Quantitative ultrastructural analysis of basket and axo-axonic cell terminals in the mouse hippocampus

Three functionally different populations of perisomatic interneurons establish GABAergic synapses on hippocampal pyramidal cells: parvalbumin (PV)-containing basket cells, type 1 cannabinoid receptor (CB1)-positive basket cells both of which target somata, and PV-positive axo-axonic cells that innervate axon initial segments. Using electron microscopic reconstructions, we estimated that a pyramidal cell body receives synapses from about 60 and 140 synaptic terminals in the CA1 and CA3 area, respectively. About 60 % of these terminals were PV positive, whereas 35-40 % of them were CB1 positive. Only about 1 % (CA1) and 4 % (CA3) of the somatic boutons were negative for both markers. Using fluorescent labeling, we showed that most of the CB1-positive terminals expressed vesicular glutamate transporter 3. Reconstruction of somatic boutons revealed that although their volumes are similar, CB1-positive boutons are more flat and the total volume of their mitochondria was smaller than that of PV-positive boutons. Both types of boutons contain dense-core vesicles and frequently formed multiple release sites on their targets and innervated an additional soma or dendrite as well. PV-positive boutons possessed small, macular synapses; whereas the total synaptic area of CB1-positive boutons was larger and formed multiple irregular-shaped synapses. Axo-axonic boutons were smaller than somatic boutons, had only one synapse and their ultrastructural parameters were closer to those of PV-positive somatic boutons. Our results represent the first quantitative measurement-using a highly reliable method-of the contribution of different cell types to the perisomatic innervation of pyramidal neurons, and may help to explain functional differences in their output properties

Az endocannabinoid-mediált szignalizáció funkciója a hippocampus neuronhálózatainak normális és kóros működéseiben = The role of endocannabinoid-mediated signaling in normal and pathological operations os hippocampal circuits

Az OTKA pályázat támogatásával az elmúlt négy évben alapvető felismeréseket tettünk egy új kémiai szignálrendszer, az endokannabinoid rendszer molekuláris és anatómiai szerveződéséről, élettani és kórélettani jelentőségéről. Kimutattuk, hogy az endokannabinoid rendszer egy specializált jelátviteli rendszer, amelynek feladata, hogy a szinapszisok működését a preszinaptikus és a posztszinaptikus idegsejt aktivitásának függvényében szabályozza. Elsőként írtuk le egy endokannabinoid molekula, a 2-arachidonilglicerol kulcsszerepét ebben a folyamatban, és feltártuk, hogy milyen molekuláris mechanizmusok szabályozzák keletkezését és lebontását. Igazoltuk, hogy ez a kémiai szignálrendszer számos agyterületen (agykéregben, hippocampusban, nucleus accumbensben, a ventrális tegmentális areában) megtalálható. Élettani kísérleteink alapján az endokannabinoidok egy negatív visszacsatolási folyamat közvetítői a szinapszisokban. Ezzel párhuzamosan felfedeztük, hogy az endokannabinoid rendszer működési zavarai kulcsszerepet játszhatnak a temporális lebeny eredetű epilepsziában, valamint a szorongásos és poszttraumatikus stressz-okozta panaszok hátterében is megtalálhatók. Ezek az eredményeink egyben új molekuláris gyógyszercélpontokat jelölnek ki, amelyek számos idegrendszeri megbetegedésben vezethetnek hatékonyabb és szelektívebb farmakoterápia kidolgozásához. | During the last four years, our research group has reached fundamental milestones in the understanding of a new chemical messenger system, the so-called endocannabinoid system. We have uncovered the molecular and anatomical organization of endocannabinoid signaling and provided clues for its physiological and pathophysiological importance. We have shown that the endocannabinoid system is a specialized signaling machinery, which controls the efficacy synaptic transmission as a function of the activity of presynaptic and postsynaptic neurons. We have described for the first time that 2-arachidonoylglycerol is a key player in this process, and uncovered the basic mechanisms in its biosynthesis and degradation. We have provided evidence that this chemical signaling mechanism is a conserved feature of several types of synapses in various brain regions, for example in the neocortex, hippocampus, nucleus accumbens and the ventral tegmental area. We have shown that this system is involved in a negative feed-back regulation of transmitter release, and described its impairment in the human epileptic hippocampus, as well as its contribution to anxiety and post-traumatic stress disorder in animal models. These findings unravel new drug targets, whereby they could open novel therapeutic approaches for a more efficient and selective treatment of several brain disorders

