Synaptic and cellular changes induced by the schizophrenia susceptibility gene G72 are rescued by N-acetylcysteine treatment.

Genetic studies have linked the primate-specific gene locus G72 to the development of schizophrenia and bipolar disorder. Transgenic mice carrying the entire gene locus express G72 mRNA in dentate gyrus (DG) and entorhinal cortex, causing altered electrophysiological properties of their connections. These transgenic mice exhibit behavioral alterations related to psychiatric diseases, including cognitive deficits that can be reversed by treatment with N-acetylcysteine, which was also found to be effective in human patients. Here, we show that G72 transgenic mice have larger excitatory synapses with an increased amount of N-methyl-d-aspartate (NMDA) receptors in the molecular layer of DG, compared with wild-type littermates. Furthermore, transgenic animals have lower number of dentate granule cells with a parallel, but an even stronger decrease in the number of excitatory synapses in the molecular layer. Importantly, we also show that treatment with N-acetylcysteine can effectively normalize all these changes in transgenic animals, resulting in a state similar to wild-type mice. Our results show that G72 transcripts induce robust alterations in the glutamatergic system at the synaptic level that can be rescued with N-acetylcysteine treatment.

Properties and dynamics of inhibitory synaptic communication within the CA3 microcircuits of pyramidal cells and interneurons expressing parvalbumin or cholecystokinin.

KEY POINTS: To understand how a network operates, its elements must be identified and characterized, and the interactions of the elements need to be studied in detail. In the present study, we describe quantitatively the connectivity of two classes of inhibitory neurons in the hippocampal CA3 area (parvalbumin-positive and cholecystokinin-positive interneurons), a key region for the generation of behaviourally relevant synchronous activity patterns. We describe how interactions among these inhibitory cells and their local excitatory target neurons evolve over the course of physiological and pathological activity patterns. The results of the present study enable the construction of precise neuronal network models that may help us understand how network dynamics is generated and how it can underlie information processing and pathological conditions in the brain. We show how inhibitory dynamics between parvalbumin-positive basket cells and pyramidal cells could contribute to sharp wave-ripple generation. ABSTRACT: Different hippocampal activity patterns are determined primarily by the interaction of excitatory cells and different types of interneurons. To understand the mechanisms underlying the generation of different network dynamics, the properties of synaptic transmission need to be uncovered. Perisomatic inhibition is critical for the generation of sharp wave-ripples, gamma oscillations and pathological epileptic activities. Therefore, we aimed to quantitatively and systematically characterize the temporal properties of the synaptic transmission between perisomatic inhibitory neurons and pyramidal cells in the CA3 area of mouse hippocampal slices, using action potential patterns recorded during physiological and pathological network states. Parvalbumin-positive (PV+) and cholecystokinin-positive (CCK+) interneurons showed distinct intrinsic physiological features. Interneurons of the same type formed reciprocally connected subnetworks, whereas the connectivity between interneuron classes was sparse. The characteristics of unitary interactions depended on the identity of both synaptic partners, whereas the short-term plasticity of synaptic transmission depended mainly on the presynaptic cell type. PV+ interneurons showed frequency-dependent depression, whereas more complex dynamics characterized the output of CCK+ interneurons. We quantitatively captured the dynamics of transmission at these different types of connection with simple mathematical models, and describe in detail the response to physiological and pathological discharge patterns. Our data suggest that the temporal propeties of PV+ interneuron transmission may contribute to sharp wave-ripple generation. These findings support the view that intrinsic and synaptic features of PV+ cells make them ideally suited for the generation of physiological network oscillations, whereas CCK+ cells implement a more subtle, graded control in the hippocampus.

Complementary synaptic distribution of enzymes responsible for synthesis and inactivation of the endocannabinoid 2-arachidonoylglycerol in the human hippocampus.

