Juan Mendizabal-Zubiaga

Acute Cannabinoids Impair Working Memory through Astroglial CB1 Receptor Modulation of Hippocampal LTD

Impairment of working memory is one of the most important deleterious effects of marijuana intoxication in humans, but its underlying mechanisms are presently unknown. Here, we demonstrate that the impairment of spatial working memory (SWM) and in vivo long-term depression (LTD) of synaptic strength at hippocampal CA3-CA1 synapses, induced by an acute exposure of exogenous cannabinoids, is fully abolished in conditional mutant mice lacking type-1 cannabinoid receptors (CB(1)R) in brain astroglial cells but is conserved in mice lacking CB(1)R in glutamatergic or GABAergic neurons. Blockade of neuronal glutamate N-methyl-D-aspartate receptors (NMDAR) and of synaptic trafficking of glutamate α-amino-3-hydroxy-5-methyl-isoxazole propionic acid receptors (AMPAR) also abolishes cannabinoid effects on SWM and LTD induction and expression. We conclude that the impairment of working memory by marijuana and cannabinoids is due to the activation of astroglial CB(1)R and is associated with astroglia-dependent hippocampal LTD in vivo.

Astroglial CB1 cannabinoid receptors regulate leptin signaling in mouse brain astrocytes

Type-1 cannabinoid (CB1) and leptin (ObR) receptors regulate metabolic and astroglial functions, but the potential links between the two systems in astrocytes were not investigated so far. Genetic and pharmacological manipulations of CB1 receptor expression and activity in cultured cortical and hypothalamic astrocytes demonstrated that cannabinoid signaling controls the levels of ObR expression. Lack of CB1 receptors also markedly impaired leptin-mediated activation of signal transducers and activators of transcription 3 and 5 (STAT3 and STAT5) in astrocytes. In particular, CB1 deletion determined a basal overactivation of STAT5, thereby leading to the downregulation of ObR expression, and leptin failed to regulate STAT5-dependent glycogen storage in the absence of CB1 receptors. These results show that CB1 receptors directly interfere with leptin signaling and its ability to regulate glycogen storage, thereby representing a novel mechanism linking endocannabinoid and leptin signaling in the regulation of brain energy storage and neuronal functions.

GABA release by hippocampal astrocytes.

Astrocytes can directly influence neuronal activity through the release of various transmitters acting on membrane receptors expressed by neurons. However, in contrast to glutamate and ATP for instance, the release of GABA (γ-amino-butyric acid) by astrocytes is still poorly documented. Here, we used whole-cell recordings in rat acute brain slices and electron microscopy to test whether hippocampal astrocytes release the inhibitory transmitter GABA. We observed that slow transient inhibitory currents due to the activation of GABAA receptors occur spontaneously in principal neurons of the three main hippocampal fields (CA1, CA3, and dentate gyrus). These currents share characteristics with the slow NMDA receptor-mediated currents previously shown to result from astrocytic glutamate release: they occur in the absence of synaptic transmission and have variable kinetics and amplitudes as well as low frequencies. Osmotic pressure reduction, known to enhance transmitter release from astrocytes, similarly increased the frequency of non-synaptic GABA and glutamate currents. Simultaneous occurrence of slow inhibitory and excitatory currents was extremely rare. Yet, electron microscopy examination of immunostained hippocampal sections shows that about 80% of hippocampal astrocytes [positive for glial fibrillary acidic protein (GFAP)] were immunostained for GABA. Our results provide quantitative characteristics of the astrocyte-to-neuron GABAergic signaling. They also suggest that all principal neurons of the hippocampal network are under a dual, excitatory and inhibitory, influence of astrocytes. The relevance of the astrocytic release of GABA, and glutamate, on the physiopathology of the hippocampus remains to be established.

