Presynaptic Kainate Receptors onto Somatostatin Interneurons Are Recruited by Activity throughout Development and Contribute to Cortical Sensory Adaptation.

Somatostatin (SST) interneurons produce delayed inhibition because of the short-term facilitation of their excitatory inputs created by the expression of metabotropic glutamate receptor 7 (mGluR7) and presynaptic GluK2-containing kainate receptors (GluK2-KARs). Using mice of both sexes, we find that as synaptic facilitation at layer (L)2/3 SST cell inputs increases during the first few postnatal weeks, so does GluK2-KAR expression. Removal of sensory input by whisker trimming does not affect mGluR7 but prevents the emergence of presynaptic GluK2-KARs, which can be restored by allowing whisker regrowth or by acute calmodulin activation. Conversely, late trimming or acute inhibition of Ca2+/calmodulin-dependent protein kinase II is sufficient to reduce GluK2-KAR activity. This developmental and activity-dependent regulation also produces a specific reduction of L4 GluK2-KARs that advances in parallel with the maturation of sensory processing in L2/3. Finally, we find that removal of both GluK2-KARs and mGluR7 from the synapse eliminates short-term facilitation and reduces sensory adaptation to repetitive stimuli, first in L4 of somatosensory cortex, then later in development in L2/3. The dynamic regulation of presynaptic GluK2-KARs potentially allows for flexible scaling of late inhibition and sensory adaptation.SIGNIFICANCE STATEMENT Excitatory synapses onto somatostatin (SST) interneurons express presynaptic, calcium-permeable kainate receptors containing the GluK2 subunit (GluK2-KARs), activated by high-frequency activity. In this study we find that their presence on L2/3 SST synapses in the barrel cortex is not based on a hardwired genetic program but instead is regulated by sensory activity, in contrast to that of mGluR7. Thus, in addition to standard synaptic potentiation and depression mechanisms, excitatory synapses onto SST neurons undergo an activity-dependent presynaptic modulation that uses GluK2-KARs. Further, we present evidence that loss of the frequency-dependent synaptic components (both GluK2-KARs and mGluR7 via Elfn1 deletion) contributes to a decrease in the sensory adaptation commonly seen on repetitive stimulus presentation.

Cell-specific retrograde signals mediate antiparallel effects of angiotensin II on osmoreceptor afferents to vasopressin and oxytocin neurons.

Homeostatic control of extracellular fluid osmolality in rats requires a parallel excitation of vasopressin (VP) and oxytocin (OT) neurosecretory neurons by osmoreceptor afferents to regulate the amount of water and sodium in the urine under normal conditions. However, during decreased blood volume (hypovolemia), natriuresis is suppressed, whereas osmotically driven antidiuresis is enhanced to promote retention of isotonic fluid. Because Angiotensin II (Ang II) is released centrally to indicate hypovolemia, we hypothesized that Ang II can evoke a state-dependent switch in circuit function. Here, we show that Ang II, a neuropeptide released centrally during hypovolemia, suppresses osmoreceptor-mediated synaptic excitation of OT neurons while potentiating excitation of VP neurons. Ang II does this by inducing cell-autonomous release of nitric oxide by VP neurons and endocannabinoids by OT neurons to respectively enhance and reduce glutamate release by osmoreceptor afferents. These findings indicate that peptide modulators such as Ang II can regulate synaptic communication to achieve a state-dependent and target-specific modulation of circuit activity.

Chemogenetic synaptic silencing of neural circuits localizes a hypothalamus→midbrain pathway for feeding behavior.

Brain function is mediated by neural circuit connectivity, and elucidating the role of connections is aided by techniques to block their output. We developed cell-type-selective, reversible synaptic inhibition tools for mammalian neural circuits by leveraging G protein signaling pathways to suppress synaptic vesicle release. Here, we find that the pharmacologically selective designer Gi-protein-coupled receptor hM4D is a presynaptic silencer in the presence of its cognate ligand clozapine-N-oxide (CNO). Activation of hM4D signaling sharply reduced synaptic release probability and synaptic current amplitude. To demonstrate the utility of this tool for neural circuit perturbations, we developed an axon-selective hM4D-neurexin variant and used spatially targeted intracranial CNO injections to localize circuit connections from the hypothalamus to the midbrain responsible for feeding behavior. This synaptic silencing approach is broadly applicable for cell-type-specific and axon projection-selective functional analysis of diverse neural circuits.

Activity maintains structural plasticity of mossy fiber terminals in the hippocampus.