Clinical and experimental evidence demonstrates that endocannabinoids play either beneficial or adverse roles in many neurological and psychiatric disorders. Their medical significance may be best explained by the emerging concept that endocannabinoids are essential modulators of synaptic transmission throughout the central nervous system. However, the precise molecular architecture of the endocannabinoid signaling machinery in the human brain remains elusive. To address this issue, we investigated the synaptic distribution of metabolic enzymes for the most abundant endocannabinoid molecule, 2-arachidonoylglycerol (2-AG), in the postmortem human hippocampus. Immunostaining for diacylglycerol lipase-α (DGL-α), the main synthesizing enzyme of 2-AG, resulted in a laminar pattern corresponding to the termination zones of glutamatergic pathways. The highest density of DGL-α-immunostaining was observed in strata radiatum and oriens of the cornu ammonis and in the inner third of stratum moleculare of the dentate gyrus. At higher magnification, DGL-α-immunopositive puncta were distributed throughout the neuropil outlining the immunonegative main dendrites of pyramidal and granule cells. Electron microscopic analysis revealed that this pattern was due to the accumulation of DGL-α in dendritic spine heads. Similar DGL-α-immunostaining pattern was also found in hippocampi of wild-type, but not of DGL-α knockout mice. Using two independent antibodies developed against monoacylglycerol lipase (MGL), the predominant enzyme inactivating 2-AG, immunostaining also revealed a laminar and punctate staining pattern. However, as observed previously in rodent hippocampus, MGL was enriched in axon terminals instead of postsynaptic structures at the ultrastructural level. Taken together, these findings demonstrate the post- and presynaptic segregation of primary enzymes responsible for synthesis and elimination of 2-AG, respectively, in the human hippocampus. Thus, molecular architecture of the endocannabinoid signaling machinery supports retrograde regulation of synaptic activity, and its similar blueprint in rodents and humans further indicates that 2-AG's physiological role as a negative feed-back signal is an evolutionarily conserved feature of excitatory synapses.

The effect of Echinacea preparations in three laboratory tests of anxiety: comparison with chlordiazepoxide.

Echinacea preparations are traditionally used to treat upper respiratory infections and inflammations. No psychotropic effects of Echinacea have been reported so far, although some recently reported active constituents are behaviorally active. Prompted by these findings, the anxiolytic potential of five different Echinacea preparations was evaluated. Three of these decreased anxiety but two of them had a very narrow effective dose range. Only one extract decreased anxiety within a wide dose-range (3-8 mg/kg). Anxiolytic effects were consistently seen in three different tests of anxiety, the elevated plus-maze, social interaction and shock-induced social avoidance tests. No locomotor suppressant effects were seen at any dose. Noteworthy, the doses that showed anxiolytic effects in the present study were much lower than those used in the laboratory models of the traditional indications. Chlordiazepoxide robustly decreased anxiety-like behavior in all tests but suppressed locomotion at higher doses. Perceived and real risks of conventional medications increase the demand for alternative therapies, provided that these are safe and efficient. Earlier evidence shows that Echinacea preparations have an excellent safety profile, while our findings suggest for the first time that certain preparations have a considerable anxiolytic potential. Further research is required to identify factors that differentiate efficient and inefficient preparations.

Relationship between neuronal vulnerability and potassium-chloride cotransporter 2 immunoreactivity in hippocampus following transient forebrain ischemia.

Cation chloride cotransporters have been reported to be expressed in neurons in the hippocampus and to regulate intracellular Cl(-) concentration. The neuron-specific K-Cl cotransporter 2 (KCC2) is necessary for maintaining the low intracellular chloride concentration required for the hyperpolarizing actions of GABA. In this study we examined the vulnerability of KCC2-containing neurons as well as the changes in the pattern of KCC2 distribution in the rat hippocampus following 15 min ischemia induced by four-vessel occlusion. Immunostaining for the 72 kDa heat shock protein (HSP-72) was used to investigate the extent of damage in neuronal populations previously shown to be vulnerable to ischemia. At 6-24 h after ischemia, when the pyramidal cells in the CA1 (subfield of cornu Ammonis) region showed no morphological signs of damage, a small rise of KCC2 immunoreactivity was already observed. After 2 days, when the CA1 pyramidal cells started to degenerate, a progressive downregulation of the KCC2 protein was visible. Interestingly, in the same areas, the parvalbumin containing interneurons showed no signs of ischemic damage, and KCC2 immunoreactivity was retained on their membrane surface. In CA1 pyramidal cells, the reduction in KCC2 expression may lead to an elevation of intracellular Cl(-) concentration, which causes a shift in equilibrium potential toward more positive levels. Consequently, the reduction of the inhibitory action of GABA through downregulation of KCC2 function may be involved in the pathomechanisms of delayed neuronal death in the CA1 subfield.