Hippocampal GABAergic Synapses Possess the Molecular Machinery for Retrograde Nitric Oxide Signaling

Nitric oxide (NO) plays an important role in synaptic plasticity as a retrograde messenger at glutamatergic synapses. Here we describe that, in hippocampal pyramidal cells, neuronal nitric oxide synthase (nNOS) is also associated with the postsynaptic active zones of GABAergic symmetrical synapses terminating on their somata, dendrites, and axon initial segments in both mice and rats. The NO receptor nitric oxide-sensitive guanylyl cyclase (NOsGC) is present in the brain in two functional subunit compositions: alpha1beta1 and alpha2beta1. The beta1 subunit is expressed in both pyramidal cells and interneurons in the hippocampus. Using immunohistochemistry and in situ hybridization methods, we describe that the alpha1 subunit is detectable only in interneurons, which are always positive for beta1 subunit as well; however, pyramidal cells are labeled only for beta1 and alpha2 subunits. With double-immunofluorescent staining, we also found that most cholecystokinin- and parvalbumin-positive and smaller proportion of the somatostatin- and nNOS-positive interneurons are alpha1 subunit positive. We also found that the alpha1 subunit is present in parvalbumin- and cholecystokinin-positive interneuron terminals that establish synapses on somata, dendrites, or axon initial segments. Our results demonstrate that NOsGC, composed of alpha1beta1 subunits, is selectively expressed in different types of interneurons and is present in their presynaptic GABAergic terminals, in which it may serve as a receptor for NO produced postsynaptically by nNOS in the very same synapse

Cellular architecture and transmitter phenotypes of neurons of the mouse median raphe region

The median raphe region (MRR, which consist of MR and paramedian raphe regions) plays a crucial role in regulating cortical as well as subcortical network activity and behavior, while its malfunctioning may lead to disorders, such as schizophrenia, major depression, or anxiety. Mouse MRR neurons are classically identified on the basis of their serotonin (5-HT), vesicular glutamate transporter type 3 (VGLUT3), and gamma-aminobutyric acid (GABA) contents; however, the exact cellular composition of MRR regarding transmitter phenotypes is still unknown. Using an unbiased stereological method, we found that in the MR, 8.5 % of the neurons were 5-HT, 26 % were VGLUT3, and 12.8 % were 5-HT and VGLUT3 positive; whereas 37.2 % of the neurons were GABAergic, and 14.4 % were triple negative. In the whole MRR, 2.1 % of the neurons were 5-HT, 7 % were VGLUT3, and 3.6 % were 5-HT and VGLUT3 positive; whereas 61 % of the neurons were GABAergic. Surprisingly, 25.4 % of the neurons were triple negative and were only positive for the neuronal marker NeuN. PET-1/ePET-Cre transgenic mouse lines are widely used to specifically manipulate only 5-HT containing neurons. Interestingly, however, using the ePET-Cre transgenic mice, we found that far more VGLUT3 positive cells expressed ePET than 5-HT positive cells, and about 38 % of the ePET cells contained only VGLUT3, while more than 30 % of 5-HT cells were ePET negative. These data should facilitate the reinterpretation of PET-1/ePET related data in the literature and the identification of the functional role of a putatively new type of triple-negative neuron in the MRR

