Pharmacology Biochemistry & Behavior, Vol. 23, pp. 153-159, 1985. Ankho International Inc. Printed in the U.S.A. 0091-3057/85 $3.00 + .00
Involvement of Brain Histamine in A 9-Tetrahydrocannabinol Tolerance
A. J. M. VERBERNE, t M. R. FENNESSY,* S. J. LEWISt AND D. A. TAYLOR$
*Department of Pharmacology, University of Melbourne, Parkville, Victoria, 3052, Australia tClinical Pharmacology and Therapeutics Unit, Department of Medicine, Austin Hospital
Heidelberg, 3084, Australia $School of Pharmacology, Victorian College of Pharmacy, Parkville, Victoria, 3052, Australia
Rece ived 29 October 1984
VERBERNE, A. J. M., M. R. FENNESSY, S. J. LEWIS AND D. A. TAYLOR. Involvement of brain histamine in Ag-tetrahydrocannabinol tolerance and withdrawal. PHARMACOL BIOCHEM BEHAV 23(2) 153-159, 1985.--The in- volvement of brain histamine (HA) in Ag-tetrahydrocannabinol (A9-THC) tolerance and dependence was studied in rats. Rats treated for 5 days with Ag-THC (2-6 mg/kg, IV) developed tolerance to the hypothermic effects of the drug. Tolerance also developed over the 5 day period to the decrease in brain regional HA concentrations observed after an acute injection of Aa-THC. Administration of the tricyclic antidepressant drug clomipramine hydrochloride to tolerant rats induced a withdrawal-like behavioural syndrome. Accompanying this behaviour was a fall in HA concentrations of the midbrain, cortex, medulla oblongata/pons and the cerebellum. Administration of Ag-THC, but not of the Aa-THC vehicle, prior to clomipramine challenge attenuated both the intensity of the withdrawal-like syndrome and the reductions in brain regional HA concentration.
Aa-Tetrahydrocannabinol Histamine Tolerance Withdrawal-like behaviour
TOLERANCE develops to many of the pharmacological ef- fects of tetrahydrocannabinols (THCs) in most species studied, including man . The degree of tolerance depends on the frequency of administration and the magnitude of the dose administered [13,20] and it appears to occur through a cellular, rather than a metabolic, mechanism. A recent de- tailed investigation has demonstrated that no major altera- tions in absorption, disposition and elimination accompany the development of tolerance to A9-tetrahydrocannabinol (Aa-THC) in man .
In rats made tolerant to Ag-THC, clomipramine and other biogenic amine reuptake inhibitors induced a characteristic pattern of behaviour, a 'withdrawal-like' syndrome [15,19], the intensity of which is dependent on the degree of tolerance . Since more recent studies have shown that brain histamine (HA) may mediate some of the pharmacolog- ical effects of Aa-THC, e.g., hypothermia [3,4], it was of interest to investigate whether tolerance also develops to the Ag-THC-induced reductions in brain HA and whether this amine is involved in the 'withdrawal-like' behaviour ob- served after clomipramine challenge.
and standard laboratory food were freely available. All ex- periments were performed in a laboratory with an ambient temperature of 21 +2C and 12 hr light-dark cycle. In total, 86 rats were used and individual group sizes are indicated in Table 1.
Permanently indwelling cannula were implanted into the external jugular veins of individually-caged rats according to a previously described method . Animals were allowed 48 hr to recover from surgery before being randomly divided into two main groups which received twice daily intravenous (IV) injections of either Ag-THC or the vehicle, polyvinyl- pyrrolidone (PVP), for up to 5 days. Doses of Ag-THC, which increased from 2 mg/kg on day 1 to 6 mg/kg on day 5, were administered according to the following dosage schedule:
METHOD Day a.m. p.m.
