Axonal CB1 Receptors Mediate Inhibitory Bouton Formation via cAMP Increase and PKA.

Experience-dependent formation and removal of inhibitory synapses are essential throughout life. For instance, GABAergic synapses are removed to facilitate learning, and strong excitatory activity is accompanied by the formation of inhibitory synapses to maintain coordination between excitation and inhibition. We recently discovered that active dendrites trigger the growth of inhibitory synapses via CB1 receptor-mediated endocannabinoid signaling, but the underlying mechanism remained unclear. Using two-photon microscopy to monitor the formation of individual inhibitory boutons in hippocampal organotypic slices from mice (both sexes), we found that CB1 receptor activation mediated the formation of inhibitory boutons and promoted their subsequent stabilization. Inhibitory bouton formation did not require neuronal activity and was independent of Gi/o-protein signaling, but was directly induced by elevating cAMP levels using forskolin and by activating Gs-proteins using DREADDs. Blocking PKA activity prevented CB1 receptor-mediated inhibitory bouton formation. Our findings reveal that axonal CB1 receptors signal via unconventional downstream pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels. Our results demonstrate an unexpected role for axonal CB1 receptors in axon-specific, and context-dependent, inhibitory synapse formation.SIGNIFICANCE STATEMENT Coordination between excitation and inhibition is required for proper brain function throughout life. It was previously shown that new inhibitory synapses can be formed in response to strong excitation to maintain this coordination, and this was mediated by endocannabinoid signaling via CB1 receptors. As activation of CB1 receptors generally results in the suppression of synaptic transmission, it remained unclear how CB1 receptors can mediate the formation of inhibitory synapses. Here we show that CB1 receptors on inhibitory axons signal via unconventional intracellular pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels and requires PKA activity. Our findings point to a central role for axonal cAMP signaling in activity-dependent inhibitory synapse formation.

Direction selectivity in retinal bipolar cell axon terminals.

The ability to encode the direction of image motion is fundamental to our sense of vision. Direction selectivity along the four cardinal directions is thought to originate in direction-selective ganglion cells (DSGCs) because of directionally tuned GABAergic suppression by starburst cells. Here, by utilizing two-photon glutamate imaging to measure synaptic release, we reveal that direction selectivity along all four directions arises earlier than expected at bipolar cell outputs. Individual bipolar cells contained four distinct populations of axon terminal boutons with different preferred directions. We further show that this bouton-specific tuning relies on cholinergic excitation from starburst cells and GABAergic inhibition from wide-field amacrine cells. DSGCs received both tuned directionally aligned inputs and untuned inputs from among heterogeneously tuned glutamatergic bouton populations. Thus, directional tuning in the excitatory visual pathway is incrementally refined at the bipolar cell axon terminals and their recipient DSGC dendrites by two different neurotransmitters co-released from starburst cells.

Presynaptic inputs to vasopressin neurons in the hypothalamic supraoptic nucleus and paraventricular nucleus in mice.

Arginine vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) are involved in important physiological behaviors, such as controling osmotic stability and thermoregulation. However, the presynaptic input patterns governing AVP neurons have remained poorly understood due to their heterogeneity, as well as intermingling of AVP neurons with other neurons both in the SON and PVN. In the present study, we employed a retrograde modified rabies-virus system to reveal the brain areas that provide specific inputs to AVP neurons in the SON and PVN. We found that AVP neurons of the SON and PVN received similar input patterns from multiple areas of the brain, particularly massive afferent inputs from the diencephalon and other brain regions of the limbic system; however, PVNAVP neurons received relatively broader and denser inputs compared to SONAVP neurons. Additionally, SONAVP neurons received more projections from the median preoptic nucleus and organum vasculosum of the lamina terminalis (a circumventricular organ), compared to PVNAVP neurons, while PVNAVP neurons received more afferent inputs from the bed nucleus of stria terminalis and dorsomedial nucleus of the hypothalamus, both of which are thermoregulatory nuclei, compared to those of SONAVP neurons. In addition, both SONAVP and PVNAVP neurons received direct afferent projections from the bilateral suprachiasmatic nucleus, which is the master regulator of circadian rhythms and is concomitantly responsible for fluctuations in AVP levels. Taken together, our present results provide a comprehensive understanding of the specific afferent framework of AVP neurons both in the SON and PVN, and lay the foundation for further dissecting the diverse roles of SONAVP and PVNAVP neurons.

