Targeting myelin lipid metabolism as a potential therapeutic strategy in a model of CMT1A neuropathy.

In patients with Charcot-Marie-Tooth disease 1A (CMT1A), peripheral nerves display aberrant myelination during postnatal development, followed by slowly progressive demyelination and axonal loss during adult life. Here, we show that myelinating Schwann cells in a rat model of CMT1A exhibit a developmental defect that includes reduced transcription of genes required for myelin lipid biosynthesis. Consequently, lipid incorporation into myelin is reduced, leading to an overall distorted stoichiometry of myelin proteins and lipids with ultrastructural changes of the myelin sheath. Substitution of phosphatidylcholine and phosphatidylethanolamine in the diet is sufficient to overcome the myelination deficit of affected Schwann cells in vivo. This treatment rescues the number of myelinated axons in the peripheral nerves of the CMT rats and leads to a marked amelioration of neuropathic symptoms. We propose that lipid supplementation is an easily translatable potential therapeutic approach in CMT1A and possibly other dysmyelinating neuropathies.

ALS-Associated Endoplasmic Reticulum Proteins in Denervated Skeletal Muscle: Implications for Motor Neuron Disease Pathology.

Alpha-motoneurons and muscle fibres are structurally and functionally interdependent. Both cell types particularly rely on endoplasmic reticulum (ER/SR) functions. Mutations of the ER proteins VAPB, SigR1 and HSP27 lead to hereditary motor neuron diseases (MNDs). Here, we determined the expression profile and localization of these ER proteins/chaperons by immunohistochemistry and immunoblotting in biopsy and autopsy muscle tissue of patients with amyotrophic lateral sclerosis (ALS) and other neurogenic muscular atrophies (NMAs) and compared these patterns to mouse models of neurogenic muscular atrophy. Postsynaptic neuromuscular junction staining for VAPB was intense in normal human and mouse muscle and decreased in denervated Nmd2J mouse muscle fibres. In contrast, VAPB levels together with other chaperones and autophagy markers were increased in extrasynaptic regions of denervated muscle fibres of patients with MNDs and other NMAs, especially at sites of focal myofibrillar disintegration (targets). These findings did not differ between NMAs due to ALS and other causes. G93A-SOD1 mouse muscle fibres showed a similar pattern of protein level increases in denervated muscle fibres. In addition, they showed globular VAPB-immunoreactive structures together with misfolded SOD1 protein accumulations, suggesting a primary myopathic change. Our findings indicate that altered expression and localization of these ER proteins and autophagy markers are part of the dynamic response of muscle fibres to denervation. The ER is particularly prominent and vulnerable in both muscle fibres and alpha-motoneurons. Thus, ER pathology could contribute to the selective build-up of degenerative changes in the neuromuscular axis in MNDs.

Loss of function of the ALS protein SigR1 leads to ER pathology associated with defective autophagy and lipid raft disturbances.

Intracellular accumulations of altered, misfolded proteins in neuronal and other cells are pathological hallmarks shared by many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Mutations in several genes give rise to familial forms of ALS. Mutations in Sigma receptor 1 have been found to cause a juvenile form of ALS and frontotemporal lobar degeneration (FTLD). We recently described altered localization, abnormal modification and loss of function of SigR1 in sporadic ALS. In order to further elucidate the molecular mechanisms underlying SigR1-mediated alterations in sporadic and familial ALS, we extended our previous studies using neuronal SigR1 knockdown cell lines. We found that loss of SigR1 leads to abnormal ER morphology, mitochondrial abnormalities and impaired autophagic degradation. Consistent with these results, we found that endosomal trafficking of EGFR is impaired upon SigR1 knockdown. Furthermore, in SigR1-deficient cells the transport of vesicular stomatitis virus glycoprotein is inhibited, leading to the accumulation of this cargo protein in the Golgi apparatus. Moreover, depletion of SigR1 destabilized lipid rafts and associated calcium mobilization, confirming the crucial role of SigR1 in lipid raft and intracellular calcium homeostasis. Taken together, our results support the notion that loss of SigR1 function contributes to ALS pathology by causing abnormal ER morphology, lipid raft destabilization and defective endolysosomal pathways.

Altered localization, abnormal modification and loss of function of Sigma receptor-1 in amyotrophic lateral sclerosis.

