-receptor signal transduction mechanisms. Two important signal transduction mechanisms in the brain are the cAMP system and the phosphoinositide (PI) system. In the PI system, agonist binding to the receptor induces activation of guanine nucleotide-binding (G) proteins to stimulate phospholipase C-mediated hydrolysis of PIs. Studies of blood platelets provide evidence of alterations in PI signalling in affective disorders. Increased 5-HT and adrenaline-stimulated PI hydrolysis has been reported in platelets of antidepressant-free depressed patients compared to controls (Mikuni et al., 1991 ; Mori et al., 1991). Antidepressants have also been shown to modify PI signalling in platelets (Pandey et al., 1991 ; Rehavi et al., 1993). While these studies imply that Address for correspondence : Professor R. W. Horton, Department of Pharmacology and Clinical Pharmacology, St. George’s Hospital Medical School, London SW17 0RE, UK. Tel. : 0208 725 5613 Fax : 0208 725 3581 E-mail : hortonr!sghms.ac.uk

depression is associated with overstimulation of the PI pathway in platelets, there are clearly limitations to the use of peripheral cells as models of central nervous system (CNS) function. More direct evidence of altered signal transduction in affective disorders comes from studies in post-mortem human brain tissue. In samples from a small group of subjects with bipolar affective disorder, GTPγS(guanosine-5h-O-(3-thio)triphosphate) stimulated [$H]PI hydrolysis was reduced by 50 % in the occipital cortex, but unaltered in the frontal and temporal cortex (Jope et al., 1996). Reduced GTPγS-stimulated [$H]PI hydrolysis has been also been reported in the frontal cortex (30 % lower) of depressed suicides, although the effects of five neurotransmitter agonists were unaltered (Pacheco et al., 1996). Lower PI-specific phospholipase C activity has also been demonstrated in the prefrontal cortex of teenage suicides (Pandey et al., 1999). While these studies provide evidence for a dysfunction in PI signalling in affective disorders, interpretation of data is hampered by small sample size and heterogeneity of psychiatric disorders, as well as inconsistency in brain areas studied. In addition, limited information on psychoactive drug treatment makes it impossible to differentiate the effects of treatment from those of the underlying illness. In the present study we have attempted to address some of these issues. We have measured basal, neuro-

110

M. A. Coull et al.

transmitter and G protein-mediated [$H]PI hydrolysis in the frontal cortex, temporal cortex, hippocampus and thalamus from suicides with a firm retrospective diagnosis of depression, and individually matched controls. This is the first study in depressed suicides to examine [$H]PI hydrolysis in subcortical regions. Suicides were divided into antidepressant-free and antidepressant-treated subjects to establish if there is impairment of the PI pathway in depression and whether antidepressants modify this pathway. [$H]PI hydrolysis was measured under basal conditions and by direct stimulation of the G protein (GTPγS) and receptor-coupled activation [5-HT and the metabotropic glutamate agonist ACPD (trans-1-aminocyclopentyl-1,3-dicarboxylic acid)]. All measurements were performed concurrently, to allow direct comparison of relative changes. We have chosen 5-HT because it is strongly implicated in depression and ACPD as a control marker because excitatory amino acids have not been shown to have a role in depression. Methods Subject selection Brain tissue was obtained at post-mortem examination from suicide deaths, as recorded at coroners’ inquests. Suicides were subjected to retrospective diagnosis by a psychiatrist (C.L.E.K.), using hospital and coroners’ records, plus interviews with the subject’s medical practitioner. Information on current and previous drug treatment was sought and blood samples obtained at