Cholinergic boutons are closely associated with excitatory cells and four subtypes of inhibitory cells in the inferior colliculus.

Acetylcholine (ACh) is a neuromodulator that has been implicated in multiple roles across the brain, including the central auditory system, where it sets neuronal excitability and gain and affects plasticity. In the cerebral cortex, subtypes of GABAergic interneurons are modulated by ACh in a subtype-specific manner. Subtypes of GABAergic neurons have also begun to be described in the inferior colliculus (IC), a midbrain hub of the auditory system. Here, we used male and female mice (Mus musculus) that express fluorescent protein in cholinergic cells, axons, and boutons to look at the association between ACh and four subtypes of GABAergic IC cells that differ in their associations with extracellular markers, their soma sizes, and their distribution within the IC. We found that most IC cells, including excitatory and inhibitory cells, have cholinergic boutons closely associated with their somas and proximal dendrites. We also found that similar proportions of each of four subtypes of GABAergic cells are closely associated with cholinergic boutons. Whether the different types of GABAergic cells in the IC are differentially regulated remains unclear, as the response of cells to ACh is dependent on which types of ACh receptors are present. Additionally, this study confirms the presence of these four subtypes of GABAergic cells in the mouse IC, as they had previously been identified only in guinea pigs. These results suggest that cholinergic projections to the IC modulate auditory processing via direct effects on a multitude of inhibitory circuits.

Brain-wide mapping of presynaptic inputs to basolateral amygdala neurons.

The basolateral amygdala (BLA), a region critical for emotional processing, is the limbic hub that is connected with various brain regions. BLA neurons are classified into different subtypes that exhibit differential projection patterns and mediate distinct emotional behaviors; however, little is known about their presynaptic input patterns. In this study, we employed projection-specific monosynaptic rabies virus tracing to identify the direct monosynaptic inputs to BLA subtypes. We found that each neuronal subtype receives long-range projection input from specific brain regions. In contrast to their specific axonal projection patterns, all BLA neuronal subtypes exhibited relatively similar input patterns. This anatomical organization supports the idea that the BLA is a central integrator that associates sensory information in different modalities with valence and sends associative information to behaviorally relevant brain regions.

Caveolin-1 deficiency impairs synaptic transmission in hippocampal neurons.

In addition to providing structural support, caveolin-1 (Cav1), a component of lipid rafts, including caveolae, in the plasma membrane, is involved in various cellular mechanisms, including signal transduction. Although pre-synaptic membrane dynamics and trafficking are essential cellular processes during synaptic vesicle exocytosis/synaptic transmission and synaptic vesicle endocytosis/synaptic retrieval, little is known about the involvement of Cav1 in synaptic vesicle dynamics. Here we demonstrate that synaptic vesicle exocytosis is significantly impaired in Cav1-knockdown (Cav1-KD) neurons. Specifically, the size of the synaptic recycled vesicle pool is modestly decreased in Cav1-KD synapses and the kinetics of synaptic vesicle endocytosis are somewhat slowed. Notably, neurons rescued by triple mutants of Cav1 lacking palmitoylation sites mutants show impairments in both synaptic transmission and retrieval. Collectively, our findings implicate Cav1 in activity-driven synaptic vesicle dynamics-both exocytosis and endocytosis-and demonstrate that palmitoylation of Cav1 is important for this activity.

Expression of the type 1 cannabinoid receptor (CB1R) in CCK-immunoreactive axon terminals in the basolateral amygdala of the rhesus monkey (Macaca mulatta).