Intracellular accumulations of mutant, misfolded proteins are major pathological hallmarks of amyotrophic lateral sclerosis (ALS) and related disorders. Recently, mutations in Sigma receptor 1 (SigR1) have been found to cause a form of ALS and frontotemporal lobar degeneration (FTLD). Our goal was to pinpoint alterations and modifications of SigR1 in ALS and to determine how these changes contribute to the pathogenesis of ALS. In the present study, we found that levels of the SigR1 protein were reduced in lumbar ALS patient spinal cord. SigR1 was abnormally accumulated in enlarged C-terminals and endoplasmic reticulum (ER) structures of alpha motor neurons. These accumulations co-localized with the 20s proteasome subunit. SigR1 accumulations were also observed in SOD1 transgenic mice, cultured ALS-8 patient's fibroblasts with the P56S-VAPB mutation and in neuronal cell culture models. Along with the accumulation of SigR1 and several other proteins involved in protein quality control, severe disturbances in the unfolded protein response and impairment of protein degradation pathways were detected in the above-mentioned cell culture systems. Furthermore, shRNA knockdown of SigR1 lead to deranged calcium signaling and caused abnormalities in ER and Golgi structures in cultured NSC-34 cells. Finally, pharmacological activation of SigR1 induced the clearance of mutant protein aggregates in these cells. Our results support the notion that SigR1 is abnormally modified and contributes to the pathogenesis of ALS.

Bortezomib-induced severe autonomic neuropathy.

Peripheral neuropathy is a known side effect of bortezomib therapy. Acute autonomic neuropathy may also follow treatment with this cytotoxic agent used for treatment of multiple myeloma. Here, we report clinical characteristics and patterns of autonomic involvement in a 75-year-old patient who presented with recurring syncopes.

Small-fiber neuropathy in patients with ALS.

OBJECTIVE: To investigate the involvement of the epidermal small sensory fibers in the neurodegenerative process in amyotrophic lateral sclerosis (ALS). METHODS: In the present study, skin biopsies of 28 patients with ALS were obtained at an average of 34 months after disease onset by history. Protein gene product 9.5 (PGP9.5) immunohistochemistry findings were compared to 17 age-matched controls. The primary endpoint of the study was to evaluate the decrease in the density of small intraepidermal nerve fibers and to compare the prevalence of small-fiber neuropathy in patients with ALS and in controls. RESULTS: We found a significant reduction in epidermal nerve fiber density in the distal calf of patients with ALS (4.8 ± 3.7 fibers/mm vs 12.2 ± 4.6 in age-matched controls, p<0.0001). The extent of fiber loss was age-dependent. Also, the number of subjects with small-fiber neuropathy was significantly higher in the ALS group than in the controls (79% vs 12%). Correspondingly, mild sensory symptoms including diffuse dysesthesias, paresthesias, and hypesthesia were found in 7 patients. In 17 biopsies of patients with ALS, but only in 2 controls, we saw larger (>1.5 µm in diameter) focal swellings of epidermal axons resembling spheroids, suggesting trafficking defects. CONCLUSIONS: These results indicate that small, distal epidermal nerve fibers are involved in this disease, supporting the concept of distal axonopathy in ALS.

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.

Interneurons are the local targets of hippocampal inhibitory cells which project to the medial septum.

A subset of GABAergic neurons projecting to the medial septum has long been described in the hippocampus. However, the lack of information about their local connectivity pattern or their correspondence with any of the well-established hippocampal interneuron types has hampered the understanding of their functional role. Retrograde tracing combined with immunostaining for neurochemical markers in the adult rat hippocampus showed that nearly all hippocampo-septal (HS) neurons express somatostatin (>95%) and, in the hilus and CA3 stratum lucidum, many contain calretinin (>45%). In contrast, in stratum oriens of the CA1 and CA3 subfields, the majority of HS neurons contain somatostatin (>86%) and calbindin (>73%), but not calretinin. Because somatostatin-positive hippocampal interneurons have been most extensively characterized in the stratum oriens of CA1, we focused our further analysis on HS cells found in this region. In 18-20-day-old rats, intracellularly filled CA1-HS cells had extensive local axon collaterals crossing subfield boundaries and innervating the CA3 region and the dentate gyrus. Electron microscopic analysis provided evidence that the axon terminals of CA1-HS cells form symmetrical synapses selectively on GABAergic interneurons, both locally and in the CA3 region. In addition, double retrograde labelling experiments revealed that many CA1-HS neurons of the dorsal hippocampus also have collateral projections to the ventral hippocampus. Thus, CA1-HS cells innervate inhibitory interneurons locally and in remote hippocampal regions, in addition to targeting mostly GABAergic neurons in the medial septum. This dual projection with striking target selectivity for GABAergic neurons may be ideally suited to synchronize neuronal activity along the septo-hippocampal axis.