post-mortem were analysed for the presence of psychoactive drugs. Tissue was obtained in accordance with the Human Tissue Act (1961). Subjects in whom there was evidence of alcohol or other substance abuse, organic brain disease, or chronic physical illness were excluded. Twenty-six male and 13 female suicides were selected for study. The suicides were subdivided into two groups. (1) Antidepressant-free suicides. Twenty-five suicides with sufficiently well-documented medical evidence to allow a firm retrospective diagnosis of depression, classified within the groups defined by Beskow et al. (1976) as belonging to group 1 (endogenous depression) and group 2 (depressive syndromes). These subjects had not been prescribed antidepressant drugs within 3 months prior to death and none were found in blood samples taken at post-mortem. Causes of death were classified as violent (hanging, n l 12 ; jumping from height, n l 2 ; and self-inflicted stab wound, n l 1), or non-violent (drug overdosage, n l 6 ; carbon monoxide poisoning, n l 4). (2) Antidepressant-treated suicides. Fourteen suicides with the same firm retrospective diagnosis of depression as above, but in whom prescription of antidepressant drugs was clearly documented. Details of drug treatment and causes of death for suicides are presented in Table 1. Those antidepressant-treated suicides who died by methods other than antidepressant overdose had therapeutic levels of antidepressants in their blood at post-mortem. Brain tissue was also obtained for 26 male and 13 female control subjects who died suddenly from causes not involving the CNS, and were without documented

Table 1. Details of antidepressant treatment and cause of death for antidepressant-treated suicides

Drug treatment

Duration (wk)

Cause of death

Dothiepin, haloperidol Tranylcypromine Amitriptyline, diazepam Dothiepin Imipramine, lithium, diazepam Trimipramine, temazepam Amitriptyline Dothiepin Trimipramine, nitrazepam

4 6 20 38 40 52 52 52 52

Overdose with antidepressant drugs Dothiepin, haloperidol Tranylcypromine, amoxapine, temazepam Amitriptyline, aspirin Dothiepin Imipramine Trimipramine Amitriptyline Dothiepin, paracetamol Trimipramine, aspirin, nitrazepam

Mianserin Clomipramine, thioridazine, procyclidine Imipramine, flupenthixol

4 16 52

Overdose without antidepressant drugs Barbiturate, temazepam Dextropropoxyphene, paracetamol Aspirin

Amitriptyline, trifluoperazine, temazepam Lofepramine

11 12

Violent methods Hanging Hanging

Brain PI hydrolysis in depressed suicides

111

Table 2. Demographic details of antidepressant-free suicides and controls

Region Frontal cortex Controls Suicides Temporal cortex Controls Suicides Hippocampus Controls Suicides Thalamus Controls Suicides

n

Age (yr)

Gender

Post-mortem delay (h)

15 15

49.4p4.2 (20–79) 47.9p4.3 (17–77)

12M\3F 12M\3F

38.5p4.6 (6–72) 37.2p4.9 (15–70)

18 18

47.5p4.0 (20–79) 47.6p3.8 (17–77)

15M\3F 15M\3F

37.9p3.7 (6–62) 39.1p4.2 (15–70)

11 11

48.9p5.6 (20–79) 48.5p5.6 (17–77)

10M\1F 10M\1F

38.4p4.9 (6–59) 38.7p5.8 (19–70)

13 13

49.0p3.8 (21–79) 48.8p4.1 (17–77)

10M\3F 10M\3F

34.1p4.3 (16–72) 32.3p4.9 (15–70)

Data expressed as meansp... with range shown in parentheses, n l number of subjects. None of the comparisons between controls and suicides reached statistical significance (unpaired t test).

evidence of mental illness, use or abuse of psychoactive drugs or alcohol abuse. Control subjects were selected on the basis of individual matching for gender, age (59 % within 2 yr, 90 % within 5 yr) and post-mortem delay (PMD ; the delay between death and freezing of the brain at k80 mC) with the suicides. Controls matched to antidepressant-free suicides died from myocardial infarction (n l 18), road traffic accident (n l 2), accidental fall (n l 1), accidental carbon monoxide poisoning (n l 1), accidental drowning (n l 1), ruptured aneurysm (n l 1) and acute asthma (n l 1). Controls matched to antidepressant-treated suicides died from myocardial infarction (n l 11), accidental drowning (n l 1), road traffic accident (n l 1), and acute asthma (n l 1). Samples were coded and assayed blind to subject classification but were arranged such that suicides and controls were assayed concurrently. Tissue availability prevented the study of all brain regions in all subjects. Tissue collection, dissection and storage Tissue collection, dissection and storage were as previously described by Cheetham et al. (1988). The brain areas studied were the frontal cortex [Brodmann area (Ba) 10], temporal cortex (Ba 21\22), hippocampus and thalamus. Membrane preparation Membranes were prepared according to the method of Wallace and Claro (1993). An aliquot was removed for protein analysis using the method of Lowry et al. (1951).