Animals 1 40 40 2-4 80 80
Male albino Wistar rats (220-300 g) were used and water 5 80 120
2 2 4 4 4 6
~The pyran numbering system for tetrahydrocannabinol was used. Ag-tetrahydrocannabinol is equivalent to Al-tetrahydrocannabinol using the monoterpenoid numbering system.
154 VERBERNE, FENNESSY, LEWIS AND TAYLOR
TABLE 1 EXPERIMENTAL PROCEDURE FOR THE TWO MAIN GROUPS OF RATS
Groups of Rats Receiving Chronic Drug Treatment Decapitation
No, of Days No. of Time after Injection of PVP A~'-THC of Treatment Injections last injection Clomipramine
A(5) I (5) 4 7 12hr B(5) J (5) 4 8 30min C(5) K (5) 5 9 12 hr D(5) L (5) 5 10 30min
*E (6) M (6) 6 10(+ saline) 12.5 hr - - F (6) N (6) 6 10(+ PVP)* 30 min ~/ G (6) P (6) 6 10(+ A~-THC)* 30 min ~/
H (5) Q (5) These groups were used for the body temperature studies
The rats which received chronic PVP treatment were divided into 8 sub-groups and are desig- nated as groups A to H. Animals receiving chronic A~-THC treatment were divided similarly and are designated as groups I to Q. The numbers in parentheses indicate the number of animals per group.
*Groups E and M are acting as controls for the groups F, N, G and P which received clomi- pramine HCI.
All rats were injected twice daily with either PVP or A 9- THC. Table 1 outlines the experimental protocol for groups of rats, designated as groups A to Q. Rats in groups A to H were injected with PVP as above, whereas groups ! to Q received injections of Ag-THC.
Body temperature. In groups H and Q (5 rats per group), body temperature was recorded for 2.5 hr after each morning injection of PVP or Aa-THC. Body temperature was re- corded every 30 min by means of a thermistor probe inserted 6 cm into the rectum.
Brain HA concentration. Groups A-D (PVP treatment) and groups I -L (Ag-THC treatment) were used for the brain HA concentration studies. There were 5 rats per group. The rats in groups A and I were decapitated at the time when their next injection was due, i.e., 12 hr after the 7th injection, on the evening of day 4. Those in groups B and J were decapitated 30 min after receiving the evening injection on day 4. The same procedure was followed on day 5 using the rats in groups C and K and D and L, respectively. Following decapitation, the brains were removed and dissected into various brain regions  and assayed for their HA content .
Withdrawal Attenuation Studies
Behaviour. On day 6, rats in groups F and N (6 rats per group) were injected with PVP (120 mg/kg, IV), whereas those in groups G and P (6 rats per group) were injected with Ag-THC (6 mg/kg, IV). These drugs were administered 30 min before the challenging injection of clomipramine HC1 (15 mg/kg, IP). The behaviour of these rats was observed for 30
min following clomipramine administration as previously de- scribed . The number of writhes, backward kicks of the hind legs, jumps, head shakes and episodes of paw tremor was counted by experienced observers who were unaware of which treatment each rat had received.
Brain regional HA concentrations. At the end of the above 30 min observation period, the rats in groups F and N, and groups G and P were decapitated. Animals in groups E and M were decapitated on the morning of day 6 when the next injection of PVP or Ag-THC, respectively, was due (i.e., 12.5 hr after the 10th injection). Again, all brains were re- moved, dissected into various brain regions and assayed for their HA content .
Drugs and Chemicals
(-)-Trans-Ag-THC (batch N780401A, NIDA, Bethesda, MD) was suspended in normal saline (0.9% NaCI w/v) using PVP (Kollidon 25, BASF) . Clomipramine HC1 (Anafranil) (Eli Lilly) was dissolved in normal saline. Rea- gents used in the assay for HA were all of analytical quality.
Student's unpaired t-test was used for the statistical analysis of biochemical and body temperature data. Be- havioural results were analysed by the x2-test.