Brain monoglyceride lipase participating in endocannabinoid inactivation.

The endogenous cannabinoids (endocannabinoids) are lipid molecules that may mediate retrograde signaling at central synapses and other forms of short-range neuronal communication. The monoglyceride 2-arachidonoylglycerol (2-AG) meets several criteria of an endocannabinoid substance: (i) it activates cannabinoid receptors; (ii) it is produced by neurons in an activity-dependent manner; and (iii) it is rapidly eliminated. 2-AG inactivation is only partially understood, but it may occur by transport into cells and enzymatic hydrolysis. Here we tested the hypothesis that monoglyceride lipase (MGL), a serine hydrolase that converts monoglycerides to fatty acid and glycerol, participates in 2-AG inactivation. We cloned MGL by homology from a rat brain cDNA library. Its cDNA sequence encoded for a 303-aa protein with a calculated molecular weight of 33,367 daltons. Northern blot and in situ hybridization analyses revealed that MGL mRNA is heterogeneously expressed in the rat brain, with highest levels in regions where CB(1) cannabinoid receptors are also present (hippocampus, cortex, anterior thalamus, and cerebellum). Immunohistochemical studies in the hippocampus showed that MGL distribution has striking laminar specificity, suggesting a presynaptic localization of the enzyme. Adenovirus-mediated transfer of MGL cDNA into rat cortical neurons increased MGL expression and attenuated N-methyl-D-aspartate/carbachol-induced 2-AG accumulation in these cells. No such effect was observed on the accumulation of anandamide, another endocannabinoid lipid. The results suggest that hydrolysis by means of MGL is a primary mechanism for 2-AG inactivation in intact neurons.

Evidence for presynaptic cannabinoid CB(1) receptor-mediated inhibition of noradrenaline release in the guinea pig lung.

Using neurochemical method, evidence was obtained that cannabinoid CB(1) receptors are localized on noradrenergic terminals and their stimulation by WIN-55,212-2 reduces the release of [3H]noradrenaline evoked by axonal activity in a frequency-dependent manner. At stimulation rates of 1 and 3 Hz, there was significant inhibition of noradrenaline release, with IC(50) of WIN-55,212-2 41.5+/-2.6 and 320.5+/-28.2 nM, for 1 and 3 Hz, respectively. Cannabinoid CB(1) receptor antagonist SR 141716A completely prevented WIN-55,212-2 from reducing the release. The release of noradrenaline is negatively modulated by presynaptic alpha(2)-adrenoceptors. Because BRL-44408, an alpha(2B)-adrenoceptor, and prazosin, an alpha(1)- and alpha(2B)-adrenoceptor antagonist, both increased the release of [(3)H]noradrenaline, it seems likely that the alpha(2B) subtype is responsible for the negative feedback modulation of noradrenaline release. In the presence of alpha(2)-adrenoceptor antagonism, cannabinoid CB(1) receptor activation by WIN-55,212-2 was much more effective in inhibiting the release of [(3)H]noradrenaline. Using a specific antibody against the C-terminus of the rat cannabinoid CB(1) receptor and also against neuropeptide Y, ultrastructural evidence was obtained that cannabinoid CB(1) receptors are exclusively localized on neuropeptide Y-positive noradrenergic varicosities. Since the sympathetic innervation of the human airway smooth muscle is sparse, and mainly the circulating adrenaline relaxes the airways via activation of beta(2)-adrenoceptor localized on the smooth muscle, it is suggested that inhibition of noradrenaline release by cannabinoids, and the subsequent bronchospasm, may be limited to those cases when noradrenaline released from sympathetic varicosities is involved in airway relaxation.

Distribution of CB1 cannabinoid receptors in the amygdala and their role in the control of GABAergic transmission.

