Julia Brill

Mechanism for Hypocretin-mediated sleep-to-wake transitions

Current models of sleep/wake regulation posit that Hypocretin (Hcrt)-expressing neurons in the lateral hypothalamus promote and stabilize wakefulness by projecting to subcortical arousal centers. However, the critical downstream effectors of Hcrt neurons are unknown. Here we use optogenetic, pharmacological, and computational tools to investigate the functional connectivity between Hcrt neurons and downstream noradrenergic neurons in the locus coeruleus (LC) during nonrapid eye movement (NREM) sleep. We found that photoinhibiting LC neurons during Hcrt stimulation blocked Hcrt-mediated sleep-to-wake transitions. In contrast, when LC neurons were optically stimulated to increase membrane excitability, concomitant photostimulation of Hcrt neurons significantly increased the probability of sleep-to-wake transitions compared with Hcrt stimulation alone. We also built a conductance-based computational model of Hcrt-LC circuitry that recapitulates our behavioral results using LC neurons as the main effectors of Hcrt signaling. These results establish the Hcrt-LC connection as a critical integrator-effector circuit that regulates NREM sleep/wake behavior during the inactive period. This coupling of distinct neuronal systems can be generalized to other hypothalamic integrator nuclei with downstream effector/output populations in the brain.

Sequential Changes in AMPA Receptor Targeting in the Developing Neocortical Excitatory Circuit

Many principal neurons undergo an early developmental switch from GluR2-lacking to GluR2-containing synaptic glutamate receptors. We tested the generality and timing of the GluR2 switch in excitatory neurons of rat somatosensory cortex. Previous studies show that the switch occurs between postnatal day 14 (P14) and P16 in layer 5 pyramidal neurons. We show, using sensitivity to intracellular spermine, that a similar switch occurs between P12 and P14 in layer 2/3 pyramidal cells and between P7 and P8 in layer 4 stellate cells. The presence of GluR2-lacking receptors in layer 2/3 pyramidal cells before P12 was confirmed by demonstrating sensitivity to blockade by 1-naphthyl-acetyl-spermine and large single-channel conductances. GluR2 and the postsynaptic protein PSD95 show progressive colocalization in tissue from P10, P14, and P24 rats, mirroring electrophysiological developments. To distinguish whether changes in GluR2 expression or targeting underlie the switch, we characterized dendritic AMPA receptor responses using focal photolysis of caged glutamate. Contrary to synaptic responses, dendritic responses at all ages studied (P6–P40) were characteristic of GluR2-containing receptors. In addition, dendritically and synaptically evoked responses showed a corresponding decrease in NMDA/AMPA ratios in pyramidal cells, suggesting parallel mechanisms that regulate neuronal calcium levels. These data suggest that the GluR2 switch results from changes in AMPA receptor targeting during early postnatal development, and that rather than following the laminar sequence of cortical development, it proceeds sequentially from layer 4 to layer 2/3 and finally to layer 5b.

Enhanced NMDA Receptor-Dependent Thalamic Excitation and Network Oscillations in Stargazer Mice

Disturbances in corticothalamic circuitry can lead to absence epilepsy. The reticular thalamic nucleus (RTN) plays a pivotal role in that it receives excitation from cortex and thalamus and, when strongly activated, can generate excessive inhibitory output and epileptic thalamocortical oscillations that depend on postinhibitory rebound. Stargazer (stg) mice have prominent absence seizures resulting from a mutant form of the AMPAR auxiliary protein stargazin. Reduced AMPAR excitation in RTN has been demonstrated previously in stg, yet the mechanisms leading from RTN hypoexcitation to epilepsy are unknown and unexpected because thalamic epileptiform oscillatory activity requires AMPARs. We demonstrate hyperexcitability in stg thalamic slices and further characterize the various excitatory inputs to RTN using electrical stimulation and laser scanning photostimulation. Patch-clamp recordings of spontaneous and evoked EPSCs in RTN neurons demonstrate reduced amplitude and increased duration of the AMPAR component with an increased amplitude NMDAR component. Short 200 Hz stimulus trains evoked a gradual approximately threefold increase in NMDAR EPSCs compared with single stimuli in wild-type (WT), indicating progressive NMDAR recruitment, whereas in stg cells, NMDAR responses were nearly maximal with single stimuli. Array tomography revealed lower synaptic, but higher perisynaptic, AMPAR density in stg RTN. Increasing NMDAR activity via reduced [Mg2+]o in WT phenocopied the thalamic hyperexcitability observed in stg, whereas changing [Mg2+]o had no effect on stg slices. These findings suggest that, in stg, a trafficking defect in synaptic AMPARs in RTN cells leads to a compensatory increase in synaptic NMDARs and enhanced thalamic excitability.

