Neuroscience Letters 365 (2004) 58–63
A novel component of cannabis extract potentiates excitatory
synaptic transmission in rat olfactory cortex in vitro
Benjamin J. Whalleya, Jonathan D. Wilkinsonb,1,
Elizabeth M. Williamsonb, Andrew Constantia,∗
aDepartment of Pharmacology, The School of Pharmacy, University of London, 29/39 Brunswick Square,
London WC1N 1AX, UK
bCentre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, 29/39 Brunswick Square,
London WC1N 1AX, UK
Received 8 March 2004; received in revised form 14 April 2004; accepted 19 April 2004
Cannabis is a potential treatment for epilepsy, although the few human studies supporting this use have proved inconclusive. Previously,
we showed that a standardized cannabis extract (SCE), isolated ?9-tetrahydrocannabinol (?9-THC), and even ?9-THC-free SCE inhibited
that although ?9-THC (1?M) significantly depressed evoked depolarizing postsynaptic potentials (PSPs) in rat olfactory cortex neurones,
both SCE and ?9-THC-free SCE significantly potentiated evoked PSPs (all results were fully reversed by the CB1 receptor antagonist
SR141716A, 1?M); interestingly, the potentiation by ?9-THC-free SCE was greater than that produced by SCE. On comparing the effects
of ?9-THC-free SCE upon evoked PSPs and artificial PSPs (aPSPs; evoked electrotonically following brief intracellular current injection),
PSPs were enhanced, whereas aPSPs were unaffected, suggesting that the effect was not due to changes in background input resistance.
Similar recordings made using CB1 receptor-deficient knockout mice (CB1−/−) and wild-type littermate controls revealed cannabinoid or
extract-induced changes in membrane resistance, cell excitability and synaptic transmission in wild-type mice that were similar to those seen
the suppressive effects of ?9-THC on excitatory neurotransmitter release, which may explain some patients’ preference for herbal cannabis
rather than isolated ?9-THC (due to attenuation of some of the central ?9-THC side effects) and possibly account for the rare incidence of
seizures in some individuals taking cannabis recreationally.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cannabis extract; Cannabinoid CB1receptor; ?9-Tetrahydrocannabinol (?9-THC); Olfactory cortex; Brain slice; Electrophysiology; Intracellular
long been consumed both for recreational (mood-altering)
and medicinal purposes. Currently, considerable research
efforts are being expended into the potential therapeu-
tic uses of cannabinoids for disorders such as HIV, can-
cer, multiple sclerosis and more recently epilepsy, with
?9-tetrahydrocannabinol (?9-THC, the major psychoactive
∗Corresponding author. Tel.: +44-20-7753-5898;
E-mail address: email@example.com (A. Constanti).
1Present address: School of Biomedical Sciences, University of Not-
tingham Medical School, Queen’s Medical Centre, Clifton Boulevard,
Nottingham, NG7 2UH, UK.
component), and some other cannabinoid CB1 receptor ag-
onists being the primary targets for investigation [6,12]. In
Canada, patients with a number of intractable diseases, in-
cluding uncontrolled epilepsy, have recently been allowed to
possess marijuana or cultivate a limited number of cannabis
plants for their own medicinal use under Section 11 of the
Marijuana Medical Access Regulations 2003. Additionally,
in the US in 1998, Washington State passed a law defining
certain conditions (including epilepsy) for which medical
cannabis may be used without prosecution , whilst a
survey covering Germany, Austria and Switzerland found
that 3.5% of patients using cannabis medicinally were doing
so to alleviate symptoms of epilepsy  and a more re-
cent German survey found 2.1% to be doing likewise .
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
B.J. Whalley et al. / Neuroscience Letters 365 (2004) 58–63
However, the few human studies conducted to date have
given equivocal results . Cannabis herb contains over
400 compounds, of which 60 are classified as cannabinoids
, but research has so far focused mainly on ?9-THC and
cannabidiol (CBD), a major non-psychotropic constituent.