Quantitative ultrastructural differences between local and medial septal GABAergic axon terminals in the rat hippocampus.

Functionally distinct subsets of hippocampal inhibitory neurons exhibit large differences in the frequency, pattern and short-term plasticity of GABA release from their terminals. Heterogeneity is also evident in the ultrastructural features of GABAergic axon terminals examined in the electron microscope, but it is not known if or how this corresponds to interneuron subtypes. We investigated the feasibility of separating morphologically distinct clusters of terminal types, using the approach of measuring several ultrastructural parameters of GABAergic terminals in the CA1 area of the rat hippocampus. Septo-hippocampal axon terminals were anterogradely labeled by biotinylated dextran amine and visualized by pre-embedding immunogold staining to delineate one homogeneous terminal population. Long series (100-150) of ultrathin sections were cut from stratum oriens and stratum radiatum of the CA1 area, and GABAergic terminals were identified by post-embedding immunogold staining. Stereologically unbiased samples of the total GABAergic axon terminal population and a random sample of the septal axon terminals were reconstructed in 3D, and several of their parameters were measured (e.g. bouton volume, synapse surface, volume occupied by vesicles, mitochondria volume). Septal terminals demonstrated significantly larger mean values for most parameters than the total population of local GABAergic terminals. There was no significant difference between terminals reconstructed in the basal and apical dendritic regions of pyramidal cells, neither for the septal nor for the local population. Importantly, almost all parameters were highly correlated, precluding the possibility of clustering the local terminals into non-overlapping subsets. Factor and cluster analysis confirmed these findings. Our results suggest that similarly to excitatory terminals, inhibitory terminals follow an "ultrastructural size principle," and that the terminals of different interneuron subtypes cannot be distinguished by ultrastructure alone.

Correlated species differences in the effects of cannabinoid ligands on anxiety and on GABAergic and glutamatergic synaptic transmission.

Cannabinoid ligands show therapeutic potential in a variety of disorders including anxiety. However, the anxiety-related effects of cannabinoids remain controversial as agonists show opposite effects in mice and rats. Here we compared the effects of the cannabinoid agonist WIN-55,212 and the CB1 antagonist AM-251 in CD1 mice and Wistar rats. Special attention was paid to antagonist-agonist interactions, which had not yet been studied in rats. In mice, WIN-55,212 decreased whereas AM-251 increased anxiety. The antagonist abolished the effects of the agonist. In contrast, WIN-55,212 increased anxiety in rats. Surprisingly, the antagonist potentiated this effect. Cannabinoids affect both GABAergic and glutamatergic functions, which play opposite roles in anxiety. We hypothesized that discrepant findings resulted from species differences in the relative responsiveness of the two transmitter systems to cannabinoids. We investigated this hypothesis by studying the effects of WIN-55,212 on evoked hippocampal inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs). IPSCs were one order of magnitude more sensitive to WIN-55,212 in mice than in rats. In mice, IPSCs were more sensitive than EPSCs to WIN-55,212. This is the first study showing that the relative cannabinoid sensitivity of GABA and glutamate neurotransmission is species-dependent. Based on behavioural and electrophysiological findings, we hypothesize that WIN-55,212 reduced anxiety in mice by affecting GABA neurotransmission whereas it increased anxiety in rats via glutamatergic mechanisms. In rats, AM-251 potentiated this anxiogenic effect by inhibiting the anxiolytic GABAergic mechanism. We suggest that the anxiety-related effects of cannabinoids depend on the relative cannabinoid responsiveness of GABAergic and glutamatergic neurotransmission.

Morphology and synaptic input of substance P receptor-immunoreactive interneurons in control and epileptic human hippocampus.