Cannabinoids are the most popular illicit drugs used for recreational purposes worldwide. However, the neurobiological substrate of their mood-altering capacity has not been elucidated so far. Here we report that CB1 cannabinoid receptors are expressed at high levels in certain amygdala nuclei, especially in the lateral and basal nuclei, but are absent in other nuclei (e.g., in the central nucleus and in the medial nucleus). Expression of the CB1 protein was restricted to a distinct subpopulation of GABAergic interneurons corresponding to large cholecystokinin-positive cells. Detailed electron microscopic investigation revealed that CB1 receptors are located presynaptically on cholecystokinin-positive axon terminals, which establish symmetrical GABAergic synapses with their postsynaptic targets. The physiological consequence of this particular anatomical localization was investigated by whole-cell patch-clamp recordings in principal cells of the lateral and basal nuclei. CB1 receptor agonists WIN 55,212-2 and CP 55,940 reduced the amplitude of GABA(A) receptor-mediated evoked and spontaneous IPSCs, whereas the action potential-independent miniature IPSCs were not significantly affected. In contrast, CB1 receptor agonists were ineffective in changing the amplitude of IPSCs in the rat central nucleus and in the basal nucleus of CB1 knock-out mice. These results suggest that cannabinoids target specific elements in neuronal networks of given amygdala nuclei, where they presynaptically modulate GABAergic synaptic transmission. We propose that these anatomical and physiological features, characteristic of CB1 receptors in several forebrain regions, represent the neuronal substrate for endocannabinoids involved in retrograde synaptic signaling and may explain some of the emotionally relevant behavioral effects of cannabinoid exposure.

Validation of the Childhood Health Assessment Questionnaire in the juvenile idiopathic myopathies. Juvenile Dermatomyositis Disease Activity Collaborative Study Group.

OBJECTIVE: To examine the validity of the Childhood Health Assessment Questionnaire (CHAQ) in patients with juvenile idiopathic inflammatory myopathy (IIM). METHODS: One hundred fifteen patients were enrolled in a multicenter collaborative study, during which subjects were assessed twice, 7-9 months apart. Physical function was measured using the CHAQ. Internal reliability was assessed using adjusted item-total correlations and item endorsement rates. Construct validity was assessed by comparing predicted and actual correlations of the CHAQ with other measures of physical function and disease activity. Responsiveness was assessed by calculating effect size (ES) and standardized response mean (SRM) in a group of a priori defined "improvers." RESULTS: Item-total correlations were high (rs range = 0.35-0.81), suggesting all items were related to overall physical function. Manual muscle testing and the Childhood Myositis Assessment Scale correlated moderate to strongly with the CHAQ (r = -0.64 and -0.75, both p < 0.001). Moderate correlations were also seen with the physician global assessment of disease activity (rs = 0.58, p < 0.001), parent global assessment of overall health (rs = -0.65, p < 0.001), Steinbrocker function class (rs = 0.69, p < 0.001), and global skin activity (rs = 0.40, p < 0.001), while global disease damage and skin damage had low correlations (rs = 0.13 and 0.07, p > or =0.17). Responsiveness of the CHAQ was high, with ES = 1.05 and SRM = 1.20. CONCLUSION: In this large cohort of patients with juvenile IIM, the CHAQ exhibited internal reliability, construct validity, and strong responsiveness. We conclude that the CHAQ is a valid measure of physical function in juvenile IIM, appropriate for use in therapeutic trials, and potentially in the clinical care of these patients.

Bidirectional control of airway responsiveness by endogenous cannabinoids.

Smoking marijuana or administration of its main active constituent, delta9-tetrahydrocannabinol (delta9-THC), may exert potent dilating effects on human airways. But the physiological significance of this observation and its potential therapeutic value are obscured by the fact that some asthmatic patients respond to these compounds with a paradoxical bronchospasm. The mechanisms underlying these contrasting responses remain unresolved. Here we show that the endogenous cannabinoid anandamide exerts dual effects on bronchial responsiveness in rodents: it strongly inhibits bronchospasm and cough evoked by the chemical irritant, capsaicin, but causes bronchospasm when the constricting tone exerted by the vagus nerve is removed. Both effects are mediated through peripheral CB1 cannabinoid receptors found on axon terminals of airway nerves. Biochemical analyses indicate that anandamide is synthesized in lung tissue on calcium-ion stimulation, suggesting that locally generated anandamide participates in the intrinsic control of airway responsiveness. In support of this conclusion, the CB1 antagonist SR141716A enhances capsaicin-evoked bronchospasm and cough. Our results may account for the contrasting bronchial actions of cannabis-like drugs in humans, and provide a framework for the development of more selective cannabinoid-based agents for the treatment of respiratory pathologies.