[3H]PI hydrolysis assay The assay was based on the method of Jope et al. (1996) and was performed in quintuplicate. Stimulants (1 and 2.5 m 5-HT, 1 and 2 m ACPD, or 3 µ GTPγS) and reagents for the reaction buffer were prepared in advance and stored frozen in individual aliquots to minimize dayto-day variation.

Statistical analysis Agonist- (5-HT or ACPD) stimulated [$H]PI hydrolysis was performed in the presence of 3 µ GTPγS to maintain receptor-coupled G protein-mediated PLC activation. Values are expressed as nmol [$H]PI hydrolysed\mg protein;h. Differences between means were tested for statistical significance using paired Student’s t test with Bonferroni correction for multiple comparisons. Bonferroni correction was calculated by multiplying the observed p value from the significance tests by the number of t tests (k), any kp which exceeded 1 was classified as not significant. Any kp less than 0.05 was classified as significant at the 0.05 level (Bland, 1995). Relationships between basal and stimulated [$H]PI hydrolysis and age and post-mortem delay were examined by Pearson’s correlations on the combined suicide and control groups. All tests were performed using GraphPad Prism version 3.00 (GraphPad Software, San Diego, California, USA).

112

M. A. Coull et al. basis of violence of death, basal and stimulated [$H]PI hydrolysis did not differ between violent and non-violent suicides and their matched controls in any of the regions studied. There was a trend for basal and stimulated [$H]PI hydrolysis to be slightly lower in violent suicides compared to controls. However, this trend did not reach significance. As antidepressant-free suicides and controls did not differ, correlations between [$H]PI hydrolysis and age and post-mortem delay were examined in a combined suicide and control group. Significant correlations were found

Results Antidepressant-free suicides and controls Age and post-mortem delay did not differ significantly between antidepressant-free suicides and their matched controls (Table 2). Basal and stimulated [$H]PI hydrolysis did not differ between antidepressant-free suicides and controls in the frontal cortex, temporal cortex, hippocampus and thalamus (Table 3). When antidepressant-free suicides were divided on the

Table 3. Basal and stimulated [$H]PI hydrolysis in antidepressant-free suicides and controls


n

Basal

GTPγS

1 m 5-HT jGTPγS

2.5 m 5-HT jGTPγS

1 m ACPD jGTPγS

2 m ACPD jGTPγS

15 15

1.37p0.14 1.30p0.15

1.53p0.15 1.41p0.16

2.06p0.20 1.86p0.19

2.89p0.26 2.58p0.24

1.85p0.18 1.73p0.16

2.20p0.20 2.03p0.19

18 18

1.13p0.06 1.10p0.06

1.23p0.06 1.18p0.06

1.71p0.08 1.66p0.07

2.33p0.09 2.26p0.10

1.52p0.06 1.50p0.06

1.78p0.06 1.73p0.06

11 11

1.45p0.09 1.49p0.10

1.55p0.07 1.53p0.11

1.77p0.09 1.79p0.09

2.27p0.10 2.24p0.09

1.82p0.11 1.73p0.12

2.00p0.13 1.89p0.13

13 13

1.44p0.16 1.34p0.15

1.43p0.16 1.39p0.14

1.61p0.16 1.51p0.15

1.88p0.20 1.84p0.17

1.60p0.15 1.52p0.12

1.80p0.17 1.77p0.14

Data are meansp... (nmol [$H]PI hydrolysed\mg protein;h). None of the comparisons between controls and suicides reached statistical significance (paired t test with Bonferroni’s multiple comparison test). Table 4. Correlations between [$H]PI hydrolysis and age and post-mortem delay