Studies in rodents have shown that interactions between cholecystokinin (CCK) and the endogenous cannabinoid system in the basolateral nuclear complex of the amygdala (BNC) modulate anxiety-like behavior and fear learning/expression. One of the main cell types implicated is a CCK-immunoreactive (CCK+) basket cell that innervates the somata of pyramidal projection neurons (PNs) and expresses the type 1 cannabinoid receptor (CB1R) in its axon terminals. Although numerous studies have elucidated the anatomy and physiology of these CCK+/CB1R + interneurons in rodents, it has not been determined if they exist in primates. The present investigation used immunohistochemical techniques in the monkey to answer this question. It was found that the monkey BNC, as in rodents, has a very high density of CB1R + axons, including CB1R + axon terminals that form basket-like plexuses contacting somata of PNs. These axons, as well as axons in the neuropil, exhibit extensive colocalization of CCK and CB1R. These findings suggest that the same synaptic mechanisms involved in CCK-CB1R interactions in rodents may also apply to primates, and that therapies that target the cannabinoid system in the BNC may be useful for treating fear and anxiety in human patients.

Extracellular pH modulation of excitatory synaptic transmission in hippocampal CA3 neurons.

In this study, the effect of extracellular pH on glutamatergic synaptic transmission was examined in mechanically dissociated rat hippocampal CA3 pyramidal neurons using a whole-cell patch-clamp technique under voltage-clamp conditions. Native synaptic boutons were isolated without using any enzymes, using a so-called "synapse bouton preparation," and preserved for the electrical stimulation of single boutons. Both the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were found to decrease and increase in response to modest acidic (~pH 6.5) and basic (~pH 8.5) solutions, respectively. These changes in sEPSC frequency were not affected by the addition of TTX but completely disappeared by successive addition of Cd2+. However, changes in sEPSC amplitude induced by acidic and basic extracellular solutions were not affected by the addition of neither TTX nor Cd2+. The glutamate-induced whole-cell currents were decreased and increased by acidic and basic solutions, respectively. Acidic pH also decreased the amplitude and increased the failure rate (Rf) and paired-pulse rate (PPR) of glutamatergic electrically evoked excitatory postsynaptic currents (eEPSCs), while a basic pH increased the amplitude and decreased both the Rf and PPR of eEPSCs. The kinetics of the currents were not affected by changes in pH. Acidic and basic solutions decreased and increased voltage-gated Ca2+ but not Na+ channel currents in the dentate gyrus granule cell bodies. Our results indicate that extracellular pH modulates excitatory transmission via both pre- and postsynaptic sites, with the presynaptic modulation correlated to changes in voltage-gated Ca2+ channel currents.NEW & NOTEWORTHY The effects of external pH changes on spontaneous, miniature, and evoked excitatory synaptic transmission in CA3 hippocampal synapses were examined using the isolated nerve bouton preparation, which allowed for the accurate regulation of extracellular pH at the synapses. Acidification generally reduced transmission, partly via effects on presynaptic Ca2+ channel currents, while alkalization generally enhanced transmission. Both pre- and postsynaptic sites contributed to these effects.

Off-Target Influences of Arch-Mediated Axon Terminal Inhibition on Network Activity and Behavior.

Archaerhodopsin (ArchT)-mediated photoinhibition of axon terminals is commonly used to test the involvement of specific long-range neural projections in behavior. Although sustained activation of this opsin in axon terminals has the unintended consequence of enhancing spontaneous vesicle release, it is unclear whether this desynchronized signaling is consequential for ArchT's behavioral effects. Here, we compare axon terminal and cell body photoinhibition of nucleus accumbens (NAc) afferents to test the utility of these approaches for uncovering pathway-specific contributions of neural circuits to behavior. First, in brain slice recordings we confirmed that ArchT photoinhibition of glutamatergic axons reduces evoked synaptic currents and increases spontaneous transmitter release. A further consequence was increased interneuron activity, which served to broadly suppress glutamate input via presynaptic GABAB receptors. In vivo, axon terminal photoinhibition increased feeding and reward-seeking behavior irrespective of the afferent pathway targeted. These behavioral effects are comparable to those obtained with broad inhibition of NAc neurons. In contrast, cell body inhibition of excitatory NAc afferents revealed a pathway-specific contribution of thalamic input to feeding behavior and amygdala input to reward-seeking under extinction conditions. These findings underscore the off-target behavioral consequences of ArchT-mediated axon terminal inhibition while highlighting cell body inhibition as a valuable alternative for pathway-specific optogenetic silencing.

Presence of substance P positive terminals on hypothalamic somatostatinergic neurons in humans: the possible morphological substrate of the substance P-modulated growth hormone secretion.