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus.

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [3H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [3H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A. This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.

Cannabinoids inhibit hippocampal GABAergic transmission and network oscillations.

Using a new antibody developed against the C-terminus of the cannabinoid receptor (CB1), the immunostaining in the hippocampus revealed additional axon terminals relative to the pattern reported previously with an N-terminus antibody. Due to a greater sensitivity of this antibody, a large proportion of boutons in the dendritic layers displaying symmetrical (GABAergic) synapses were also strongly immunoreactive for CB1 receptors, as were axon terminals of perisomatic inhibitory cells containing cholecystokinin. Asymmetrical (glutamatergic) synapses, however, were always negative for CB1. To investigate the effect of presynaptic CB1 receptor activation on hippocampal inhibition, we recorded inhibitory postsynaptic currents (IPSCs) from principal cells. Bath application of CB1 receptor agonists (WIN55,212-2 and CP55,940) suppressed IPSCs evoked by local electrical stimulation, which could be prevented or reversed by the CB1 receptor antagonist SR141716A. Action potential-driven IPSCs, evoked by pharmacological stimulation of a subset of interneurons, were also decreased by CB1 receptor activation. We also examined the effects of CB1 receptor agonists on Ca2+-independent miniature IPSCs (mIPSC). Both agonists were without significant effect on the frequency or amplitude of mIPSCs. Synchronous gamma oscillations induced by kainic acid in the CA3 region of hippocampal slices were reversibly reduced in amplitude by the CB1 receptor agonist CP 55,940, which is consistent with an action on IPSCs. We used CB1-/- knock-out mice to confirm the specificity of the antibody and of the agonist (WIN55,212-2) action. We conclude that activation of presynaptic CB1 receptors decreases Ca2+-dependent GABA release, and thereby reduces the power of hippocampal network oscillations.

Unusual target selectivity of perisomatic inhibitory cells in the hilar region of the rat hippocampus.

Perisomatic inhibitory innervation of all neuron types profoundly affects their firing characteristics and vulnerability. In this study we examined the postsynaptic targets of perisomatic inhibitory cells in the hilar region of the dentate gyrus where the proportion of potential target cells (excitatory mossy cells and inhibitory interneurons) is approximately equal. Both cholecystokinin (CCK)- and parvalbumin-immunoreactive basket cells formed multiple contacts on the somata and proximal dendrites of mossy cells. Unexpectedly, however, perisomatic inhibitory terminals arriving from these cell types largely ignored hilar GABAergic cell populations. Eighty-ninety percent of various GABAergic neurons including other CCK-containing basket cells received no input from CCK-positive terminals. Parvalbumin-containing cells sometimes innervated each other but avoided 75% of other GABAergic cells. Overall, a single mossy cell received 40 times more CCK-immunoreactive terminals and 15 times more parvalbumin-positive terminals onto its soma than the cell body of an average hilar GABAergic cell. In contrast to the pronounced target selectivity in the hilar region, CCK- and parvalbumin-positive neurons innervated each other via collaterals in stratum granulosum and moleculare. Our observations indicate that the inhibitory control in the hilar region is qualitatively different from other cortical areas at both the network level and the level of single neurons. The paucity of perisomatic innervation of hilar interneurons should have profound consequences on their action potential generation and on their ensemble behavior. These findings may help explain the unique physiological patterns observed in the hilus and the selective vulnerability of the hilar cell population in various pathophysiological conditions.

Cholinergic innervation of mossy cells in the rat fascia dentata.

Hilar mossy cells are the main cells of origin of the commissural/associational projection to the inner molecular layer of the rat fascia dentata. In order to analyze the cholinergic innervation of hilar mossy cells, a light and electron microscopic double-labeling technique was used. Immunolabeling for calcitonin gene-related peptide (CGRP) was employed to identify mossy cells and immunocytochemistry for choline acetyltransferase (ChAT) was used to label cholinergic septohippocampal fibers. Cholinergic boutons were abundant around mossy cell somata and on their proximal dendrites. Electron microscopy confirmed that many of these boutons formed synapses with the CGRP-positive mossy cells. These data demonstrate a direct innervation of hilar mossy cells by cholinergic septohippocampal afferents. This connectivity could contribute to the electrophysiological behavior of mossy cells during theta oscillations.

Presynaptically located CB1 cannabinoid receptors regulate GABA release from axon terminals of specific hippocampal interneurons.