Substance P (SP) is known to be a peptide that facilitates epileptic activity of principal cells in the hippocampus. Paradoxically, in other models, it was found to be protective against seizures by activating substance P receptor (SPR)-expressing interneurons. Thus, these cells appear to play an important role in the generation and regulation of epileptic seizures. The number, distribution, morphological features and input characteristics of SPR-immunoreactive cells were analyzed in surgically removed hippocampi of 28 temporal lobe epileptic patients and eight control hippocampi in order to examine their changes in epileptic tissues. SPR is expressed in a subset of inhibitory cells in the control human hippocampus, they are multipolar interneurons with smooth dendrites, present in all hippocampal subfields. This cell population is considerably different from SPR-positive cells of the rat hippocampus. The CA1 (cornu Ammonis subfield 1) region was chosen for the detailed morphological analysis of the SPR-immunoreactive cells because of its extreme vulnerability in epilepsy. The presence of various neurochemical markers identifies functionally distinct interneuron types, such as those responsible for perisomatic, dendritic or interneuron-selective inhibition. We found considerable colocalization of SPR with calbindin but not with parvalbumin, calretinin, cholecystokinin and somatostatin, therefore we suppose that SPR-positive cells participate mainly in dendritic inhibition. In the non-sclerotic CA1 region they are mainly preserved, whereas their number is decreased in the sclerotic cases. In the epileptic samples their morphology is considerably altered, they possessed more dendritic branches, which often became beaded. Analyses of synaptic coverage revealed that the ratio of symmetric synaptic input of SPR-immunoreactive cells has increased in epileptic samples. Our results suggest that SPR-positive cells are preserved while principal cells are present in the CA1 region, but show reactive changes in epilepsy including intense branching and growth of their dendritic arborization.

Subcellular localization of type 1 cannabinoid receptors in the rat basal ganglia.

Endocannabinoids, acting via type 1 cannabinoid receptors (CB1), are known to be involved in short-term synaptic plasticity via retrograde signaling. Strong depolarization of the postsynaptic neurons is followed by the endocannabinoid-mediated activation of presynaptic CB1 receptors, which suppresses GABA and/or glutamate release. This phenomenon is termed depolarization-induced suppression of inhibition (DSI) or excitation (DSE), respectively. Although both phenomena have been reported to be present in the basal ganglia, the anatomical substrate for these actions has not been clearly identified. Here we investigate the high-resolution subcellular localization of CB1 receptors in the nucleus accumbens, striatum, globus pallidus and substantia nigra, as well as in the internal capsule, where the striato-nigral and pallido-nigral pathways are located. In all examined nuclei of the basal ganglia, we found that CB1 receptors were located on the membrane of axon terminals and preterminal axons. Electron microscopic examination revealed that the majority of these axon terminals were GABAergic, giving rise to mostly symmetrical synapses. Interestingly, preterminal axons showed far more intense staining for CB1, especially in the globus pallidus and substantia nigra, whereas their terminals were only faintly stained. Non-varicose, thin unmyelinated fibers in the internal capsule also showed strong CB1-labeling, and were embedded in bundles of myelinated CB1-negative axons. The majority of CB1 receptors labeled by immunogold particles were located in the axonal plasma membrane (92.3%), apparently capable of signaling cannabinoid actions. CB1 receptors in this location cannot directly modulate transmitter release, because the release sites are several hundred micrometers away. Interestingly, both the CB1 agonist, WIN55,212-2, as well as its antagonist, AM251, were able to block action potential generation, but via a CB1 independent mechanism, since the effects remained intact in CB1 knockout animals. Thus, our electrophysiological data suggest that these receptors are unable to influence action potential propagation, thus they may not be functional at these sites, but are likely being transported to the terminal fields. The present data are consistent with a role of endocannabinoids in the control of GABA, but not glutamate, release in the basal ganglia via presynaptic CB1 receptors, but also call the attention to possible non-CB1-mediated effects of widely used cannabinoid ligands on action potential generation.

CB1 cannabinoid receptors are enriched in the perisynaptic annulus and on preterminal segments of hippocampal GABAergic axons.