Substance P is an undecapeptide affecting the gastrointestinal, cardiovascular, and urinary systems. In the central nervous system, substance P participates in the regulation of pain, learning, memory, and sexual homeostasis. In addition to these effects, previous papers provided solid evidence that substance P exhibits regulatory effects on growth. Indeed, our previous study revealed that growth hormone-releasing hormone (GHRH) neurons appear to be densely innervated by substance P fibers in humans. Since growth hormone secretion is regulated by the antagonistic actions of both GHRH and somatostatin, in the present paper we have examined the possibility that SP may also affect growth via the somatostatinergic system. Therefore, we have studied the putative presence of juxtapositions between the substance P-immunoreactive (IR) and somatostatinergic systems utilizing double label immunohistochemistry combined with high magnification light microscopy with oil immersion objective. In the present study, we have revealed a dense network of substance P-IR axonal varicosities contacting the majority of somatostatin-IR neurons in the human hypothalamus. Somatostatinergic perikarya are often covered by these fiber varicosities that frequently form basket-like encasements with multiple en passant type contacts, particularly in the infundibular nucleus/median eminence and in the basal periventricular area of the tuberal region. In addition, numerous substance-P-somatostatinergic juxtapositions can be found in the basal perifornical zone of the tuberal area. If these contacts are indeed functional synapses, they may represent the morphological substrate of the control of substance P on growth. Indeed, the frequency and density of these juxtapositions indicate that in addition to the regulatory action of substance P on GHRH secretion, substance P also influences growth by regulating hypothalamic somatostatinergic system via direct synaptic contacts.

Glutamate, GABA, and Presynaptic Markers Involved in Neurotransmission Are Differently Affected by Age in Distinct Mouse Brain Regions.

Molecular synaptic aging perturbs neurotransmission and decreases the potential for neuroplasticity. The direction and degree of changes observed in aging are often region or cell specific, hampering the generalization of age-related effects. Using real-time PCR and Western blot analyses, we investigated age-related changes in several presynaptic markers (Vglut1, Vglut2, Gad65, Gad67, Vgat, synaptophysin) involved in the initial steps of glutamatergic and GABAergic neurotransmission, in several cortical regions, in young (3-4 months old), middle-aged (1 year old), and old (2 years old) mice. We found age-related changes mainly in protein levels while, apart from the occipital cortex, virtually no significant changes in mRNA levels were detected, which suggests that aging acts on the investigated markers mainly through post-transcriptional mechanisms depending on the brain region. Principal component analysis (PCA) of protein data revealed that each brain region possessed a type of "biochemical distinctiveness" (each analyzed brain region was best characterized by higher variability level of a particular synaptic marker) that was lost with age. Analysis of glutamate and γ-aminobutyric acid (GABA) levels in aging suggested that mechanisms keeping an overall balance between the two amino acids in the brain are weakened in the hippocampus. Our results unravel the differences in mRNA/protein interactions in the aging brain.

The physiological role of α-synuclein and its relationship to Parkinson's Disease.

The protein α-synuclein has a central role in the pathogenesis of Parkinson's disease (PD). In this review, we discuss recent results concerning its primary function, which appears to be on cell membranes. The pre-synaptic location of synuclein has suggested a role in neurotransmitter release and it apparently associates with synaptic vesicles because of their high curvature. Indeed, synuclein over-expression inhibits synaptic vesicle exocytosis. However, loss of synuclein has not yet been shown to have a major effect on synaptic transmission. Consistent with work showing that synuclein can promote as well as sense membrane curvature, recent analysis of synuclein triple knockout mice now shows that synuclein accelerates dilation of the exocytic fusion pore. This form of regulation affects primarily the release of slowly discharged lumenal cargo such as neural peptides, but presumably also contributes to maintenance of the release site. This article is part of the Special Issue "Synuclein".

DiI-mediated analysis of presynaptic and postsynaptic structures in human postmortem brain tissue.