The underside of the cerebral cortex: layer V/VI spiny inverted neurons

This paper presents an account of past and current research on spiny inverted neurons – alternatively also known as ‘inverted pyramidal neurons’– in rats, rabbits and cats. In our laboratory, we have studied these cells with a battery of techniques suited for light and electron microscopy, including Nissl staining, Golgi impregnation, dye intracellular filling and axon retrograde track‐tracing. Our results show that spiny inverted neurons make up less than 8.5 and 5.5% of all cortical neurons in the primary and secondary rabbit visual cortex, respectively. Infragranular spiny inverted neurons constitute 15 and 8.5% of infragranular neurons in the same animal and areas. Spiny inverted neurons congregate at layers V–VI in all studied species. Studies have also revealed that spiny inverted neurons are excitatory neurons which furnish axons for various cortico‐cortical, cortico‐claustral and cortico‐striatal projections, but not for non‐telencephalic centres such as the lateral and medial geniculate nuclei, the colliculi or the pons. As a group, each subset of inverted cells contributing to a given projection is located below the pyramidal neurons whose axons furnish the same centre. Spiny inverted neurons are particularly conspicuous as a source of the backward cortico‐cortical projection to primary visual cortex and from this to the claustrum. Indeed, they constitute up to 82% of the infragranular cells that furnish these projections. Spiny inverted neurons may be classified into three subtypes according to the point of origin of the axon on the cell: the somatic basal pole which faces the cortical outer surface, the somatic flank and the reverse apical dendrite. As seen with electron microscopy, the axon initial segments of these subtypes are distinct from one another, not only in length and thickness, but also in the number of received synaptic boutons. All of these anatomical features together may support a synaptic‐input integration which is peculiar to spiny inverted neurons. In this way, two differently qualified streams of axonal output may coexist in a projection which arises from a particular infragranular point within a given cortical area; one stream would be furnished by the typical pyramidal neurons, whereas spiny inverted neurons would constitute the other source of distinct information flow.

GABAergic and cortical and subcortical glutamatergic axon terminals contain CB1 cannabinoid receptors in the ventromedial nucleus of the hypothalamus.

Background Type-1 cannabinoid receptors (CB1R) are enriched in the hypothalamus, particularly in the ventromedial hypothalamic nucleus (VMH) that participates in homeostatic and behavioral functions including food intake. Although CB1R activation modulates excitatory and inhibitory synaptic transmission in the brain, CB1R contribution to the molecular architecture of the excitatory and inhibitory synaptic terminals in the VMH is not known. Therefore, the aim of this study was to investigate the precise subcellular distribution of CB1R in the VMH to better understand the modulation exerted by the endocannabinoid system on the complex brain circuitries converging into this nucleus. Methodology/Principal Findings Light and electron microscopy techniques were used to analyze CB1R distribution in the VMH of CB1R-WT, CB1R-KO and conditional mutant mice bearing a selective deletion of CB1R in cortical glutamatergic (Glu-CB1R-KO) or GABAergic neurons (GABA-CB1R-KO). At light microscopy, CB1R immunolabeling was observed in the VMH of CB1R-WT and Glu-CB1R-KO animals, being remarkably reduced in GABA-CB1R-KO mice. In the electron microscope, CB1R appeared in membranes of both glutamatergic and GABAergic terminals/preterminals. There was no significant difference in the percentage of CB1R immunopositive profiles and CB1R density in terminals making asymmetric or symmetric synapses in CB1R-WT mice. Furthermore, the proportion of CB1R immunopositive terminals/preterminals in CB1R-WT and Glu-CB1R-KO mice was reduced in GABA-CB1R-KO mutants. CB1R density was similar in all animal conditions. Finally, the percentage of CB1R labeled boutons making asymmetric synapses slightly decreased in Glu-CB1R-KO mutants relative to CB1R-WT mice, indicating that CB1R was distributed in cortical and subcortical excitatory synaptic terminals. Conclusions/Significance Our anatomical results support the idea that the VMH is a relevant hub candidate in the endocannabinoid-mediated modulation of the excitatory and inhibitory neurotransmission of cortical and subcortical pathways regulating essential hypothalamic functions for the individuals survival such as the feeding behavior.