The remaining cannabinoids, although present in smaller
quantities and generally lacking psychoactivity, have not
been pharmacologically investigated to the same extent .
We have recently shown that a standardized extract of
herbal cannabis (SCE) was more potent at inhibiting sus-
tained muscarinic agonist-induced epileptiform seizures
in in vitro rat olfactory (piriform) cortical brain slices (a
useful model of human status epilepticus limbic epilepsy;
) than an equivalent dose of isolated ?9-THC, both
acting via a CB1 receptor-mediated mechanism; moreover,
suppression of epileptiform bursting even occurred when a
?9-THC-free cannabis extract was used. In contrast, CBD,
which has also been reported to have anticonvulsant activity
in a number of animal models  but has little or no affin-
ity for CB1 receptors in comparison , did not inhibit
seizures, nor did it modulate the activity of ?9-THC in our
G-protein-coupled CB1 receptors are found in neocorti-
cal, limbic sensory and motor areas of the mammalian brain
including the piriform cortex region  and are activated
by endogenous endocannabinoids such as anandamide ;
this lipophilic ligand is released from depolarized postsy-
naptic neurones in a Ca2+-dependent manner and diffuses
retrogradely to suppress both excitatory [14,31] and in-
hibitory [13,27] neurotransmission, in vitro via a presynap-
tic CB1 receptor mechanism, believed to involve a direct
G-protein inhibition of voltage-dependent N- (and possibly
also P/Q-type) Ca2+channels [14,31]. This effect has also
been demonstrated in vivo, where ?9-THC has been shown
to inhibit hippocampal acetylcholine release . Activation
of CB1 receptors is additionally known to modulate a num-
ber of intrinsic neuronal membrane conductances via post-
synaptic effector mechanisms . In the present report, we
describe a novel potentiation of intrinsic excitatory synaptic
transmission in the rat olfactory cortex in vitro  by a com-
ponent (or components) contained in a ?9-THC-SCE. We
believe that this effect is also mediated via a CB1 receptor
interaction since it was blocked by a specific CB1 antagonist
and was not observed in olfactory cortical slices prepared
from mutant CB1 receptor knockout mice (CB1−/−) .
Cannabis extracts were prepared and analysed as pre-
viously reported . Briefly, the freshly dried herb was
obtained by Soxhlet extraction using hexane, and assayed
by HPLC to identify and quantify the ?9-THC content; it
was then dried under vacuum and re-dissolved in ethanol
to produce a solution of extract containing 20% ?9-THC,
and designated as the SCE. This sample contained very lit-
tle CBD and cannabinol (CBN, a decomposition product of
?9-THC). A ?9-THC-free SCE was then prepared by sub-
jecting a proportion of the SCE to preparative HPLC, during
which the ?9-THC was removed from the eluate of succes-
sive injections. Confirmation that the ?9-THC had been ef-
fectively removed and the composition otherwise unchanged
was made by comparison of spectra on analytical HPLC
For preparation of brain slices, adult (P ≥ 40) Wistar
rats of either sex and normal adult (P ≥ 40) mice or mu-
tant CB1−/−knockout mice (kindly supplied by Drs. D.
Baker and G. Pryce, Department of Neuroinflammation,
Institute of Neurology, University College London) were
deeply anaesthetised with halothane in accordance with the
Home Office Animals (Scientific Procedures) Act 1986, de-
capitated and the brain rapidly removed. Transverse slices
of the olfactory cortex (∼450?m thick) were prepared as
previously described  and stored in oxygenated Krebs
solution at 32◦C for at least 30min before being transferred
to the recording chamber and constantly superfused with
pre-warmed (29–30◦C), oxygenated Krebs at ∼10ml/min.
The Krebs composition was (mM): 118 NaCl, 3 KCl, 1.5
CaCl2, 25 NaHCO3, 1 MgCl2·6H2O and 11 d-glucose, and
was continuously bubbled with 95% O2/5% CO2, pH 7.4.