Cannabinoids have been shown to modulate the inhibitory effect of cholecystokinin-containing GABAergic interneurons in the hippocampus via type 1 cannabinoid receptors (CB1 receptor). Although immunohistochemical studies, using pre-embedding techniques, have demonstrated that these receptors are abundant on GABAergic axon terminals, little is known about their exact location relative to the synapse. Here we used two recently developed antibodies against the CB1 receptor to study this question with the postembedding immunogold method, which allows the quantitative examination of receptor distribution along the axonal membrane, even within the synaptic active zone. CB1 receptor positive terminals target both the dendritic and somatic surface of neurons in the CA1 area of the rat hippocampus. We found no difference between these two populations of terminals either in their CB1 receptor density or in the distribution of receptors on their membrane. Recent studies suggest that endocannabinoids play a role in retrograde signaling at these synapses, i.e. signaling molecules diffuse from the postsynaptic membrane to nearby presynaptic terminals. Therefore, we examined the distribution of CB1 receptors on the terminal membranes. We found that they are rare in the synaptic active zone, but are enriched in the perisynaptic annulus, where they can directly influence synaptic calcium channels. Perisynaptic CB1 receptors represent about one tenth of all CB1 receptors in a terminal. In contrast, CB1 receptors have a lower density on the extrasynaptic membrane of terminals far from the postsynaptic cell. We estimated that these terminals contain exceptionally large numbers of CB1 receptors, i.e. a single axon terminal was usually labeled with more than 450 particles. An unexpected finding was that the density of CB1 receptors was significantly higher on preterminal axons than on synaptic terminals. These observations suggest that endocannabinoid signaling may subserve roles other than simply reducing transmitter release from axon terminals.

Cannabinoid CB1 receptor dependent effects of the NMDA antagonist phencyclidine in the social withdrawal model of schizophrenia.

Clinical and laboratory findings suggest that cannabinoid signalling is implicated in schizophrenia. However, the interaction remains poorly understood, as data are often contradictory. Here we investigated wild-type (WT) and cannabinoid CB1 receptor-knockout (CB1-KO) mice in the phencyclidine-induced social withdrawal model of schizophrenia. N-methyl-D-aspartate (NMDA) antagonists (including phencyclidine) induce psychotic symptoms in humans, and are used to model schizophrenia in a variety of experimental conditions. In WTs, 5 mg/kg phencyclidine increased locomotion and stereotyped behaviours, and decreased social interactions. These changes are consistent with a schizophrenia-like effect. In CB1-KOs, phencyclidine decreased locomotion, enhanced ataxia and stereotypy more markedly than in WTs, but did not affect social interactions. Locomotion showed a significant negative correlation with both ataxia and stereotypy, suggesting that in CB1-KOs, the locomotor suppressive effect of phencyclidine was secondary to changes in these variables. Our findings demonstrate that CB1 gene disruption dramatically alters the behavioural effects of the NMDA antagonist phencyclidine, suggesting that the CB1 receptor is involved in schizophrenia. As social disruption and stereotypy respectively are believed to model negative and positive symptoms of schizophrenia, our findings tentatively suggest that cannabinoids are differentially involved in these two symptom categories. These findings require verification by experiments involving CB1 receptor blockers, as the genetic and pharmacological blockade of receptors may not always provide similar results.

Altering cannabinoid signaling during development disrupts neuronal activity.

In adult cortical tissue, recruitment of GABAergic inhibition prevents the progression of synchronous population discharges to epileptic activity. However, at early developmental stages, GABA is excitatory and thus unable to fulfill this role. Here, we report that retrograde signaling involving endocannabinoids is responsible for the homeostatic control of synaptic transmission and the resulting network patterns in the immature hippocampus. Blockade of cannabinoid type 1 (CB1) receptor led to epileptic discharges, whereas overactivation of CB1 reduced network activity in vivo. Endocannabinoid signaling thus is able to keep population discharge patterns within a narrow physiological time window, balancing between epilepsy on one side and sparse activity on the other, which may result in impaired developmental plasticity. Disturbing this delicate balance during pregnancy in either direction, e.g., with marijuana as a CB1 agonist or with an antagonist marketed as an antiobesity drug, can have profound consequences for brain maturation even in human embryos.

Surviving CA1 pyramidal cells receive intact perisomatic inhibitory input in the human epileptic hippocampus.