Neural activity plays an important role in organizing and optimizing neural circuits during development and in the mature nervous system. However, the cellular events that underlie this process still remain to be fully understood. In this study, we investigated the role of neural activity in regulating the structural plasticity of presynaptic terminals in the hippocampal formation. We designed a virus to drive the Drosophila Allatostatin receptor in individual dentate granule neurons to suppress activity of complex mossy fiber terminals 'on-demand' in organotypic slices and used time-lapse confocal imaging to determine the impact on presynaptic remodeling. We found that activity played an important role in maintaining the structural plasticity of the core region of the mossy fiber terminal (MFT) that synapses onto CA3 pyramidal cell thorny excrescences but was not essential for the motility of terminal filopodial extensions that contact local inhibitory neurons. Short-term suppression of activity did not have an impact on the size of the MFT, however, longer-term suppression reduced the overall size of the MFT. Remarkably, global blockade of activity with tetrodotoxin (TTX) interfered with the ability of single cell activity deprivation to slow down terminal dynamics suggesting that differences in activity levels among neighboring synapses promote synaptic remodeling events. The results from our studies indicate that neural activity plays an important role in maintaining structural plasticity of presynaptic compartments in the central nervous system and provide new insight into the time-frame during which activity can affect the morphology of synaptic connections.

Neurophysiology of supraoptic neurons in C57/BL mice studied in three acute in vitro preparations.

Osmotic control of arginine vasopressin (AVP) and oxytocin (OXT) release from magnocellular neurosecretory cells (MNCs) of the supraoptic (SON) and paraventricular (PVN) nuclei is essential for body fluid homeostasis. The electrical activity of MNCs, which is regulated by intrinsic and extrinsic osmosensitive factors, is a primary determinant of blood AVP and OXT levels. Although we now understand many of the cellular mechanisms that mediate the osmotic control of electrical activity and secretion from MNCs, further insight is likely to emerge from a molecular analysis of these mechanisms. An important step towards this goal could be made through the use of mouse genetic models. However, the electrophysiological properties of MNCs in mice have not been characterized, making direct comparisons with the rat model somewhat difficult. In this study, we examined the electrical properties of MNCs from the mouse SON. Extracellular recordings from neurons in superfused explants revealed modes of basal and osmotically modulated firing very similar to those observed previously in rats. Recordings in hypothalamic slices confirmed that SON neurons receive kynurenic-acid-sensitive excitatory synaptic inputs from the organum vasculosum laminae terminalis (OVLT). Current-clamp recordings from acutely dissociated SON neurons showed proportional changes in membrane cation conductance during changes in fluid osmolality. We conclude, therefore, that MNCs in the mouse SON display intrinsic osmosensitive properties and firing patterns that are very similar to those reported in the rat. Mouse MNCs therefore represent a useful model for the study of molecular factors contributing to the osmotic control of AVP and OXT release.

Neurophysiological characterization of mammalian osmosensitive neurones.

In mammals, the osmolality of the extracellular fluid is maintained near a predetermined set-point through a negative feedback regulation of thirst, diuresis, salt appetite and natriuresis. This homeostatic control is believed to be mediated by osmosensory neurones which synaptically regulate the electrical activity of command neurones that mediate each of these osmoregulatory effector responses. Our present understanding of the molecular, cellular and network basis that underlies the central control of osmoregulation is largely derived from studies on primary osmosensory neurones in the organum vasculosum lamina terminalis (OVLT) and effector neurones in the supraoptic nucleus (SON), which release hormones that regulate diuresis and natriuresis. Primary osmosensory neurones in the OVLT exhibit changes in action potential firing rate that vary in proportion with ECF osmolality. This effect results from the intrinsic depolarizing receptor potential which these cells generate via a molecular transduction complex that may comprise various members of the transient receptor potential vanilloid (TRPV) family of cation channel proteins, notably TRPV1 and TRPV4. Osmotically evoked changes in the firing rate of OVLT neurones then regulate the electrical activity of downstream neurones in the SON through graded changes in glutamate release.

Visually guided whole cell patch clamp of mouse supraoptic nucleus neurons in cultured and acute conditions.

Recent advances in neuronal culturing techniques have supplied a new set of tools for studying neural tissue, providing effective means to study molecular aspects of regulatory elements in the supraoptic nucleus of the hypothalamus (SON). To combine molecular biology techniques with electrophysiological recording, we modified an organotypic culture protocol to permit transfection and whole cell patch-clamp recordings from SON cells. Neonatal mouse brain coronal sections containing the SON were dissected out, placed on a filter insert in culture medium, and incubated for at least 4 days to allow attachment to the insert. The SON was identifiable using gross anatomical landmarks, which remained intact throughout the culturing period. Immunohistochemical staining identified both vasopressinergic and oxytocinergic cells present in the cultures, typically appearing in well-defined clusters. Whole cell recordings from these cultures demonstrated that certain properties of the neonatal mouse SON were comparable to adult mouse magnocellular neurons. SON neurons in both neonatal cultures and acute adult slices showed similar sustained outward rectification above -60 mV and action potential broadening during evoked activity. Membrane potential, input resistance, and rapidly inactivating potassium current density (IA) were reduced in the cultures, whereas whole cell capacitance and spontaneous synaptic excitation were increased, perhaps reflecting developmental changes in cell physiology that warrant further study. The use of the outlined organotypic culturing procedures will allow the study of such electrophysiological properties of mouse SON using whole cell patch-clamp, in addition to various molecular, techniques that require longer incubation times.