All chemicals were obtained from BDH, UK. Conventional
intracellular current-clamp recordings were made from pre-
sumed ‘deep’ pyramidal cells in layers II–III of the piriform
cortex using 4M K acetate-filled glass microelectrodes
(tip resistance: 50–80M?) connected to an Axoclamp 2A
sample-and-hold preamplifier (Axon Instruments, Foster
City, CA). Current and voltage signals were recorded on
a storage oscilloscope, a Gould RS3200 chart recorder
and using pCLAMP6.1 data capture software (Axon In-
struments). Cell input resistance was calculated from the
steady-state electrotonic voltage response (≤10mV) to a
0.25nA, 160ms hyperpolarizing current pulse. Focal or-
thodromic synaptic stimuli (subthreshold) were applied
using a bipolar nichrome electrode (25?m diameter) placed
in layers II–III of the piriform cortex (to predominantly
activate local association and intrinsic fibre terminals pro-
jecting to layers II–III neurones), driven by an external
Digitimer DS-2 isolated stimulator (Digitimer Ltd., Wel-
wyn Garden, UK) (stimulus strength: 3–5V; 0.2ms). The
degree of inhibition or potentiation of the evoked depolar-
izing postsynaptic potentials (PSPs) in the presence of drug
were expressed as a mean percentage of the control value
(±S.E.; n = number of cells) and statistical differences
assessed by a non-parametric Wilcoxon signed rank test.
All other data were analysed using Student’s t-tests, with
significance accepted at P < 0.05. Subthreshold stimuli
producing a response of ∼60% of the level required for
spike firing (∼6–10mV) were used in control conditions
to provide a response that was easily measurable for de-
grees of inhibition or potentiation in the presence of drug,
whilst minimizing polysynaptic events. ?9-THC, the syn-
thetic, high affinity, cannabinoid agonist R-(+)-[2,3-dih-
(WIN-55,212-2; Tocris, UK) or the cannabinoid CB1 recep-
tor antagonist SR141716A (kindly provided by NIDA/NIH,
B.J. Whalley et al. / Neuroscience Letters 365 (2004) 58–63
Bethesda, USA) were pre-dissolved in the minimum re-
quired amount of ethanol and stored at 4◦C; dilutions of
these drugs or the cannabis extracts (SCE or ?9THC-free
SCE) were then prepared freshly in Krebs’ solution as
required and bath-applied by superfusion (bath-exchange
time ∼30s). The final bath concentration of ethanol did not
exceed 0.001%, and this concentration had no noticeable
effect on membrane or synaptic properties when applied
alone in control tests.
Stable intracellular recordings were obtained from 19
adult rat, 7 adult CB1−/−knockout mouse and 4 wild-type
adult mouse piriform cortical neurones. The average rest-
ing membrane potential and input resistance of recorded
cells was −84±1mV, 44±2M? (adult rat), −80±2mV,
41 ± 4M? (CB1−/−mouse) and −83 ± 1mV, 51 ± 5M?
(wild-type mouse), respectively. No significant differ-
ences between mean parameter values obtained in rat
and wild-type mouse or between CB1−/−knockout and
wild-type mouse neurones were found (P > 0.5 for each
comparison). Application of 1?M ?9-THC (30min) pro-
duced a small reversible membrane hyperpolarization and
decrease in input resistance (3 ± 1mV, 12 ± 2.1% rat,
n = 6) (cf. Refs. [14,16]), without any obvious changes in
spike amplitude (∼100mV measured from rest potential)
or firing threshold (typically between ∼−60 and −65mV).
