Changes in levels of D1, D2, or NMDA receptors during withdrawal from brief or extended daily access to IV cocaine

We previously reported that brief (1 h), but not extended (6 h), daily access to cocaine results in a sensitized locomotor response to cocaine and in elevated c-Fos immunoreactivity and DAT binding in the nucleus accumbens (N.Acc) core. In order to better our understanding of the neural adaptations mediating the transition from controlled drug use to addiction, the current experiments were set to further explore the neural adaptations resulting from these two access conditions. Rats received either brief daily access to saline or cocaine, or brief daily access followed by extended daily access to cocaine. Subjects were then sacrificed either 20 min, or 14 or 60 days, after the last self-administration session. Samples of the ventral tegmental area (VTA), N.Acc core and shell, dorsal striatum, and medial prefrontal cortex (mPFC) were taken for analysis of D1 ([3H]SCH-23390), D2 ([3H]Spiperone), and NMDA ([3H]MK-801) receptor binding (using the method of receptor autoradiography). At 20 min into withdrawal, D2 receptors were elevated and NMDA receptors were reduced in the mPFC of the brief access animals while D1 receptors were elevated in the N.Acc shell of the extended access animals, compared to saline controls. D2 receptors were reduced in the N.Acc shell of the brief access animals compared to saline controls after 14 days, and compared to extended access animals after 60 days of withdrawal. In summary, extended access to cocaine resulted in only transient changes in D1 receptors binding. These results suggest that the development of compulsive drug use is largely unrelated to changes in total binding of D2 or NMDA receptors.     

One hour, but not six hours, of daily access to self-administered cocaine results in elevated levels of the dopamine transporter

We have previously shown that brief (1 h) and extended (6 h) daily access to IV cocaine self-administration produce different behavioral and neural consequences following 2 weeks of drug withdrawal. Brief daily access produced stable consumption of the drug and, after withdrawal, a sensitized locomotor response and an enhanced c-Fos labeling to a single cocaine challenge. In contrast, extended daily cocaine self-administration produced escalation of drug consumption over trials but no enhanced behavioral or neurochemical response after withdrawal. Cocaine affects dopaminergic (DA) function by binding to the presynaptic transporter and thereby preventing reuptake of the neurotransmitter-an action thought to be responsible for the drug's reinforcing properties. In an extension of our previous work, the current study, using receptor autoradiography, compared binding (by [3H]WIN35428) of the dopamine transporter (DAT) in animals having experienced either brief or extended daily access to cocaine over 8 days, followed by 14 days of withdrawal. DAT densities were found to increase in the nucleus accumbens core (N.Acc Core) and the dorsal striatum (but not in the N.Acc shell, medial prefrontal cortex (mPFC), or ventral tegmental area (VTA)) of the 1-h, but not 6-h, subjects. In other words, elevations in DAT density were not associated with the 6-h access group, the group that models patterns of drug-use in human addicts, and therefore are likely to be independent of the neuroadaptations that occur in the "addictive" process. Such conclusions are also consistent with brain-imaging studies of human cocaine addicts. Additional research will be needed to identify the specific neural changes relevant to addiction.     

The motivation to self-administer is increased after a history of spiking brain levels of cocaine

Recent attempts to model the addiction process in rodents have focused on cocaine self-administration procedures that provide extended daily access. Such procedures produce a characteristic loading phase during which blood levels rapidly rise and then are maintained within an elevated range for the duration of the session. The present experiments tested the hypothesis that multiple fast-rising spikes in cocaine levels contribute to the addiction process more robustly than constant, maintained drug levels. Here, we compared the effects of various cocaine self-administration procedures that produced very different patterns of drug intake and drug dynamics on Pmax, a behavioral economic measure of the motivation to self-administer drug. Two groups received intermittent access (IntA) to cocaine during daily 6-h sessions. Access was limited to twelve 5-min trials that alternated with 25-min timeout periods, using either a hold-down procedure or a fixed ratio 1 (FR1). Cocaine levels could not be maintained with this procedure; instead the animals experienced 12 fast-rising spikes in cocaine levels each day. The IntA groups were compared with groups given 6-h FR1 long access and 2-h short access sessions and two other control groups. Here, we report that cocaine self-administration procedures resulting in repeatedly spiking drug levels produce more robust increases in Pmax than procedures resulting in maintained high levels of cocaine. These results suggest that rapid spiking of brain-cocaine levels is sufficient to increase the motivation to self-administer cocaine.     