Temporal lobe epilepsy (TLE) is known to be linked to an impaired balance of excitation and inhibition. Whether inhibition is decreased or preserved in the human epileptic hippocampus, beside the excess excitation, is still a debated question. In the present study, quantitative light and electron microscopy has been performed to analyse the distribution, morphology and input-output connections of parvalbumin (PV)-immunopositive interneurons, together with the entire perisomatic input of pyramidal cells, in the human control and epileptic CA1 region. Based on the degree of cell loss, the patients with therapy-resistant TLE formed four pathological groups. In the non-sclerotic CA1 region of TLE patients, where large numbers of pyramidal cells are preserved, the number of PV-immunopositive cell bodies decreased, whereas axon terminal staining, and the distribution of their postsynaptic targets was not altered. The synaptic coverage of CA1 pyramidal cell axon initial segments (AISs) remained unchanged in the epileptic tissue. The somatic inhibitory input is also preserved; it has been decreased only in the cases with patchy pyramidal cell loss in the CA1 region (control, 0.637; epileptic with mild cell loss, 0.642; epileptic with patchy cell loss, 0.424 microm synaptic length/100 microm soma perimeter). The strongly sclerotic epileptic CA1 region, where pyramidal cells can hardly be seen, contains a very small number of PV-immunopositive elements. Our results suggest that perisomatic inhibitory input is preserved in the epileptic CA1 region as long as pyramidal cells are present. Basket and axo-axonic cells survive in epilepsy if their original targets are present, although many of them lose their PV content or PV immunoreactivity. An efficient perisomatic inhibition is likely to take part in the generation of abnormal synchrony in the non-sclerotic epileptic CA1 region, and thus participate in the maintenance of epileptic seizures driven, for example, by hyperactive afferent input.

Differential distribution of the KCl cotransporter KCC2 in thalamic relay and reticular nuclei.

In the thalamus of the rat the reversal potential of GABA-induced anion currents is more negative in relay cells than in neurones of the reticular nucleus (nRt) due to different chloride extrusion mechanisms operating in these cells. The distribution of KCl cotransporter type 2 (KCC2), the major neuronal chloride transporter that may underlie this effect, is unknown in the thalamus. In this study the precise regional and ultrastructural localization of KCC2 was examined in the thalamus using immunocytochemical methods. The neuropil of all relay nuclei was found to display intense KCC2 immunostaining to varying degrees. In sharp contrast, the majority of the nRt was negative for KCC2. In the anterior and dorsal part of the nRt, however, KCC2 immunostaining was similar to relay nuclei and parvalbumin and calretinin were found to colocalize with KCC2. At the ultrastructural level, KCC2 immunoreactivity was mainly located in the extrasynaptic membranes of thick and thin dendrites and the somata of relay cells but was also found in close association with asymmetrical synapses formed by cortical afferents. Quantitative evaluation of KCC2 distribution at the electron microscopic level demonstrated that the density of KCC2 did not correlate with dendritic diameter or synaptic coverage but is 1.7 times higher on perisynaptic membrane surfaces than on extrasynaptic membranes. Our data demonstrate that the regional distribution of KCC2 is compatible with the difference in GABA-A reversal potential between relay and reticular nuclei. At the ultrastructural level, abundant extrasynaptic KCC2 expression will probably play a role in the regulation of extrasynaptic GABA-A receptor-mediated inhibition.

Distinct properties of carbachol- and DHPG-induced network oscillations in hippocampal slices.

The aim of this study was to compare and contrast the properties of gamma oscillations induced by activation of muscarinic acetylcholine or metabotropic glutamate receptors in the CA3 region of rat hippocampal slices. Both carbachol and the group I metabotropic glutamate receptor agonist, (RS)-3,5-dihydroxyphenylglycine (DHPG), induced network oscillations in the gamma-frequency range (30-100 Hz). The M1 muscarinic receptor antagonist, pirenzepine, blocked carbachol-, but enhanced DHPG-induced oscillations, whereas LY 341495, an antagonist at metabotropic glutamate receptors, abolished DHPG-, but left carbachol-induced oscillations unchanged. There were significant differences in the peak frequency, maximal power, and spectral width of the two oscillations. Pharmacological experiments showed that both types of oscillation depend on fast excitatory and inhibitory synaptic transmission. Interestingly, activation of neurokinin-1 receptors by substance P fragment or enhancement of inhibitory synaptic currents by the benzodiazepine ligand, zolpidem, boosted DHPG-, but reduced the power of carbachol-induced oscillations. These results suggest that, although carbachol and DHPG might activate similar conductances in individual pyramidal cells, the oscillations they induce in slices involve different network mechanisms, most likely by recruiting distinct types of GABAergic interneuron.