Body temperature. Body temperature data recorded on days 1-5 of PVP (group H) and Ag-THC (group Q) adminis- tration are graphed in Fig. 1. On day 1 Ag-THC induced significant hypothermia (o
Ag-THC, H ISTAMINE, TOLERANCE AND WITHDRAWAL 155
hod fllmpllroture (~ C)
DAY I 39
I I 1 I I I I
-30 0 30 60 90 120 150
I I I I I I I
-30 0 30 60 90 120 150 TIME (rain)
body temperoture (*C)
I I I I I I I -30 0 30 60 90 120 150
t I I I I I
-30 0 30 60 90 120 TIME (rain)
I 1 I 1 I I
-30 0 30 60 90 120 TIME(rnin)
FIG. 1. The effect of Ag-THC (group Q, closed circles) and PVP (group H, open circles) on body temperature in rats treated with Ag-THC or PVP for 1-5 days (see text for dosage schedule). Ag-THC and PVP were injected at t=0 min. Each point is the means.e .m, of 5 determinations. *p
156 VERBERNE, FENNESSY, LEWIS AND TAYLOR
TABLE 3 THE EFFECTS OF PVP OR AU-THC ON REGIONAL BRAIN HISTAMINE (HA) CONCENTRATIONS AFTER 5 DAYS
Time Brain Region (min) PVPt Group 2t~'-THC~ Group % PVP*
Midbra in 0 23.2 3.4 (5) C 29.3 3.2 (5) K 126.5 _+ 23.0 30 24.8 3.1 (5) D 31.7 5.3 (5) L 127.7 30.1
Cor tex 0 13.3 0.8 (5) C 14.6 1.1 (5) K 110.4 10.6 30 13.4 1.3 (5) D 14.4 2.9 (5) L 107.6 25.2
Hypotha lamus 0 509.4 _+ 53.1 (5) C 541.6 73.8 (5) K 106.3 _+ 18.6 30 440.6 49.1 (5) D 554.0 76.8 (5) L 125.7 24.6
Cerebe l lum + 0 36.5 11.4 (4) C 30.9 6.2 (4) K 84.7 39.4 medul la 30 38.8 7.6(5) D 33.0 _+ 9.1 (4) L 85.1 27.9
The groups of rats were decapitated at the time of, or 30 min after, the evening injection of PVP or A~'-THC. *No significant differences were detected between the levels of histamine at 0 and 30 min. tNumbers in parentheses refer to the numbers of rats in each group.
~o c 125 ol m
o 100 > m J~ G) .Q
= 50 z
F N G P F N G P F N G P F N G P F N G P F N G P
wr i thes backward jumps head shakes paw t remor to ta l s igns k icks
bahav loura l s igns
FIG. 2. The effect of acute injection of Ag-THC (6 mg/kg, IV) or PVP (120 mg/kg, IV) on the intensity of withdrawal-like behavioural signs observed after administration of clomipramine hydrochloride (15 mg/kg, IP) to rats treated with Ag-THC or PVP for 5 days. Six animals were in each group. Open columns (group F) and horizontally striped columns (group N) represent chronic PVP- and chronic Aa-THC-treated rats, respectively, which received acute PVP (120 mg/kg, IP) 30 min prior to clomipramine (15 mg/kg, IP). Diagonally-striped columns (group G) and closed columns (group P) represent chronic PVP- and chronic Aa-THC-treated rats, respectively, which received acute 2tg-THC (6 mg/kg, IP) 30 min prior to clomip- ramine (15 mg/kg, IP). *p
Aa-THC, HISTAMINE, TOLERANCE AND WITHDRAWAL 157
EFFECTS OF 5 DAY TREATMENT WITH Ag-THC (GROUP M) AND PVP GROUP E) ON BRAIN REGIONAL CONCENTRATIONS OF HA
HA concentration (ng/g)
Brain region PVP (E) A"-THC (M) % PVP?