Both glutamate receptor antagonists and prefrontal cortex lesions prevent induction of cocaine sensitization and associated neuroadaptations.

Behavioral sensitization to psychomotor stimulants is accompanied by a number of alterations in the mesoaccumbens dopamine (DA) system, including DA autoreceptor subsensitivity in the ventral tegmental area (VTA) and DA D1 receptor supersensitivity in the nucleus accumbens (NAc). We investigated the role of excitatory amino acid (EAA) transmission in the induction of cocaine sensitization and these accompanying DA receptor alterations. To do so, we used three glutamate receptor antagonists, the noncompetitive NMDA receptor antagonist MK-801 (0.1 mg/kg), the competitive NMDA receptor antagonist CGS 19755 (10.0 mg/kg), and the AMPA receptor antagonist NBQX (12.5 mg/kg). Rats received daily double injections of either one of these antagonists or saline with either cocaine (15.0 mg/kg) or saline for 5 days. Cocaine sensitization was defined as an increase in horizontal locomotor activity in response to cocaine challenge (7.5 mg/kg) on the third day of withdrawal. All three antagonists prevented the induction of cocaine sensitization. Extracellular single cell recordings revealed that these antagonists also prevented the induction of DA autoreceptor subsensitivity in the VTA and DA D1 receptor supersensitivity in the NAc. To determine whether the relevant glutamate receptors were under regulation by medial prefrontal cortex (mPFC) EAA efferents, we next lesioned the mPFC bilaterally with ibotenic acid at least 7 days before repeated cocaine treatment began. These lesions also prevented the induction of cocaine sensitization and the associated neuroadaptations. Our findings indicate that glutamate transmission from mPFC to the mesoaccumbens DA system is critical for the induction of cocaine sensitization and its cellular correlates.     

Intracerebroventricular administration of NMDA-R1 antisense oligodeoxynucleotide significantly alters the activity of ventral tegmental area dopamine neurons: an electrophysiological study

In this study, we determined the activity of midbrain dopamine (DA) neurons in male albino rats following the intracerebroventricular (i.c.v.) administration of antisense oligodeoxynucleotide (aODN) against the mRNA for the NR1 subunit of the NMDA receptor. In addition, the effect of aODN on the specific binding of the NMDA receptor ligand [(3)H]MK-801 was also examined in various brain areas, including the midbrain. Antisense ODN against the NR1 mRNA, the corresponding sense ODN (sODN) or saline was continuously administered into the right ventricle of rats by osmotic minipumps for 7 days (20 nmol/day). Autoradiographic binding studies indicated that aODN significantly reduced the density of [(3)H]MK-801 binding by an average of 20-30% in several forebrain regions, including the anterior cingulate cortex, caudate putamen, and nucleus accumbens. However, [(3)H]MK-801 binding was not significantly altered in the ventral tegmental area (VTA) or substantia nigra pars compacta (SNC). Subsequently, using the technique of extracellular single-unit recording, the number, as well as the firing pattern, of spontaneously active DA neurons was determined in the VTA and SNC. The administration of aODN did not significantly alter the number of spontaneously active VTA and SNC DA neurons compared to saline- of sODN-treated animals. Furthermore, the firing pattern of spontaneously active SNC DA neurons was not significantly altered. However, for spontaneously active VTA DA neurons, the administration of aODN significantly decreased the percent events in bursts, number of bursts, and percentage of DA neurons exhibiting a bursting pattern compared to saline- and sODN-treated animals, i.e., neurons show less bursting activity. The present results suggest that subchronic aODN treatment against the mRNA for the NR1 subunit of the NMDA receptors can reduce NMDA receptor number and can result in an altered activity of spontaneously active VTA DA neurons in anesthetized rats.     

Repeated cocaine and stress increase dopamine clearance in the rat medial prefrontal cortex

The effects of repeated footshock stress or cocaine on the kinetics of dopamine clearance in the medial prefrontal cortex (mPFC) were measured by rotating disk electrode voltammetry (RDEV). Five groups of rats were used: animals were either naive (non-handled), pre-treated with five daily saline (1 ml/kg i.p.) or cocaine (15 mg/kg i.p.) injections, or pre-treated with five daily 20-min sessions of sham shock or footshock (0.05 mA/200 ms/s). Dopamine clearance was measured after a 1-week withdrawal period. No difference in K_m values was present among the treatment groups, with the mean K_m value at ≈0.5 μM for all groups. However, V_max values were ≈50% higher in daily sham shock-, footshock- and cocaine-pre-treated animals compared to naive rats. The increased ability to remove dopamine in these animals suggests that altered dopamine clearance may serve an adaptive mechanism in the mPFC.     