Antidepressant-free suicides and controls (n l 36) Age

r

Region

Condition

Frontal cortex

1 m 5-HT 2.5 m 5-HT 2 m ACPD 1 m 5-HT 2.5 m 5-HT 1 m ACPD 2 m ACPD 1 m 5-HT 2.5 m 5-HT 1 m ACPD

k0.42* k0.45** k0.36* k0.59*** k0.38* k0.44** k0.62*** 0.48** 0.37* k0.45**

2.5 m 5-HT 2.5 m 5-HT

k0.53* k0.39*

Age

Temporal cortex

Post-mortem delay

Frontal cortex

Post-mortem delay Temporal cortex Antidepressant-treated suicides and controls (n l 26) Age Frontal cortex Age Temporal cortex

r, correlation coefficient. Significant correlations are denoted by * p ** p 0.01 ; *** p 0.001 (Pearson’s correlation).

0.05 ;

Brain PI hydrolysis in depressed suicides

113

Table 5. Demographic details of antidepressant-treated suicides and controls


n

Age (yr)

Gender

Post-mortem delay (h)

11 11

47.4p3.6 (27–66) 47.0p3.6 (27–62)

6M\5F 6M\5F

45.8p4.9 (26–72) 38.3p4.8 (12–70)

13 13

44.1p3.9 (18–66) 43.5p3.9 (16–62)

7M\6F 7M\6F

46.3p4.3 (26–72) 37.5p4.2 (12–70)

11 11

47.4p3.6 (27–66) 47.4p3.4 (27–62)

6M\5F 6M\5F

45.8p4.9 (26–72) 38.3p4.8 (12–70)

13 13

43.8p3.8 (18–66) 43.1p3.9 (16–62)

7M\6F 7M\6F

43.5p3.8 (26–63) 38.1p4.0 (16–70)

Data expressed as meansp... with range shown in parentheses, n l number of subjects. None of the comparisons between controls and suicides reached statistical significance (unpaired t test). Table 6. Basal and stimulated [$H]PI hydrolysis in antidepressant-treated depressed suicides and controls


n

Basal

GTPγS

1 m 5-HT jGTPγS

2.5 m 5-HT jGTPγS

1 m ACPD jGTPγS

2 m ACPD jGTPγS

11 11

1.38p0.11 1.31p0.10

1.47p0.12 1.38p0.11

2.13p0.16 1.94p0.16

3.50p0.27 3.36p0.25

1.79p0.13 1.76p0.13

2.30p0.16 2.18p0.17

13 13

1.27p0.08 1.25p0.07

1.48p0.10 1.42p0.08

2.06p0.16 1.94p0.10

3.28p0.29 3.11p0.15

1.74p0.12 1.67p0.09

2.35p0.18 2.15p0.11

11 11

1.21p0.04 1.25p0.09

1.27p0.05 1.37p0.12

1.60p0.09 1.54p0.11

2.33p0.13 2.44p0.24

1.51p0.07 1.63p0.10

1.96p0.08 2.10p0.19

13 13

1.24p0.11 1.19p0.09

1.46p0.17 1.24p0.09

1.61p0.17 1.40p0.11

2.15p0.26 1.94p0.19

1.64p0.19 1.42p0.11

2.03p0.26 1.72p0.12

Data are meansp... (nmol [$H]PI hydrolysed\mg protein;h). None of the comparisons between controls and suicides reached statistical significance (paired t test with Bonferroni’s multiple comparison test).