Hypothalamus 612.9 _+ 69.2 516.0 _+ 40.9 84.2 _+ 12.6" Midbrain 76.8 _+ 6.5 86.3 +- 8.6 112.4 -+ 15.2" Cortex 59.6 -+ 3.4 68.4 -+ 6.8 114.8 -+ 14.3" Medulla 31.8 _+ 1.1 32.1 _+ 3.6 100.9 _+ 11.9" oblongata/pons
Cerebellum 37.2 _+ 4.7 28.6 _+ 1.2 76.9 -+ 12.9"
The rats were decapitated on the morning of day 6 when the next injection was due. Each number is the mean _+ s.e.m, of 6 determi- nations.
*p>0.05. tThe results are expressed as percentages of the PVP control
value +_ relative s.e.
ring at 60 min. On day 2, significant hypothermia was in- duced after Aa-THC (p
158 VERBERNE, FENNESSY, LEWIS AND TAYLOR
regions examined (p >0.05 compared to that of the respective PVP control; Table 4).
Figure 3 indicates that clomipramine causes a significant reduction in the HA concentrations of the midbrain (47.8 + 12.2% of PVP control, p0.05) and the cerebellum (92.9+5.9% of PVP control, p>0.05) (group P compared to group G).
Recent experiments in which brain regional HA concen- trations were measured after an acute injection of A9-THC indicate that A9-THC may release HA in various parts of the rat brain . These observations might suggest that cerebral HA is involved in the neuropharmacological actions (e.g., hypothermia) of A9-THC. In the present study, tolerance de- velops to the effects of A9-THC on brain regional HA con- centrations with repeated twice daily IV doses over a 5-day period. Tolerance developed to this effect in the cortex and midbrain prior to the day 4 sampling time, while in the hypo- thalamus and combined cerebellum/medulla oblongata/pons regions it occurred between day 4 and day 5. In addition, the body temperature studies indicate that tolerance develops to the hypothermic effects of Ag-THC within the 5 day treat- ment period. These observations suggest that a relationship may exist between hypothalamic and brain stem HA concen- trations, and hypothermic responses to A9-THC, since their respective time-courses for the development of tolerance are similar.
Recent studies  have suggested that Ag-THC may act to produce its thermoregulatory effects via a site in the caudal brain stem, a region known to contain the origins of an as- cending histaminergic neuronal projection which passes through the hypothalamus via the medial forebrain bundle . This histaminergic projection then innervates the telen- cephalon and the hippocampus . It is conceivable that
stimulation of these histaminergic neurones would also cause a release of HA in the cortex, midbrain and hypothalamus. Since neuronally released HA is not taken up by a high- affinity reuptake mechanism , a decrease in the HA con- centrations of these brain regions should result.
The present study shows that Ag-THC attenuates the be- havioural syndrome induced by clomipramine in A9-THC - tolerant rats. When the two chronic PVP-treated groups are compared, only one of the behavioural parameters differed. An increase in the jumping response in the group treated acutely with A9-THC (group G) was observed. It is well documented that Ag-THC, injected acutely, induces spon- taneous jumping in naive rats [ 14,16]. Comparison of the two chronic A9-THC treatment groups (N and P) showed that acutely-administered A9-THC markedly decreased the number of behavioural signs observed after the clomip- ramine injection. This finding supports our contention that the clomipramine-precipitated behavioural syndrome in Ag-THC tolerant rats may be a manifestation of a withdrawal-like syndrome [15, 17, 19]. In addition, reduc- tions in the HA concentration of the midbrain, cortex, medulla oblongata/pons and the cerebellum, but not in the hypothalamus, occurred during the clomipramine- precipitated behavioural syndrome in these Ag-THC tolerant rats. Administration of an acute dose of Ag-THC before the clomipramine injection, which results in attenuation of the intensity of the behavioural syndrome, attenuates the reduc- tions in brain regional HA concentrations.
Certain midbrain structures, particularly the hippocam- pus, may play an important role in the mediation of withdr...