Cocaine increases medial prefrontal cortical glutamate overflow in cocaine-sensitized rats: a time course study

Excitatory amino acid transmission within mesocorticolimbic brain pathways is thought to play an important role in behavioural sensitization to psychomotor stimulants. The current studies evaluated a time course of the effects of cocaine on extracellular glutamate levels within the medial prefrontal cortex (mPFC) following increasing periods of withdrawal from repeated cocaine exposure. Male Sprague-Dawley rats underwent stereotaxic surgeries and were pretreated daily with saline (1 mL/kg/day x 4 days, i.p.) or cocaine (15 mg/kg/day x 4 days, i.p.) and withdrawn for 1, 7 or 30 days. After withdrawal rats were challenged with the same dose of saline or cocaine and in vivo microdialysis of the mPFC was conducted with concurrent analysis of locomotor activity. Animals that were withdrawn from repeated daily cocaine for 1 day and 7 days displayed an augmentation in cocaine-induced mPFC glutamate levels compared to saline and acute control subjects, which were similarly unaffected by cocaine challenge. At the 7 day time point, a subset of animals that received repeated cocaine did not express behavioural sensitization, nor did these animals exhibit the enhancement in mPFC glutamate in response to cocaine challenge. In contrast to these early effects, 30 days of withdrawal resulted in no significant changes in cocaine-induced mPFC glutamate levels regardless of the pretreatment or behavioural response. These data suggest that repeated cocaine administration transiently increases cocaine-induced glutamate levels in the mPFC during the first week of withdrawal, which may play an important role in the development of behavioural sensitization to cocaine.     

Acute brain metabolic effects of cocaine in rhesus monkeys with a history of cocaine use

Cocaine addiction involves an escalation in drug intake which alters many brain functions. The present study documented cocaine-induced changes in brain metabolic activity as a function of cocaine self-administration history. Experimentally naive rhesus monkeys (N?=?6) were given increasing access to cocaine under a fixed-ratio schedule of intravenous (i.v.) drug self-administration. PET imaging with F-18 labeled fluorodeoxyglucose (FDG) was used to measure acute intramuscular (i.m.) cocaine-induced changes in brain metabolism in the cocaine-naïve state, following 60 sessions under limited-access conditions (1 h/day), following 60 sessions under extended-access conditions (4 h/day), and following 4 weeks of drug withdrawal. In the cocaine-naïve state, cocaine-induced increases in brain metabolism were restricted to the prefrontal cortex. As cocaine exposure increased from limited to extended access, metabolic effects expanded throughout the frontal cortex and were induced within the striatum. Conversely, cocaine-induced activation was far less robust following withdrawal. The results highlight a progressive expansion of the metabolic effects of cocaine to include previously unaffected dopamine innervated brain regions as a consequence of cocaine self-administration history. The identification of brain regions progressively influenced by drug exposure may be highly relevant toward efforts to develop treatments for cocaine addiction.     

Withdrawal from extended‐access cocaine self‐administration results in dysregulated functional activity and altered locomotor activity in rats

Much work has focused on determining the consequences of cocaine self‐administration on specific neurotransmitter systems, thus neglecting the global changes that occur. Previous imaging studies have focused on the effects of cocaine self‐administration in the presence of high blood levels of cocaine, but have not determined the functional effects of cocaine self‐administration after cocaine has cleared. Extended‐access cocaine self‐administration, where animals administer cocaine for 6 h each day, results in escalation in the rate of cocaine intake and is believed to model the transition from recreational use to addiction in humans. We aimed to determine the functional changes following acute (48 h) withdrawal from an extended‐access, defined‐intake self‐administration paradigm (5 days, 40 injections/day, 6 h/day), a time point when behavioral changes are present. Using the 2‐[¹⁴C]deoxyglucose method to measure rates of local cerebral glucose metabolism, an indicator of functional activity, we found reductions in circuits related to learning and memory, attention, sleep, and reward processing, which have important clinical implications for cocaine addiction. Additionally, lower levels of functional activity were found in the dorsal raphe and locus coeruleus, suggesting that cocaine self‐administration may have broader effects on brain function than previously noted. These widespread neurochemical reductions were concomitant with substantial behavioral differences in these animals, highlighted by increased vertical activity and decreased stereotypy. These data demonstrate that behavioral and neurochemical impairments following cocaine self‐administration are present in the absence of drug and persist after cocaine has been cleared.     