Uridine release during aminopyridine-induced epilepsy.

Uridine, like adenosine, is released under sustained depolarization and it can inhibit hippocampal neuronal activity, suggesting that uridine may be released during seizures and can be involved in epileptic mechanisms. In an in vivo microdialysis study, we measured the extracellular changes of nucleoside and amino acid levels and recorded cortical EEG during 3-aminopyridine-induced epilepsy. Applying silver impregnation and immunohistochemistry, we examined the degree of hippocampal cell loss. We found that extracellular concentration of uridine, adenosine, inosine, and glutamate increased significantly, while glutamine level decreased during seizures. The release of uridine correlated with seizure activity. Systemic and local uridine application was ineffective. The number of parvalbumin- and calretinin-containing interneurons of dorsal hippocampi decreased. We conclude that uridine is released during epileptic activity, and suggest that as a neuromodulator, uridine may contribute to epilepsy-related neuronal activity changes, but uridine analogues having slower turnover would be needed for further investigation of physiological role of uridine.

CB1 cannabinoid receptors mediate anxiolytic effects: convergent genetic and pharmacological evidence with CB1-specific agents.

Cannabinoids are known to modulate GABAergic and glutamatergic transmission in cortical areas, the former via CB1 and the latter via a novel receptor. Pharmacological data demonstrate that several widely used cannabinoid ligands bind to both receptors, which may explain the inconsistencies in their behavioural effects. Earlier we showed that the cannabinoid antagonist SR-141716A affected behaviour in both CB1 knockout and wild-type animals, and its effect (anxiolysis) was different from that of CB1 gene disruption (anxiogenesis). In the present experiments, we studied the effects of the CB1 antagonist AM-251, and the cannabinoid agonist WIN-55,212-2 in wild-type as well as in CB1 knockout mice. CB1 knockout mice showed higher scores of anxiety-like behaviour than the wild-type animals in the elevated plus-maze. Selective blockade of CB1 receptors by AM-251 (0.3, 1 and 3 mg/kg) increased anxiety-like behaviour dose-dependently in the wild-type mice but had no effect in the knockouts. In wild types, the cannabinoid agonist WIN-55,212-2 (1 and 3 mg/kg) caused a decrease in anxiety-like behaviour, which was abolished by the CB1-selective antagonist AM-251 (3 mg/kg). The same agonist did not change plus-maze behaviour in CB1 knockout animals. These data demonstrate at the behavioural level that AM-251 and, at low concentrations, WIN-55,212-2, are selective ligands of the CB1 cannabinoid receptor in mice. Our studies on the behavioural effects of the cannabinoid antagonist SR-141716A and the CB1 antagonist AM-251 show that the CB1 and the novel cannabinoid receptor mediate anxiolytic and anxiogenic effects, respectively. This suggests that agonists of the former, or antagonists of the latter, are promising new compounds in the pharmacotherapy of anxiety.

Segregation of two endocannabinoid-hydrolyzing enzymes into pre- and postsynaptic compartments in the rat hippocampus, cerebellum and amygdala.