Most cognitive and psychiatric disorders are thought to be disorders of the synapse, yet the precise synapse defects remain unknown. Because synapses are highly specialized anatomical structures, defects in synapse formation and function can often be observed as changes in microscale neuroanatomy. Unfortunately, few methods are available for accurate analysis of synaptic structures in human postmortem tissues. Here, we present a methodological pipeline for assessing presynaptic and postsynaptic structures in human postmortem tissue that is accurate, rapid, and relatively inexpensive. Our method uses small tissue blocks from postmortem human brains, immersion fixation, lipophilic dye (DiI) labeling, and confocal microscopy. As proof of principle, we analyzed presynaptic and postsynaptic structures from hippocampi of 13 individuals aged 4 months to 71 years. Our results indicate that postsynaptic CA1 dendritic spine shape and density do not change in adults, while presynaptic DG mossy fiber boutons undergo significant structural rearrangements with normal aging. This suggests that mossy fiber synapses, which play a major role in learning and memory, may remain dynamic throughout life. Importantly, we find that human CA1 spine densities observed using this method on tissue that is up to 28 h postmortem is comparable to prior studies using tissue with much shorter postmortem intervals. Thus, the ease of our protocol and suitability on tissue with longer postmortem intervals should facilitate higher-powered studies of human presynaptic and postsynaptic structures in healthy and diseased states.

Proportional loss of parvalbumin-immunoreactive synaptic boutons and granule cells from the hippocampus of sea lions with temporal lobe epilepsy.

One in 26 people develop epilepsy and in these temporal lobe epilepsy (TLE) is common. Many patients display a pattern of neuron loss called hippocampal sclerosis. Seizures usually start in the hippocampus but underlying mechanisms remain unclear. One possibility is insufficient inhibition of dentate granule cells. Normally parvalbumin-immunoreactive (PV) interneurons strongly inhibit granule cells. Humans with TLE display loss of PV interneurons in the dentate gyrus but questions persist. To address this, we evaluated PV interneuron and bouton numbers in California sea lions (Zalophus californianus) that naturally develop TLE after exposure to domoic acid, a neurotoxin that enters the marine food chain during harmful algal blooms. Sclerotic hippocampi were identified by the loss of Nissl-stained hilar neurons. Stereological methods were used to estimate the number of granule cells and PV interneurons per dentate gyrus. Sclerotic hippocampi contained fewer granule cells, fewer PV interneurons, and fewer PV synaptic boutons, and the ratio of granule cells to PV interneurons was higher than in controls. To test whether fewer boutons was attributable to loss versus reduced immunoreactivity, expression of synaptotagmin-2 (syt2) was evaluated. Syt2 is also expressed in boutons of PV interneurons. Sclerotic hippocampi displayed proportional losses of syt2-immunoreactive boutons, PV boutons, and granule cells. There was no significant difference in the average numbers of PV- or syt2-positive boutons per granule cell between control and sclerotic hippocampi. These findings do not address functionality of surviving synapses but suggest reduced granule cell inhibition in TLE is not attributable to anatomical loss of PV boutons.

Presynaptic α2δ-2 Calcium Channel Subunits Regulate Postsynaptic GABAA Receptor Abundance and Axonal Wiring.

Presynaptic α2δ subunits of voltage-gated calcium channels regulate channel abundance and are involved in glutamatergic synapse formation. However, little is known about the specific functions of the individual α2δ isoforms and their role in GABAergic synapses. Using primary neuronal cultures of embryonic mice of both sexes, we here report that presynaptic overexpression of α2δ-2 in GABAergic synapses strongly increases clustering of postsynaptic GABAARs. Strikingly, presynaptic α2δ-2 exerts the same effect in glutamatergic synapses, leading to a mismatched localization of GABAARs. This mismatching is caused by an aberrant wiring of glutamatergic presynaptic boutons with GABAergic postsynaptic positions. The trans-synaptic effect of α2δ-2 is independent of the prototypical cell-adhesion molecules α-neurexins (α-Nrxns); however, α-Nrxns together with α2δ-2 can modulate postsynaptic GABAAR abundance. Finally, exclusion of the alternatively spliced exon 23 of α2δ-2 is essential for the trans-synaptic mechanism. The novel function of α2δ-2 identified here may explain how abnormal α2δ subunit expression can cause excitatory-inhibitory imbalance often associated with neuropsychiatric disorders.SIGNIFICANCE STATEMENT Voltage-gated calcium channels regulate important neuronal functions such as synaptic transmission. α2δ subunits modulate calcium channels and are emerging as regulators of brain connectivity. However, little is known about how individual α2δ subunits contribute to synapse specificity. Here, we show that presynaptic expression of a single α2δ variant can modulate synaptic connectivity and the localization of inhibitory postsynaptic receptors. Our findings provide basic insights into the development of specific synaptic connections between nerve cells and contribute to our understanding of normal nerve cell functions. Furthermore, the identified mechanism may explain how an altered expression of calcium channel subunits can result in aberrant neuronal wiring often associated with neuropsychiatric disorders such as autism or schizophrenia.