Effects of cocaine-induced behavioural sensitization on GABA transmission within rat medial prefrontal cortex

Studies support the involvement of mPFC dopaminergic and glutamatergic systems in the development of cocaine sensitization. GABA is known to modulate dopamine and glutamatergic systems in the mPFC. In addition, recent reports have suggested that cocaine sensitization might be associated with a decrease in GABAB receptor responsiveness in the mPFC. Hence, in vivo microdialysis of the mPFC was performed to examine the effects on extracellular GABA levels within the mPFC of a cocaine challenge subsequent to repeated cocaine administration. Male Sprague-Dawley rats were given four daily injections of saline (1.0 mL/kg, i.p.) or cocaine (15 mg/kg, i.p.) and challenged with the same dose of saline or cocaine 1, 7 or 28 days later. Acute cocaine produced a motor-stimulant response that was significantly augmented in repeated cocaine animals at all withdrawal time points. Moreover, sensitized animals exhibited a significant increase in extracellular GABA levels after 1 and 7 days but not 28 days following repeated cocaine exposure. These data suggest that cocaine-induced sensitization is associated with a transient increase in mPFC GABA transmission.     

Age- and Hormone-Regulation of N-Methyl-d-Aspartate Receptor Subunit NR2b in the Anteroventral Periventricular Nucleus of the Female Rat: Implications for Reproductive Senescence

Glutamate, acting through its N‐methyl‐d ‐aspartate (NMDA) and non‐NMDA receptors in the hypothalamus, regulates reproductive neuroendocrine functions via direct and indirect actions upon gonadotrophin‐releasing hormone (GnRH) neurones. Previous studies indicate that the NMDA receptor subunit NR2b undergoes changes in protein and gene expression in the hypothalamus in general, and on GnRH neurones in particular, during reproductive ageing. In the present study, we examined whether the NR2b‐expressing cell population, both alone and in association with the NR1 subunit (i.e. the latter subunit is necessary for a functional NMDA receptor), is altered as a function of age and/or steroid hormone treatment. Studies focused on the anteroventral periventricular (AVPV) nucleus of the hypothalamus, a region critically involved in the control of reproduction. Young (3–5 months), middle‐aged (9–12 months), and aged (approximately 22 months) female rats were ovariectomised and, 1 month later, they were treated sequentially with oestradiol plus progesterone, oestradiol plus vehicle, or vehicle plus vehicle, then perfused. Quantitative stereologic analysis of NR2b‐immunoreactive cell numbers in the AVPV showed an age‐associated decrease in the density of NR2b‐immunoreactive cells, but no effect of hormone treatment. In a second study, immunofluorescent double labelling of NR2b and NR1 was analysed by confocal microscopy of fraction volume, a semi‐quantitative measure of fluorescence intensity. No effect of ageing was detected for immunofluorescent NR1 or NR2b alone, whereas the NR2b fraction volume increased in the oestradiol plus vehicle group. With ageing, the fraction volume of the NR2b/NR1‐colocalised subunits increased. Together with the stereology results, this suggests that, although fewer cells express the NR2b subunit in the ageing AVPV, a greater percentage of these subunits are co‐expressed with NR1. Our results suggest that the subunit composition of NMDA receptors in the AVPV undergo both age‐ and hormonal‐regulation, which may be related to previous observations of changes in functional responses of reproductive neuroendocrine systems to NMDA receptor modulators with ageing.     

Coincidence in dendritic clustering and synaptic targeting of homer proteins and NMDA receptor complex proteins NR2B and PSD95 during development of cultured hippocampal neurons

Homer is a scaffold protein that binds glutamate receptor complexes and actin cytoskeleton in postsynapses. The present study analyzed developmental changes in subcellular localization of Homer proteins in cultured hippocampal neurons. All three Homer family proteins, Homer 1b/c, Cupidin/Homer 2, and Homer 3, not only form heteromeric coclusters, but also localize close to the NMDA receptor complex including the NR2B subunit and PSD95 throughout dendritic and synaptic differentiation. Synaptic clustering of Homer proteins is enhanced by simultaneous blockade of NMDA receptor and cAMP phosphodiesterase activities, as is clustering of NMDA receptors. Homer proteins colocalize with actin-cytoskeletal proteins F-actin and Drebrin partially during the middle stage and to a greater extent in the late stage, and with the GluR1 subunit of AMPA receptors only in the late stage. Clustering sites of Homer are not synaptic in early-middle stages, but become synaptic in the late stage, as deduced from synaptic targeting of Bassoon, Synaptophysin, and N-cadherin. Our results indicate a coincidence in dendritic clustering in addition to developmental and activity-regulated synaptic targeting between Homer and the NMDA receptor complex.     