between [$H]PI hydrolysis and age and post-mortem delay in the frontal cortex and age and post-mortem delay in the temporal cortex (Table 4). No significant correlations were found in the hippocampus or thalamus. Antidepressant-treated suicides and controls Age and post-mortem delay did not differ significantly between antidepressant-treated suicides and their matched controls (Table 5). Basal and stimulated [$H]PI hydrolysis did not differ between antidepressant-treated suicides and controls in the frontal cortex, temporal cortex, hippocampus and

thalamus (Table 6). When only those subjects who had received antidepressant treatment for 11 wk or longer were considered, basal and stimulated [$H]PI hydrolysis again did not differ between suicides and controls in any of the regions studied. Correlations were examined between [$H]PI hydrolysis and age and post-mortem delay in the combined groups of antidepressant-treated suicides and controls (Table 4). Significant correlations were found between stimulated [$H]PI hydrolysis and age in the frontal cortex and temporal cortex. No significant correlations were found between [$H]PI hydrolysis and age or post-mortem delay in the hippocampus and thalamus.

114

M. A. Coull et al.

Discussion The most important finding of this study was the lack of difference in basal and stimulated [$H]PI hydrolysis in 4 brain regions of a group of carefully selected depressed suicides, free of psychoactive drugs and well-matched controls. This lack of difference is in broad agreement with the only comparable study to date (Pacheco et al., 1996). These authors examined 5-HT-, carbachol-, 2methylthioadenosine triphosphate- (2mATP), ACPD-, histamine- and fluoride-stimulated [$H]PI turnover in two cortical areas (Ba 10 and Ba 8\9) from retrospectively diagnosed depressed suicides and controls. They found no differences between suicides and controls. The results may be influenced by the method of suicide. This is traditionally examined by the division of suicides into those dying by violent and non-violent methods. However, no significant differences were found when suicides were divided on this basis. There was a trend for 5-HT-stimulated [$H]PI hydrolysis to be lower in the frontal cortex of violent suicides than in controls. A similar trend was also seen with ACPD stimulation. These findings are also compatible with those of Pacheco et al. (1996). The second most important finding of this study was that basal and stimulated [$H]PI turnover was not altered by antidepressants. This is the first study to examine the effects of antidepressants on [$H]PI turnover in postmortem tissue. Interpretation of the results needs to be treated with caution. The nature of the treatment was varied, though mostly involving tricyclic antidepressants. The duration of treatment was also variable, ranging from 4 wk to over 1 yr. It could be argued that treatment for as little as 4 wk may have been insufficient for maximum effects to develop. However, when the subjects were restricted to those that had received treatment for 11 wk or longer, again no differences were found between suicides and controls. The potential confounding effects of medications other than antidepressants cannot be excluded. Pacheco et al. (1996) reported significantly lower (by 30 %) GTPγS-stimulated [$H]PI hydrolysis in depressed suicides than controls in Ba 10. In contrast we found no differences in the frontal cortex or the other regions studied. Possible reasons for these different findings include age, post-mortem delay and drug treatment. Subjects in the present study were on average 10 yr younger than those studied by Pacheco et al. (1996), although in both studies subjects were well matched for age. The post-mortem delays were much shorter (16p2 h) in the study of Pacheco et al. (1996) than the present study (39p4 h). It is unlikely that these two factors account for the difference between the studies as