Specificity of Primate Amygdalar Pathways to Hippocampus.

The amygdala projects to hippocampus in pathways through which affective or social stimuli may influence learning and memory. We investigated the still unknown amygdalar termination patterns and their postsynaptic targets in hippocampus from system to synapse in rhesus monkeys of both sexes. The amygdala robustly innervated the stratum lacunosum-moleculare layer of cornu ammonis fields and uncus anteriorly. Sparser terminations in posterior hippocampus innervated the radiatum and pyramidal layers at the prosubicular/CA1 juncture. The terminations, which were larger than other afferents in the surrounding neuropil, position the amygdala to influence hippocampal input anteriorly, and its output posteriorly. Most amygdalar boutons (76-80%) innervated spines of excitatory hippocampal neurons, and most of the remaining innervated presumed inhibitory neurons, identified by morphology and label with parvalbumin or calretinin, which distinguished nonoverlapping neurochemical classes of hippocampal inhibitory neurons. In CA1, amygdalar axons innervated some calretinin neurons, which disinhibit pyramidal neurons. By contrast, in CA3 the amygdala innervated both calretinin and parvalbumin neurons; the latter strongly inhibit nearby excitatory neurons. In CA3, amygdalar pathways also made closely spaced dual synapses on excitatory neurons. The strong excitatory synapses in CA3 may facilitate affective context representations and trigger sharp-wave ripples associated with memory consolidation. When the amygdala is excessively activated during traumatic events, the specialized innervation of excitatory neurons and the powerful parvalbumin inhibitory neurons in CA3 may allow the suppression of activity of nearby neurons that receive weaker nonamygdalar input, leading to biased passage of highly charged affective stimuli and generalized fear.SIGNIFICANCE STATEMENT Strong pathways from the amygdala targeted the anterior hippocampus, and more weakly its posterior sectors, positioned to influence a variety of emotional and cognitive functions. In hippocampal field CA1, the amygdala innervated some calretinin neurons, which disinhibit excitatory neurons. By contrast, in CA3 the amygdala innervated calretinin as well as some of the powerful parvalbumin inhibitory neurons and may help balance the activity of neural ensembles to allow social interactions, learning, and memory. These results suggest that when the amygdala is hyperactive during emotional upheaval, it strongly activates excitatory hippocampal neurons and parvalbumin inhibitory neurons in CA3, which can suppress nearby neurons that receive weaker input from other sources, biasing the passage of stimuli with high emotional import and leading to generalized fear.

Morphological diversity and connectivity of hippocampal interneurons.

The mammalian forebrain is constructed from ensembles of neurons that form local microcircuits giving rise to the exquisite cognitive tasks the mammalian brain can perform. Hippocampal neuronal circuits comprise populations of relatively homogenous excitatory neurons, principal cells and exceedingly heterogeneous inhibitory neurons, the interneurons. Interneurons release GABA from their axon terminals and are capable of controlling excitability in every cellular compartment of principal cells and interneurons alike; thus, they provide a brake on excess activity, control the timing of neuronal discharge and provide modulation of synaptic transmission. The dendritic and axonal morphology of interneurons, as well as their afferent and efferent connections within hippocampal circuits, is central to their ability to differentially control excitability, in a cell-type- and compartment-specific manner. This review aims to provide an up-to-date compendium of described hippocampal interneuron subtypes, with respect to their morphology, connectivity, neurochemistry and physiology, a full understanding of which will in time help to explain the rich diversity of neuronal function.

Neuroanatomical characterization of perineuronal net components in the human cochlear nucleus and superior olivary complex.