However, on testing cell firing properties by injecting a brief
(160ms, +1.2nA) positive current pulse (after correcting
for change in membrane potential), ?9-THC induced a
small but significant reduction in the number of action po-
tentials elicited during the pulse indicating a decreased cell
excitability (mean number of control spikes = 4.2 ± 0.2
decreased to 3.1 ± 0.2, P < 0.01, n = 6, ∼27% reduction;
Fig. 1A and D). Similar effects of ?9-THC were observed
in recordings from control wild-type mice (n = 4, not
shown). By contrast, application of 0.3?g/ml SCE (≡1?M
?9-THC) or 0.3?g/ml ?9-THC-free SCE (30 min) signif-
icantly increased input resistance relative to control (SCE:
24 ± 3%, P < 0.05, n = 4; ?9-THC-free SCE: 22 ± 4%,
P < 0.05, n = 4; all results for rat) along with a significant
increase in the number of evoked spikes (control mean =
3.8 ± 0.6, increased to 5.0 ± 0.5, ∼33% change in SCE;
control = 4.3 ± 0.4, increased to 6.5 ± 0.5, ∼51% change
in ?9-THC-free SCE; P < 0.05, n = 4 in both cases),
without affecting the membrane potential (Fig. 1B–D).
Interestingly, these opposing effects of ?9-THC, or SCE
and ?9-THC-free SCE (n = 3 experiments in each case)
on membrane properties and excitability were blocked
in the presence of the CB1-receptor specific antagonist
SR141716A (1?M; pre-applied for 20min)  show-
ing a dependence on CB1 cannabinoid receptor activation.
1?M SR141716A applied alone (n = 3) had no effect on
As reported in other brain areas following application
of CB1 receptor agonists [14,31], 1?M ?9-THC pro-
duced a significant depression (control mean amplitude =
6.8 ± 0.5mV, decreased to 2.5 ± 0.4mV, P < 0.05,
Fig. 1. (A) Superimposed electrotonic potentials elicited from a rat ol-
factory cortical neurone by injecting brief (160ms) depolarizing or hy-
perpolarizing current pulses (0.25nA steps; range of −1.2 to +1.0nA).
Traces show recordings made in control conditions and following applica-
tion of 1?M ?9-THC (30min); note slight decrease in membrane input
resistance. (B) Electrotonic responses elicited from an olfactory cortical
neurone in control conditions and following application of 0.3?g/ml SCE
(30min). (C) Electrotonic responses evoked in a neurone in control and
following application of 0.3?g/ml ?9-THC-free SCE (30min); note the
increase in cell input resistance and excitability produced by both SCE
and ?9-THC-free SCE. All current stimuli were applied from a hold-
ing membrane potential of −80mV. Traces in A–C were obtained from
different neurones. (D) Histogram summarizing the effects of ?9-THC
(n = 6), SCE (n = 4) and ?9-THC-free SCE (n = 4) on number of
spikes evoked by a 160ms, +1.2nA depolarizing current pulse (ordinate)
(mean data ± S.E.). (*) Significantly different from corresponding con-
trol means obtained for each experiment type. For convenience, control
histogram (C) shows pooled data from all 14 experiments.
n = 6; ∼65% change) of the evoked depolarizing PSP
complex (comprised of a subthreshold superimposed fast
EPSP/IPSP sequence) that was fully reversed on applying
1?M SR141716A (30min; Fig. 2A); this antagonist applied
alone had no effect on synaptic transmission (n = 3). In-
B.J. Whalley et al. / Neuroscience Letters 365 (2004) 58–63
Fig. 2. (A) Depolarizing postsynaptic potentials (PSPs) elicited from an olfactory cortical neurone in control (left), 1?M ?9-THC (30min; middle) and
1?M ?9-THC plus 1?M SR141716A (20min; right). (B) PSPs elicited from a neurone in control (left), 1?g/ml SCE (30 min; middle) and 1?g/ml SCE
plus 1?M SR141716A (20min; right). (C) Synaptic responses elicited from a neurone in control (left), 1?g/ml ?9-THC-free SCE (30min; middle) and
1?g/ml ?9-THC-free SCE plus 1?M SR141716A (20min; right). Note that whereas ?9-THC typically depressed synaptic transmission, both SCE and
?9-THC-free SCE enhanced the PSP responses in a CB1 receptor-dependent manner. All synaptic stimuli (subthreshold; 5V, 0.2ms) were elicited from
a holding membrane potential of −90mV. Traces in A–C are averages of a minimum of three sweeps, and were obtained from different neurones. (D)
Histogram summarizing the effects of ?9-THC (n = 6), SCE (n = 4) and ?9-THC-free SCE (n = 4) on evoked depolarizing PSP amplitude (ordinate)
(mean data ± S.E.). (*) Significantly different from corresponding control means obtained for each experiment type. For convenience, control histogram
(C) shows pooled data from all 14 experiments.