Neuroplasticity in cholinergic neurons of the laterodorsal tegmental nucleus contributes to the development of cocaine addiction

The laterodorsal tegmental nucleus (LDT) is a brainstem nucleus that sends cholinergic, glutamatergic, and gamma‐aminobutyric acid (GABA)‐ergic projections to the ventral tegmental area (VTA), a key brain region associated with reward information processing and reinforcement learning, and thus, with addiction induced by drugs of abuse, including cocaine. Recent studies have revealed that the LDT, in addition to the VTA, plays important roles in the development and expression of cocaine‐induced addiction and stress‐induced enhancement of addictive behaviors. Additionally, neuroplasticity induced in LDT cholinergic neurons by repeated cocaine administration critically contributes to these behaviors. Elucidation of the underlying mechanisms of cocaine‐induced neuroplasticity in the LDT that influences reward circuit activity may lead to the development of therapeutic strategies to treat cocaine addiction and stress‐induced reinstatement of cocaine use. This review summarizes recent progress in the study of the LDT, specifically neuroplasticity in LDT cholinergic neurons induced by cocaine and its functional roles in the development and modulation of addictive behaviors associated with cocaine.     

Initial rewarding effects of cocaine and amphetamine assessed in a day using the single‐exposure place preference protocol

In rodents, only a single dose of cocaine or amphetamine is required to cause a marked increase in extracellular dopamine, induce hyperlocomotion and cause persistent plasticity changes within dopaminergic neurons of the ventral tegmental area (VTA). The initial drug experience is suggested to predict vulnerability of developing addiction, but only few studies have assessed the perception of reward accompanying this initial exposure. We recently presented an approach to assess the initial rewarding effects of cocaine in mice with a single‐exposure place preference (sePP) protocol, avoiding repeated drug injections. Here, we demonstrate a condensed version of the sePP, allowing assessment of initial subjective reward‐perception within a day. By using this protocol, we demonstrate that a single exposure to both cocaine and amphetamine is sufficient to induce place preference. Furthermore, we use chemogenetics ( Designer Receptors Exclusively Activated by Designer Drugs [DREADD]) to show that both inhibitory and stimulatory modulation of VTA DA signalling disrupts cocaine‐induced place preference in the condensed sePP. Our findings support the presence of initial reward‐perception of both cocaine and amphetamine, and the formation of drug‐context association. In addition, our data support that VTA DA signalling prior to drug exposure affects either reward‐perception or the time during which associations are formed, thereby preventing induction of cocaine‐induced place preference in the sePP. The easy and timesaving sePP protocol should form a critical basis for further deciphering the complex mechanisms underlying the progression from the initial drug experience to escalating drug intake and addiction.     

Cocaine enhances medial prefrontal cortex neuron response to ventral tegmental area activation

Extracellular single unit recording techniques were used to characterize the effect of cocaine on the response of identified medial prefrontal cortex (mPFC) neurons to electrical stimulation of the ventral tegmental area (VTA) in chloral hydrate anesthetized rats. The select population of neurons studied were identified as cortical efferent cells by action potential characteristics and antidromic activation from the VTA. Stimulation of the VTA also induced a synaptically mediated inhibition of the spontaneous activity of the mPFC neurons. Administration of 2.0 mg/kg cocaine (IV) produced an increase in the duration of the VTA stimulus-evoked inhibition that differed significantly from the effect of 4.0 mg/kg procaine (IV). In contrast, microiontophoretic cocaine and procaine produced no significant changes in the duration of the VTA stimulus-evoked inhibition. This study provides evidence that systemic but not microiontophoretic cocaine administration enhances dopamine receptor-mediated inhibitory VTA input to a select population of mPFC efferent neurons.     