no significant correlations were found in the present study between age or post-mortem delay and GTPγS-stimulated [$H]PI hydrolysis. Pacheco et al. (1996) studied 7 subjects who had been receiving psychoactive medication (mostly antidepressants) prior to death. The present study was designed to specifically examine the effects of antidepressant treatment, and no effects on GTPγS stimulation were found. Therefore the difference is unlikely to be due to the inclusion of drug- treated subjects in the study of Pacheco et al. (1996). Thus the reason for the different findings in respect of GTPγS-stimulated [$H]PI turnover remains unclear. Consistent negative correlations were found in the present study between age and 5-HT-, and ACPDstimulated [$H]PI hydrolysis in cortical but not subcortical areas. These findings agree with those of Greenwood et al. (1995) who reported significant negative correlations between age (19–100 yr) and agonist- (5-HT and ACPD) stimulated [$H]PI hydrolysis in the prefrontal cortex from control subjects. The relationship between age and [$H]PI hydrolysis may in part be due to age-related changes in the numbers of receptors (e.g. see Lowther et al., 1994). Age-related changes in the concentration of Gαq/ may "" also contribute to the negative correlation between age and [$H]PI hydrolysis (Greenwood et al., 1995). It is unlikely that age-related effects will have influenced the interpretation of the results since our subjects were closely matched for age on an individual basis (90 % within 5 yr). In conclusion, the current study provides no support for the view that neurotransmitter (at least 5-HT acting via 5-HT receptors and glutamate acting by metabotropic # receptors) mediated PI hydrolysis is altered in depressed suicides. The present findings are in broad agreement with the only comparable study (Pacheco et al., 1996). The present study also offers no support for the view that antidepressant treatment modifies PI-mediated signal transduction. Acknowledgements We gratefully acknowledge a research award from the Theodore and Vada Stanley Foundation. M.A.C. acknowledges a Research Studentship from the Medical Research Council. References Beskow J, Gottfries CG, Roos BE, Winblad B (1976). Determination of monoamines and monoamine metabolites in the human brain : post-mortem studies in a group of suicides and in a control group. Acta Psychiatrica Scandinavica 53, 7–20.

Brain PI hydrolysis in depressed suicides Bland M (1995). An Introduction to Medical Statistics (pp. 144–146). Oxford : Oxford Medical Publications. Cheetham SC, Crompton MR, Katona CLE, Horton RW (1988). Brain 5-HT receptor binding sites in depressed # suicide victims. Brain Research 443, 272–280. Greenwood AF, Powers RE, Jope RS (1995). Phosphoinositide hydrolysis, Gαq, phospholipase C, and protein kinase C in post mortem human brain : effects of post mortem interval, subject age, and Alzheimer’s disease. Neuroscience 69, 125–138. Jope RS, Song L, Li PP, Young LT, Kish SJ, Pacheco MA, Warsh JJ (1996). The phosphoinositide signal transduction system is impaired in bipolar affective disorder. Journal of Neurochemistry 66, 2402–2409. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265–275. Lowther S, De Paermentier F, Crompton MR, Katona CLE, Horton RW (1994). Brain 5-HT receptors in suicide # victims : violence of death, depression and effects of antidepressant treatment. Brain Research 642, 281–289. Mikuni M, Kusumi I, Kagaya A, Kuroda Y, Mori H, Takahashi K (1991). Increased 5-HT-2 receptor function as measured by serotonin-stimulated phosphoinositide hydrolysis in platelets of depressed patients. Progress in NeuroPsychopharmacology and Biological Psychiatry 15, 49–61.

115

Mori H, Koyama T, Yamashita I (1991). Platelet alpha-2 adrenergic receptor-mediated phosphoinositide responses in endogenous depression. Life Sciences 48, 741–748. Pacheco MA, Stockmeier C, Meltzer HY, Overholser JC, Dilley GE, Jope RS (1996). Alterations in phosphoinositide signaling and G-protein levels in depressed suicide brain. Brain Research 723, 37–45. Pandey SC, Davis JM, Schwertz DW, Pandey GN (1991). Effect of antidepressants and neuroleptics on phosphoinositide metabolism in human platelets. Journal of Pharmacological and Experimental Therapeutics 256, 1010–1018. Pandey GN, Dwivedi Y, Pandey SC, Teas SS, Conley RR, Roberts RC, Tamminga CA (1999). Low phosphoinositidespecific phospholipase C activity and expression of prospholipase C β protein in the prefrontal cortex of " teenage suicide subjects. American Journal of Psychiatry 156, 1895–1901. Rehavi M, Jerushalemi Z, Aviv A, Laor N, Podliszeski E, Karp L, Shavit S, Weizman R (1993). Interaction between antidepressants and phosphoinositide signal transduction system in human platelets. Biological Psychiatry 33, 40–44. Wallace MA, Claro E (1993). Transmembrane signaling through phospholipase C in human cortical membranes. Neurochemical Research 18, 139–145.