To understand the functional significance and mechanisms of action in the CNS of endogenous and exogenous cannabinoids, it is crucial to identify the neural elements that serve as the structural substrate of these actions. We used a recently developed antibody against the CB1 cannabinoid receptor to study this question in hippocampal networks. Interneurons with features typical of basket cells showed a selective, intense staining for CB1 in all hippocampal subfields and layers. Most of them (85.6%) contained cholecystokinin (CCK), which corresponded to 96.9% of all CCK-positive interneurons, whereas only 4.6% of the parvalbumin (PV)-containing basket cells expressed CB1. Accordingly, electron microscopy revealed that CB1-immunoreactive axon terminals of CCK-containing basket cells surrounded the somata and proximal dendrites of pyramidal neurons, whereas PV-positive basket cell terminals in similar locations were negative for CB1. The synthetic cannabinoid agonist WIN 55,212-2 (0.01-3 microM) reduced dose-dependently the electrical field stimulation-induced [3H]GABA release from superfused hippocampal slices, with an EC50 value of 0. 041 microM. Inhibition of GABA release by WIN 55,212-2 was not mediated by inhibition of glutamatergic transmission because the WIN 55,212-2 effect was not reduced by the glutamate blockers AP5 and CNQX. In contrast, the CB1 cannabinoid receptor antagonist SR 141716A (1 microM) prevented this effect, whereas by itself it did not change the outflow of [3H]GABA. These results suggest that cannabinoid-mediated modulation of hippocampal interneuron networks operate largely via presynaptic receptors on CCK-immunoreactive basket cell terminals. Reduction of GABA release from these terminals is the likely mechanism by which both endogenous and exogenous CB1 ligands interfere with hippocampal network oscillations and associated cognitive functions.

Postsynaptic targets of somatostatin-immunoreactive interneurons in the rat hippocampus.

Two characteristic interneuron types in the hippocampus, the so-called hilar perforant path-associated cells in the dentate gyrus and stratum oriens/lacunosum-moleculare neurons in the CA3 and CA1 regions, were suggested to be involved in feedback circuits. In the present study, interneurons identical to these cell populations were visualized by somatostatin-immunostaining, then reconstructed, and processed for double-immunostaining and electron microscopy to establish their postsynaptic target selectivity. A combination of somatostatin-immunostaining with immunostaining for GABA or other interneuron markers revealed a quasi-random termination pattern. The vast majority of postsynaptic targets were GABA-negative dendritic shafts and spines of principal cells (76%), whereas other target elements contained GABA (8%). All of the examined neurochemically defined interneuron types (parvalbumin-, calretinin-, vasoactive intestinal polypeptide-, cholecystokinin-, substance P receptor-immunoreactive neurons) received innervation from somatostatin-positive boutons. Recent anatomical and electrophysiological data showed that the main excitatory inputs of somatostatin-positive interneurons originate from local principal cells. The present data revealed a massive GABAergic innervation of distal dendrites of local principal cells by these feedback driven neurons, which are proposed to control the efficacy and plasticity of entorhinal synaptic input as a function of local principal cell activity and synchrony.

Characterization of early activation events in cord blood B cells after stimulation with T cell-independent activators.

Human neonates are immunologically immature, particularly in their humoral antibody responses to T cell-independent antigens, as exemplified by their increased susceptibility to infections with polysaccharide-encapsulated bacteria. To clarify the mechanism(s) underlying the unresponsiveness of neonates to polysaccharide antigens, we used an in vitro model with neonatal cord blood cells that has been shown to mimic surface Ig-dependent signaling in the adult by T cell-independent antigens. We studied the ability of cord blood human B cells to become activated after ligation of their surface Ig by unconjugated anti-Ig, dextran-conjugated anti-Ig, and Staphylococcus aureus Cowan A1, and compared their response with that of adult B cells. After the addition of nanogram concentrations of anti-Ig-dextran, neonatal cord blood B cells proliferated at levels comparable to that observed with adult B cells. The majority of cord blood B cells showed a marked rise in intracellular calcium, increased surface expression of human leukocyte antigen DR, and an increase in cell size. Direct activation of protein kinase C by phorbol esters in neonatal B cells led to cellular proliferation, and when combined with anti-Ig, a synergistic effect on proliferation was observed. These data suggest that the unresponsiveness of human neonates to polysaccharide antigens does not represent an inability of these antigens to induce early activation events in circulating B cells.