terestingly, the high affinity synthetic cannabinoid agonist,
WIN-55,212-2  at 500nM concentration (n = 3) had a
similar effect to ?9-THC upon evoked spike firing (∼25%
decrease in spike number), and PSP amplitude (∼59% in-
crease). In contrast, 0.3?g/ml SCE or ?9-THC-free SCE
caused a significant potentiation of evoked PSP ampli-
tude (control = 6.3 ± 0.6mV, increased to 8.0 ± 0.7mV,
P < 0.05, n = 4, ∼28% change in SCE and control =
6.8±0.5mV increased to 10.5±0.9mV, P < 0.05, n = 4,
∼55% change in ?9-THC-free SCE) that was also reversed
following application of the CB1 antagonist (Fig. 2B–D).
It is worth noting that the synaptic effect of ?9-THC-free
SCE was consistently greater than that produced by SCE. To
determine whether the observed increase in background in-
for this enhancement, we compared the effect of 0.3?g/ml
?9-THC-free SCE on the evoked postsynaptic potentials
and on artificial PSPs (aPSPs) evoked electrotonically in the
same neurone by brief somatic current injection (+0.3nA,
1ms). In each experiment (n = 4), the synaptic potentials
were clearly enhanced by the extract, whereas the aPSPs re-
mained unaffected, suggesting that the enhancement was not
primarily due to a change in neuronal electrical membrane
properties (Fig. 3).
In further support of our proposal that the membrane
and synaptic effects of applied ?9-THC or the cannabis
extracts observed in rat olfactory neurones were CB1
receptor-mediated, we made similar recordings using ol-
Fig. 3. (A) Artificial (electrotonic) postsynaptic potentials (aPSPs) elicited
from an olfactory cortical neurone held at −90mV membrane potential by
injection of brief depolarizing current pulses (+0.3nA; 1ms) in control
conditions (left) and 0.3?g/ml ?9-THC-free SCE (right); note absence of
effect. (B) By comparison, subthreshold depolarizing PSPs (5V; 0.2ms
stimulus) elicited in the same neurone from −90mV membrane potential
in control (left) and 0.3?g/ml ?9-THC-free SCE (right) show a clear
potentiation. All traces are averages of a minimum of three sweeps.
B.J. Whalley et al. / Neuroscience Letters 365 (2004) 58–63
factory cortex slices prepared from CB1 receptor-deficient
knockout mice (CB1−/−) . Experiments in wild-type
littermate controls (n = 4) confirmed that the changes in
membrane resistance, cell excitability and synaptic trans-
mission produced by 1?M ?9-THC or 0.3?g/ml of the
cannabis extracts were similar in direction and magnitude
to those observed in rat neurones. However in CB1−/−
olfactory cortical cells, 1?M ?9-THC (n = 2), 0.3?g/ml
SCE (n = 2) or 0.3?g/ml ?9-THC-free SCE (n = 3) (all
applied for 30min) produced no observable changes in cell
membrane properties or synaptic amplitude.
Our previous work has shown that although ?9-THC has
anti-seizure effects in the rat piriform cortex brain slice, the
cannabis extract SCE was even more effective at abolishing
muscarinic agonist-induced epileptiform bursting [24,30].