Anatomical and pharmacological characterization of catecholamine transients in the medial prefrontal cortex evoked by ventral tegmental area stimulation

Voltammetric measurements of catecholamines in the medial prefrontal cortex (mPFC) are infrequent because of lack of chemical selectivity between dopamine and norepinephrine and their overlapping anatomical inputs. Here, we examined the contribution of norepinephrine to the catecholamine release in the mPFC evoked by electrical stimulation of the ventral tegmental area (VTA). Initially, electrical stimulation was delivered in the midbrain at incremental depths of ?5 to ?9.4 mm from bregma while catecholamine release was monitored in the mPFC. Although catecholamine release was observed at dorsal stimulation sites that may correspond to the dorsal noradrenergic bundle (DNB, containing noradrenergic axonal projections to the mPFC), maximal release was evoked by stimulation of the VTA (the source of dopaminergic input to the mPFC). Next, VTA‐evoked catecholamine release was monitored in the mPFC before and after knife incision of the DNB, and no significant changes in the evoked catecholamine signals were found. These data indicated that DNB fibers did not contribute to the VTA‐evoked catecholamine release observed in the mPFC. Finally, while the D2‐receptor antagonist raclopride significantly altered VTA‐evoked catecholamine release, the α₂‐adrenergic receptor antagonist idazoxan did not. Specifically, raclopride reduced catecholamine release in the mPFC, opposite to that observed in the striatum, indicating differential autoreceptor regulation of mesocortical and mesostriatal neurons. Together, these findings suggest that the catecholamine release in the mPFC arising from VTA stimulation was predominately dopaminergic rather than noradrenergic. Synapse 68:131–143, 2014. © 2013 Wiley Periodicals, Inc.     

Involvement of NMDA receptors in ryanodine receptor expression in dopaminergic neurons in the ventral tegmental area of mice with intermittent methamphetamine treatment

Excitatory synapses on dopaminergic neurons of the ventral tegmental area (VTA) represent an important role in psychostimulant-induced rewarding effect. This study investigated the regulation of ryanodine receptor (RyR) and N-methyl-D-aspartate (NMDA) receptor expression in mice under intermittent methamphetamine (METH) treatment using a place preference procedure. RyR-1 and -2 significantly increased in the VTA of mice with METH-induced place preference, whereas RyR-3 showed no changes. In addition, the levels of NR1, NR2A, and NR2B subunits were increased in the VTA. The METH-induced place preference was inhibited by intracerebroventricular pretreatment with MK-801, a noncompetitive NMDA receptor antagonist, and ifenprodil, a selective NR2B subunit-containing NMDA receptor antagonist, in a dose-dependent manner. Under these conditions, the increase of RyR-1 and -2 in the VTA was significantly blocked by ifenprodil. The immunohistochemical analysis revealed the colocalization of RyR-1 and -2 with NR2B subunits in dopaminergic neurons in the mouse VTA. These findings suggest that RyRs could be involved in the development of METH-induced place preference and that NR2B subunit-containing NMDA receptors in mice showing METH-induced place preference play an important role in expression of RyRs.     

Suppressing calcium/calmodulin-dependent protein kinase II activity in the ventral tegmental area enhances the acute behavioural response to cocaine but attenuates the initiation of cocaine-induced behavioural sensitization in rats

In the present experiments we administered an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist (CNQX), N-methyl-D-aspartate (NMDA) receptor antagonist (AP-5), or l-type calcium channel blocker (diltiazem) directly into the ventral tegmental area (VTA) before each of four daily systemic cocaine injections in order to assess their influence on the initiation phase of behavioural sensitization. Results indicated that pretreatment with CNQX or AP-5 impaired the initiation of cocaine-induced behavioural sensitization. Intra-VTA administration of diltiazem significantly increased the behavioural activation induced by an acute cocaine injection, but impaired the development of cocaine-induced behavioural sensitization. Because AMPA and NMDA receptors, as well as l-type calcium channels are calcium permeable, we also investigated the role of the calcium-activated second messenger calcium/calmodulin-dependent protein kinase II (CaM-KII). Similar to the results obtained with diltiazem, administration of the CaM-KII inhibitor KN-93 into the VTA enhanced the acute behavioural response to cocaine but prevented the augmentation of cocaine-induced behavioural hyperactivity following repeated injections. Consistent with this finding, the behavioural hyperactivity produced by cocaine was markedly enhanced among homozygous alpha-CaM-KII knockout mice but the initiation of behavioural sensitization to cocaine was attenuated relative to wild-type mice. Separate experiments performed in rats demonstrated an increase in total protein levels of CaM-KII in the VTA 24 h after the last of seven daily injections of cocaine. Taken together, these results indicate that blocking l-type calcium channels or impairing CaM-KII activity in the VTA augments the acute behavioural hyperactivity induced by cocaine. The present findings also suggest that increased calcium influx through AMPA receptors, NMDA receptors and l-type calcium channels on dopaminergic neurons in the VTA contributes significantly to the initiation of behavioural sensitization by amplifying calcium signalling through CaM-KII.