The transient receptor potential vanilloid-1 is localized at excitatory synapses in the mouse dentate gyrus

The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel that plays an important role in pain perception and modulates neurotransmitter release and synaptic plasticity in the brain. TRPV1 function must lay on its anatomical distribution in the peripheral and central nervous system regions involved in the physiological roles of the channel. However, the anatomical localization of TRPV1 is well established in the periphery, but in the brain it is a matter of debate. While some studies support the presence of TRPV1 in several brain regions, recent evidences suggest a restricted distribution of the channel in the central nervous system. To investigate to what extent central TRPV1 function stands on a precise brain distribution of the channel, we examined the mouse hippocampal dentate molecular layer (ML) where TRPV1 mediates long-term synaptic plasticity. Using pre-embedding immunocytochemistry for high resolution electron microscopy, we show that TRPV1 immunoparticles are highly concentrated in postsynaptic dendritic spines to asymmetric perforant path synapses in the outer 2/3 of the ML. However, TRPV1 is poorly expressed at the excitatory hilar mossy cell synapses in the inner 1/3 of this layer. Importantly, the TRPV1 pattern distribution disappeared in the ML of TRPV1-knockout mice. Taken together, these findings support the notion of the presence of TRPV1 in a brain region where the channel has been shown to have a functional role, such as the perforant path synapses in the hippocampal dentate ML.

Precise localization of the voltage-gated potassium channel subunits Kv3.1b and Kv3.3 revealed in the molecular layer of the rat cerebellar cortex by a pre-embedding immunogold method

A proper motor activity relies on a correct cerebellar function. The Kv3.1 and Kv3.3 voltage-gated potassium channels are key proteins involved in cerebellar function and dysfunction, as the lack of these causes severe motor deficits. Both channel subunits are coexpressed in granule cells and are rapidly activated at relatively positive potentials to support the generation of fast action potentials. However, the contribution of each subunit to the molecular architecture of the parallel fibers, the granule cell axons, is so far unknown. The goal of this study was to elucidate the relative distribution of Kv3.1b and Kv3.3 in specific compartments of the rat parallel fibers by using a pre-embedding immunocytochemical method for electron microscopy. Numerous Kv3.1b and Kv3.3 silver-intensified gold particles were associated with membranes of parallel fiber synaptic terminals and their intervaricose segments. Kv3.1b was found in about 85% of parallel fiber synaptic terminals and in about 47% of their intervaricose portions. However, only 28% of intervaricosities and 23% of parallel fiber presynaptic boutons were Kv3.3 immunopositive. The analysis also revealed that 54% of Purkinje cell dendritic spines localized Kv3.3. Although both potassium channel subunits share localization in the same presynaptic parallel fiber compartments, the present results with the method used indicate that there are a higher percentage of parallel fibers labeled for Kv3.1b than for Kv3.3, and that the labeling intensity for each subunit is higher in specific subcompartments analyzed than in others.

Neuroepithelial origin of the insular and endopiriform parts of the claustrum

The lateral and ventral pallia have been proposed as the source of neurons for the insular and endopiriform claustra, respectively. However, this correlation is controversial. Here, we analysed this relationship by labelling radial glia in coronal slices of the telencephalon of paraformaldehyde-fixed rabbit embryos (E18-E28) and newborn rabbits with an anti-vimentin antibody or with the fluorescent dye DiI. The radial glia that crossed the claustrum was anchored to the neuroepithelium of the lateral ventricular angle (LVA) at all ages studied. The LVA was deep at E18, but it subsequently become shallower, because of the apposition of the portion of its walls proximal to the vertex of the LVA. At E18, the radial glia that crossed most of the insular claustrum extended from the lateral wall of the LVA (presumptive lateral pallium), and the radial glia that crossed either the most ventral part of the insular claustrum or the endopiriform claustrum proceeded from the medial wall of the LVA (presumptive ventral pallium). These results suggest that although the endopiriform claustrum originates from the ventral pallium, the insular claustrum originates from both the lateral and the ventral pallial portions.