The human auditory brainstem, especially the cochlear nucleus (CN) and the superior olivary complex (SOC) are characterized by a high density of neurons associated with perineuronal nets (PNs). PNs build a specific form of extracellular matrix surrounding the neuronal somata, proximal dendrites and axon initial segments. They restrict synaptic plasticity and control high-frequency synaptic activity, a prominent characteristic of neurons of the auditory brainstem. The distribution of PNs within the auditory brainstem has been investigated in a number of mammalian species. However, much less is known regarding PNs in the human auditory brainstem. The present study aimed at the immunohistochemical identification of PNs in the cochlear nucleus (CN) and superior olivary complex (SOC) in the human brainstem. We focused on the complex nature and molecular variability of PNs in the CN and SOC by using specific antibodies against the main PN components (aggrecan, brevican, neurocan and hyaluronan and proteoglycan link protein 1). Virtually all subnuclei within the ventral CN and SOC were found to be associated with PNs. Direct comparison between gerbil and human yielded similar fine structure of PNs and confirmed the typical tight interdigitation of PNs with synaptic terminals in both species. Noticeably, an elaborate combination of immunohistochemical labelings clearly supports the still debated existence of the medial nucleus of trapezoid body (MNTB) in the human brain. In conclusion, the present study demonstrates that PNs form a prominent extracellular structure on CN and SOC neurons in the human brain, potentially stabilizing synaptic contacts, which is in agreement with many other mammalian species.

Neurexin-Neuroligin 1 regulates synaptic morphology and functions via the WAVE regulatory complex in Drosophila neuromuscular junction.

Neuroligins are postsynaptic adhesion molecules that are essential for postsynaptic specialization and synaptic function. But the underlying molecular mechanisms of neuroligin functions remain unclear. We found that Drosophila Neuroligin 1 (DNlg1) regulates synaptic structure and function through WAVE regulatory complex (WRC)-mediated postsynaptic actin reorganization. The disruption of DNlg1, DNlg2, or their presynaptic partner neurexin (DNrx) led to a dramatic decrease in the amount of F-actin. Further study showed that DNlg1, but not DNlg2 or DNlg3, directly interacts with the WRC via its C-terminal interacting receptor sequence. That interaction is required to recruit WRC to the postsynaptic membrane to promote F-actin assembly. Furthermore, the interaction between DNlg1 and the WRC is essential for DNlg1 to rescue the morphological and electrophysiological defects in dnlg1 mutants. Our results reveal a novel mechanism by which the DNrx-DNlg1 trans-synaptic interaction coordinates structural and functional properties at the neuromuscular junction.

Targeting hypersensitive corticostriatal terminals in restless legs syndrome.

OBJECTIVE: The first aim was to demonstrate a previously hypothesized increased sensitivity of corticostriatal glutamatergic terminals in the rodent with brain iron deficiency (BID), a pathogenetic model of restless legs syndrome (RLS). The second aim was to determine whether these putative hypersensitive terminals could constitute a significant target for drugs effective in RLS, including dopamine agonists (pramipexole and ropinirole) and α2 δ ligands (gabapentin). METHODS: A recently introduced in vivo optogenetic-microdialysis approach was used, which allows the measurement of the extracellular concentration of glutamate upon local light-induced stimulation of corticostriatal glutamatergic terminals. The method also allows analysis of the effect of local perfusion of compounds within the same area being sampled for glutamate. RESULTS: BID rats showed hypersensitivity of corticostriatal glutamatergic terminals (lower frequency of optogenetic stimulation to induce glutamate release). Both hypersensitive and control glutamatergic terminals were significant targets for locally perfused pramipexole, ropinirole, and gabapentin, which significantly counteracted optogenetically induced glutamate release. The use of selective antagonists demonstrated the involvement of dopamine D4 and D2 receptor subtypes in the effects of pramipexole. INTERPRETATION: Hypersensitivity of corticostriatal glutamatergic terminals can constitute a main pathogenetic mechanism of RLS symptoms. Selective D4 receptor agonists, by specifically targeting these terminals, should provide a new efficient treatment with fewer secondary effects. Ann Neurol 2017;82:951-960.