Furthermore, even when testing the ?9-THC-free SCE, the
effect was indistinguishable from that produced by SCE,
indicating that ?9-THC was not essential for anti-seizure
activity. In the present report, we have shown that there
exists a component (or components) in cannabis extract,
which in contrast with ?9-THC, increases cell input resis-
tance and excitability, and potentiates intrinsic fibre-evoked
synaptic transmission in the olfactory cortex via a CB1
ing that all the induced effects of the SCE and ?9-THC-free
SCE were not observed in the presence of SR141716A, a
CB1 antagonist  or in CB1 receptor-deficient (CB1−/−)
knockout mice. Additional work will be required to de-
termine the nature of this component(s), by bioassay-led
isolation of various cannabis extract fractions and pharma-
cological characterization. Such studies may provide a lead
for investigation into novel therapeutic agents that could
be used for enhancement of neurotransmission in clinical
disorders such as mild dementia  or depression .
Although some reports suggest that SR141716A can act
as both a CB1 receptor antagonist and as an inverse ag-
onist depending upon concentration and the tissue being
examined [19,22], we observed no intrinsic activity of this
agent in our experiments (cf. facilitation of acetylcholine
release produced by SR141716A in hippocampal slices;
) which would also suggest that there was no detectable
endocannabinoid ‘tone’ affecting membrane properties,
transmission or cannabinoid effects in our preparation .
Nevertheless, we consider it possible that the observed ex-
citability increase and potentiation of synaptic transmission
produced by the cannabis extracts was due to such an inverse
agonist action of the unknown component(s) on constitu-
tively active olfactory cortical pre-and/or postsynaptic CB1
receptors; the precise mechanisms underlying these novel
effects however, remain to be determined. A small decrease
in neuronal membrane resistance produced by CB1 receptor
agonists was reported also by Kirby et al.  and Huang
et al.  in hippocampal and striatal neurones, respectively,
most likely involving several ionic processes. It is known
that cannabinoids can activate G-protein-gated inwardly
rectifying K+channels (GIRK)  which are important
for regulating neuronal cell excitability and resting poten-
tial . We suggest that a similar opposing modulation
of postsynaptic GIRK (and/or other background ‘leak’ K+)
conductances by ?9-THC and the cannabis extracts could
account for our observed changes in cell input resistance.
The lack of effect of the cannabis extract on electrotoni-
cally-generated aPSPs suggested that the potentiating effect
on the evoked PSP was not the result of an input resistance
change (cf. increase in amplitude of EPSPs by ?9-THC
in cat spinal motoneurons; ), therefore was most likely
mediated presynaptically. Since CB1 activation does not af-
fect neuronal responses to applied AMPA , it is unlikely
that any changes in excitatory synaptic potential ampli-
tude were mediated via a change in postsynaptic glutamate
receptor sensitivity; however, we cannot exclude the pos-
sibility that a component of the enhanced PSP involved a
positive modulation of postynaptic GABA receptor respon-
siveness. In the hippocampus, inhibitory neurotransmitter
(GABA) release has been shown to be far more sensitive
to cannabinoid-mediated modulation than excitatory trans-
mitter (glutamate) release . In our experiments, since
a clear potentiation of depolarizing synaptic transmission
was produced by the cannabis extracts, it is possible that
both excitatory (glutamate) and inhibitory (GABA) neuro-
transmitter release was being concurrently affected.
Finally, our present novel findings emphasize the im-
portance of characterising the composition of cannabis
extract for medicinal use, and have some interesting im-
plications for the possible wider therapeutic usefulness of
plant-derived medicinal cannabis extracts in the future .
Thus, the excitability-enhancing and potentiating effects
of the unknown extract constituent(s) on neurotransmis-
sion could actually over-ride the predominantly suppressive
effects of ?9-THC on certain types of neurotransmitter
release, and may therefore attenuate some of its central
side effects (e.g. sedation, or cognitive deficits; ). The
enhancement of neurotransmission may even explain the
preference by patients for herbal cannabis rather than iso-
lated ?9-THC in some instances, and possibly also account
for the rare incidence of seizure episodes in some individ-
uals taking cannabis, for whatever purpose . It remains
to be seen whether these novel neuromodulatory effects of
the cannabis extract component(s) would be apparent on
neuronal circuits in other brain areas.
This work was supported by a School of Pharmacy Stu-
dentship to B.J.W.
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