Divergent in vivo activity of non-serotonergic and serotonergic VGluT3-neurones in the median raphe region

KEY POINTS: *Median raphe is a key subcortical modulatory centre involved in several brain functions e.g. regulation of sleep-wake cycle, emotions and memory storage. *A large proportion of median raphe neurones are glutamatergic and implement a radically different mode of communication than serotonergic cells, but their in vivo activity is unknown. *We provide the first description of the in vivo, brain state-dependent firing properties of median raphe glutamatergic neurones identified by immunopositivity for the vesicular glutamate transporter type 3 (VGluT3) and serotonin (5HT). Glutamatergic populations (VGluT3+/5HT- and VGluT3+/5HT+)were compared to the purely serotonergic (VGluT3-/5HT+) and VGluT3-/5HT- neurones. *VGluT3+/5HT+ neurones fired similar to VGluT3-/5HT+ cells, whereas significantly diverged from the VGluT3+/5HT- population. Activity of the latter subgroup resembled the spiking of VGluT3-/5HT- cells, except their diverging response to sensory stimulation. *The VGluT3+ population of the median raphe may broadcast rapidly varying signals on top of a state-dependent, tonic modulation. ABSTRACT: Subcortical modulation is crucial for information processing in the cerebral cortex. Besides the canonical neuromodulators, glutamate has recently been identified as a key cotransmitter of numerous monoaminergic projections. In the median raphe, a pure glutamatergic neurone population projecting to limbic areas was also discovered with a possibly novel, yet undetermined function. Here, we report the first functional description of the vesicular glutamate transporter type 3 (VGluT3)-expressing median raphe neurones. Since there is no appropriate genetic marker for the separation of serotonergic (5HT+) and non-serotonergic (5HT-) VGluT3+ neurones, we utilised immunohistochemistry after recording and juxtacellular labelling in anaesthetised rats. VGluT3+/5HT- neurones fired faster, more variably and were permanently activated during sensory stimulation, as opposed to the transient response of the slow firing VGluT3-/5HT+ subgroup. VGluT3+/5HT- cells were also more active during hippocampal theta. In addition, the VGluT3-/5HT- population - putative GABAergic cells - resembled the firing of VGluT3+/5HT- neurones, but without significant reaction to the sensory stimulus. Interestingly, the VGluT3+/5HT+ group - spiking slower than the VGluT3+/5HT- population - exhibited a mixed response i.e. the initial transient activation was followed by sustained elevation of firing. Phase coupling to hippocampal and prefrontal slow oscillations was found in VGluT3+/5HT- neurones, also differentiating them from the VGluT3+/5HT+ subpopulation. Taken together, glutamatergic neurones in the median raphe may implement multiple, highly divergent forms of modulation in parallel: a slow, tonic mode interrupted by sensory-evoked rapid transients and a fast one, capable of conveying complex patterns influenced by sensory inputs. This article is protected by copyright. All rights reserved

Differential Roles of the Two Raphe Nuclei in Amiable Social Behavior and Aggression - An Optogenetic Study.

Serotonergic mechanisms hosted by raphe nuclei have important roles in affiliative and agonistic behaviors but the separate roles of the two nuclei are poorly understood. Here we studied the roles of the dorsal (DR) and median raphe region (MRR) in aggression by optogenetically stimulating the two nuclei. Mice received three 3 min-long stimulations, which were separated by non-stimulation periods of 3 min. The stimulation of the MRR decreased aggression in a phasic-like manner. Effects were rapidly expressed during stimulations, and vanished similarly fast when stimulations were halted. No carryover effects were observed in the subsequent three trials performed at 2-day intervals. No effects on social behaviors were observed. By contrast, DR stimulation rapidly and tonically promoted social behaviors: effects were present during both the stimulation and non-stimulation periods of intermittent stimulations. Aggressive behaviors were marginally diminished by acute DR stimulations, but repeated stimulations administered over 8 days considerably decreased aggression even in the absence of concurrent stimulations, indicating the emergence of carryover effects. No such effects were observed in the case of social behaviors. We also investigated stimulation-induced neurotransmitter release in the prefrontal cortex, a major site of aggression control. MRR stimulation rapidly but transiently increased serotonin release, and induced a lasting increase in glutamate levels. DR stimulation had no effect on glutamate, but elicited a lasting increase of serotonin release. Prefrontal serotonin levels remained elevated for at least 2 h subsequent to DR stimulations. The stimulation of both nuclei increased GABA release rapidly and transiently. Thus, differential behavioral effects of the two raphe nuclei were associated with differences in their neurotransmission profiles. These findings reveal a surprisingly strong behavioral task division between the two raphe nuclei, which was associated with a nucleus-specific neurotransmitter release in the prefrontal cortex