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) catalyse the hydrolysis of the endocannabinoids anandamide and 2-arachidonoyl glycerol. We investigated their ultrastructural distribution in brain areas where the localization and effects of cannabinoid receptor activation are known. In the hippocampus, FAAH was present in somata and dendrites of principal cells, but not in interneurons. It was located mostly on the membrane surface of intracellular organelles known to store Ca(2+) (e.g. mitochondria, smooth endoplasmic reticulum), less frequently on the somatic or dendritic plasma membrane. MGL immunoreactivity was found in axon terminals of granule cells, CA3 pyramidal cells and some interneurons. In the cerebellum, Purkinje cells and their dendrites are intensively immunoreactive for FAAH, together with a sparse axon plexus at the border of the Purkinje cell/granule cell layers. Immunostaining for MGL was complementary, the axons in the molecular layer were intensively labelled leaving the Purkinje cell dendrites blank. FAAH distribution in the amygdala was similar to that of the CB(1) cannabinoid receptor: evident signal in neuronal somata and proximal dendrites in the basolateral nucleus, and hardly any labelling in the central nucleus. MGL staining was restricted to axons in the neuropil, with similar relative signal intensities seen for FAAH in different nuclei. Thus, FAAH is primarily a postsynaptic enzyme, whereas MGL is presynaptic. FAAH is associated with membranes of cytoplasmic organelles. The differential compartmentalization of the two enzymes suggests that anandamide and 2-AG signalling may subserve functional roles that are spatially segregated at least at the stage of metabolism.

Context-dependent effects of CB1 cannabinoid gene disruption on anxiety-like and social behaviour in mice.

Contrasting data were reported regarding the effects of cannabinoids on anxiety and social behaviour in both animals and humans. The cognitive effects of cannabinoids and their interactions with the HPA-axis raise the possibility that cannabinoid effects are context but not behaviour specific. To assess this hypothesis, we submitted CB1 receptor knock-out (CB1-KO) and wild-type (WT) mice to tests, which involved similar behaviours, but the behavioural context was different. The elevated plus-maze test was performed under less and more anxiogenic conditions, i.e. under low and high light, respectively. We also compared the social behaviour of the two genotypes in the resident/intruder and social interaction tests. Both tests represent a social challenge and induce similar behaviours, but involve different contexts. The behaviour of CB1-KO and WT mice was similar under low light, but CB1 gene disruption increased anxiety-like behaviour under the high light condition. CB1 gene disruption promoted aggressive behaviour in the home-cage, whereas it inhibited social behaviour in the unfamiliar cage. Thus, the anxiogenic-like effect was restricted to the more stressful unfamiliar environment. These data suggest that the effects of CB1 gene disruption were context and not behaviour specific. Novelty stress resulted in higher ACTH levels in CB1-KOs than in WTs, which suggests that context dependency occurred in conjunction with an altered HPA axis function. The present data at least partly explain contrasting effects of cannabinoids in different contexts as well as in different species and strains that show differential stress responses and coping strategies.

Postnatal development and migration of cholecystokinin-immunoreactive interneurons in rat hippocampus.

The development of cholecystokinin-immunoreactive (CCK-IR) interneurons in the rat hippocampus was studied using immunocytochemical methods at the light and electron microscopic levels from early (P0-P8) to later postnatal (P12-P20) periods. The laminar distribution of CCK-IR cell bodies changed considerably during the studied period, which is suggested to be due to migration. CCK-IR cells appear to move from the molecular layer of the dentate gyrus to their final destination at the stratum granulosum/hilus border, and tend to concentrate in the distal third of stratum radiatum in CA1-3. The density of CCK-IR cells is rapidly decreasing during the first 4 postnatal days without any apparent reduction in their total number, therefore it is due to the pronounced growth of hippocampal volume in this period. Axons of CCK-IR interneurons formed symmetrical synapses already at P0, and by far the predominant targets were dendrites of presumed principal cells in all subfields of the hippocampus. These axon arbors began to concentrate around pyramidal cell bodies only at P8, at earlier ages CCK-IR axons crossed stratum pyramidale at right angles, and gave rise to varicose collaterals only outside this layer. The dendrites and somata of CCK-IR cells received synapses already at P0, but those were mostly symmetrical, apart from a few immature asymmetrical synapses. At P4, mature asymmetrical synapses with considerable amounts of synaptic vesicles were already commonly encountered. Thus, the innervation of CCK-IR interneurons apparently develops later than their output synapses, suggesting that they may be able to release transmitter before receiving any considerable excitatory drive. We conclude that CCK-IR cells represent one, if not the major, interneuron type that assists in the maturation of glutamatergic synapses (activation of N-methyl-D-aspartate receptors) via GABAergic depolarization of principal cell dendrites, and may contribute to the generation of giant depolarizing potentials. CCK-IR cells will change their function to perisomatic hyperpolarizing inhibition, as glutamatergic transmission in the network becomes operational.