Cannabinoid CB1 Receptors Are Localized in Striated Muscle Mitochondria and Regulate Mitochondrial Respiration.

The cannabinoid type 1 (CB1) receptor is widely distributed in the brain and peripheral organs where it regulates cellular functions and metabolism. In the brain, CB1 is mainly localized on presynaptic axon terminals but is also found on mitochondria (mtCB1), where it regulates cellular respiration and energy production. Likewise, CB1 is localized on muscle mitochondria, but very little is known about it. The aim of this study was to further investigate in detail the distribution and functional role of mtCB1 in three different striated muscles. Immunoelectron microscopy for CB1 was used in skeletal muscles (gastrocnemius and rectus abdominis) and myocardium from wild-type and CB1-KO mice. Functional assessments were performed in mitochondria purified from the heart of the mice and the mitochondrial oxygen consumption upon application of different acute delta-9-tetrahydrocannabinol (Δ9-THC) concentrations (100 nM or 200 nM) was monitored. About 26% of the mitochondrial profiles in gastrocnemius, 22% in the rectus abdominis and 17% in the myocardium expressed CB1. Furthermore, the proportion of mtCB1 versus total CB1 immunoparticles was about 60% in the gastrocnemius, 55% in the rectus abdominis and 78% in the myocardium. Importantly, the CB1 immunolabeling pattern disappeared in muscles of CB1-KO mice. Functionally, acute 100 nM or 200 nM THC treatment specifically decreased mitochondria coupled respiration between 12 and 15% in wild-type isolated mitochondria of myocardial muscles but no significant difference was noticed between THC treated and vehicle in mitochondria isolated from CB1-KO heart. Furthermore, gene expression of key enzymes involved in pyruvate synthesis, tricarboxylic acid (TCA) cycle and mitochondrial respiratory chain was evaluated in the striated muscle of CB1-WT and CB1-KO. CB1-KO showed an increase in the gene expression of Eno3, Pkm2, and Pdha1, suggesting an increased production of pyruvate. In contrast, no significant difference was observed in the Sdha and Cox4i1 expression, between CB1-WT and CB1-KO. In conclusion, CB1 receptors in skeletal and myocardial muscles are predominantly localized in mitochondria. The activation of mtCB1 receptors may participate in the mitochondrial regulation of the oxidative activity probably through the relevant enzymes implicated in the pyruvate metabolism, a main substrate for TCA activity.

Expression of calcium-binding proteins in the proliferative zones around the corticostriatal junction of rabbits during pre- and postnatal development

Herein we asked whether cells expressing calcium-binding proteins around the corticostriatal junction are of pallial or subpallial origin. Brains of rabbit embryos between embryonic day E18 and E28 and postnatal day 0-P22 were immunoreacted with monoclonal antibodies raised against calretinin, calbindin and parvalbumin. At E18-E21, calbindin- and calretinin-immunoreactive cells were seen in distinct proliferative zones in the vicinity of the corticostriatal junction. Whereas calbindin-immunoreactive neurons were in the ventricular zone of the ventral pallium (the medial wall of the lateral ventricular angle), calretinin-immunoreactive cells were, nearby, in the subventricular zone of the subpallium at the lateral edge of the lateral ganglionic eminence. From E25 to P22, both calbindin- and calretinin-immunoreactive cells appeared in the pallial ventricular and subventricular zones around the lateral ventricular angle. Some of these cells resembled migratory neuroblasts. Parvalbumin-immunoreactive cells appeared at P5-P10, albeit they were almost negligible in the proliferative zones around the corticostriatal junction and the lateral ventricular angle. The results suggest that a number of the calbindin-expressing neurons that are generated in mid-gestation and postnatally are of pallial origin. They also indicate that only a few of the late-generated calretinin-immunoreactive cells may have a pallial source. The origin of the parvalbumin-immunoreactive cells was not ascertained in the present study.