Co-transmission of acetylcholine and GABA regulates hippocampal states

The basal forebrain cholinergic system is widely assumed to control cortical functions via non-synaptic transmission of a single neurotransmitter. Yet, we find that mouse hippocampal cholinergic terminals invariably establish GABAergic synapses, and their cholinergic vesicles dock at those synapses only. We demonstrate that these synapses do not co-release but co-transmit GABA and acetylcholine via different vesicles, whose release is triggered by distinct calcium channels. This co-transmission evokes composite postsynaptic potentials, which are mutually cross-regulated by presynaptic autoreceptors. Although postsynaptic cholinergic receptor distribution cannot be investigated, their response latencies suggest a focal, intra- and/or peri-synaptic localisation, while GABAA receptors are detected intra-synaptically. The GABAergic component alone effectively suppresses hippocampal sharp wave-ripples and epileptiform activity. Therefore, the differentially regulated GABAergic and cholinergic co-transmission suggests a hitherto unrecognised level of control over cortical states. This novel model of hippocampal cholinergic neurotransmission may lead to alternative pharmacotherapies after cholinergic deinnervation seen in neurodegenerative disorders

Chronic Amyloid beta Oligomer Infusion Evokes Sustained Inflammation and Microglial Changes in the Rat Hippocampus via NLRP3

Microglia are instrumental for recognition and elimination of amyloid beta1-42 oligomers (AbetaOs), but the long-term consequences of AbetaO-induced inflammatory changes in the brain are unclear. Here, we explored microglial responses and transciptome-level inflammatory signatures in the rat hippocampus after chronic AbetaO challenge. Middle-aged Long Evans rats received intracerebroventricular infusion of AbetaO or vehicle for 4weeks, followed by treatment with artificial CSF or MCC950 for the subsequent 4weeks. AbetaO infusion evoked a sustained inflammatory response including activation of NF-kappaB, triggered microglia activation and increased the expression of pattern recognition and phagocytic receptors. Abeta1-42 plaques were not detectable likely due to microglial elimination of infused oligomers. In addition, we found upregulation of neuronal inhibitory ligands and their cognate microglial receptors, while downregulation of Esr1 and Scn1a, encoding estrogen receptor alpha and voltage-gated sodium-channel Na(v)1.1, respectively, was observed. These changes were associated with impaired hippocampus-dependent spatial memory and resembled early neurological changes seen in Alzheimer's disease. To investigate the role of inflammatory actions in memory deterioration, we performed MCC950 infusion, which specifically blocks the NLRP3 inflammasome. MCC950 attenuated AbetaO-evoked microglia reactivity, restored expression of neuronal inhibitory ligands, reversed downregulation of ERalpha, and abolished memory impairments. Furthermore, MCC950 abrogated AbetaO-invoked reduction of serum IL-10. These findings provide evidence that in response to AbetaO infusion microglia change their phenotype, but the resulting inflammatory changes are sustained for at least one month after the end of AbetaO challenge. Lasting NLRP3-driven inflammatory alterations and altered hippocampal gene expression contribute to spatial memory decline