Chronic Valproic Acid Treatment Triggers Increased Neuropeptide Y Expression and Signaling in Rat Nucleus Reticularis Thalami

Valproate (VPA) can suppress absence and other seizures, but its precise mechanisms of action are not completely understood. We investigated whether VPA influences the expression of neuropeptide Y (NPY), an endogenous anticonvulsant. Chronic VPA administration to young rats (300–600 mg · kg−1 · d−1 in divided doses over 4 d) resulted in a 30–50% increase in NPY mRNA and protein expression in the nucleus reticularis thalami (nRt) and hippocampus, but not in the neocortex, as shown by real-time PCR, radioimmunoassay, and immunohistochemistry. No increased expression was observed after a single acute dose of VPA. Chronic treatment with the pharmacologically inactive VPA analog octanoic acid did not elicit changes in NPY expression. No significant expression changes could be shown for the mRNAs of the Y1 receptor or of the neuropeptides somatostatin, vasoactive intestinal polypeptide, and choleocystokinin. Fewer synchronous spontaneous epileptiform oscillations were recorded in thalamic slices from VPA-treated animals, and oscillation duration as well as the period of spontaneous and evoked oscillations were decreased. Application of the Y1 receptor inhibitor N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-d-arginine-amide (BIBP3226) enhanced thalamic oscillations, indicating that NPY is released during those oscillations and acts to downregulate oscillatory strength. Chronic VPA treatment significantly potentiated the effect of BIBP3226 on oscillation duration but not on oscillation period. These results demonstrate a novel mechanism for the antiepileptic actions of chronic VPA therapy.

Enhanced Infragranular and Supragranular Synaptic Input onto Layer 5 Pyramidal Neurons in a Rat Model of Cortical Dysplasia

Cortical dysplasias frequently underlie neurodevelopmental disorders and epilepsy. Rats with a neonatally induced cortical microgyrus [freeze-lesion (FL)], a model of human polymicrogyria, display epileptiform discharges in vitro. We probed excitatory and inhibitory connectivity onto neocortical pyramidal neurons in layers 2/3 and 5 of postnatal day 16-22 rats, approximately 1-2 mm lateral of the lesion, using laser scanning photostimulation (LSPS)/glutamate uncaging. Excitatory input from deep and supragranular layers to layer 5 pyramidal cells was greater in FL cortex, while no significant differences were seen in layer 2/3 cells. The increased input was due to a greater number of LSPS-evoked excitatory postsynaptic currents (EPSCs), without differences in amplitude or kinetics. Inhibitory input was increased in a region-specific manner in pyramidal cells in FL cortex, due to an increased inhibitory postsynaptic current (IPSC) amplitude. Connectivity within layer 5, parts of which are destroyed during lesioning, was more severely affected than connectivity in layer 2/3. Thus, we observed 2 distinct mechanisms of altered synaptic input: 1) increased EPSC frequency suggesting an increased number of excitatory synapses and 2) higher IPSC amplitude, suggesting an increased strength of inhibitory synapses. These increases in both excitatory and inhibitory connectivity may limit the extent of circuit hyperexcitability.

Robust Short-Latency Perisomatic Inhibition onto Neocortical Pyramidal Cells Detected by Laser-Scanning Photostimulation

Inhibitory connectivity onto neocortical pyramidal cells was mapped using LSPS (laser-scanning photostimulation/glutamate uncaging). The average onset latency of IPSCs was shorter than that of EPSCs recorded in the same cells, indicating a specific mechanism for rapid network recruitment of inhibition. The majority of strong inhibitory synaptic inputs originated within 300 μm of the recorded cells soma, had onset latencies between 4 and 10 ms, and high amplitude [short-latency IPSCs (slIPSCs)]. slIPSCs were GABAA receptor- mediated chloride currents that were evoked in an all-or-none manner. We tested whether slIPSCs resulted from somatic depolarization of presynaptic interneurons or from direct excitation of inhibitory presynaptic terminals via kainate receptors. Our evidence supports the former hypothesis: (1) slIPSCs had similar sensitivity to kainate and AMPA receptor blockers as electrically evoked EPSCs. (2) slIPSCs frequently had an notched rising phase suggestive of summated IPSCs resulting from repetitive firing of presynaptic neurons. (3) Latencies and interevent intervals were consistent with spike latencies and interspike intervals in fast-spiking (FS) interneurons. (4) slIPSCs were frequently evoked at spots where the recorded cell was also excited directly, but ∼15% of spots from which slIPSCs were evoked did not overlap with the recorded neurons cell body. We propose that slIPSCs from FS interneurons represent a pool of powerful inhibitory signals that can be recruited by local excitation. Because of their magnitude, progressive recruitment, and short latency, slIPSCs are a effective mechanism of regulating excitability in neocortical circuits.

Frequency-Dependent, Cell Type-Divergent Signaling in the Hippocamposeptal Projection

Hippocampal oscillations are critical for information processing, and are strongly influenced by inputs from the medial septum. Hippocamposeptal neurons provide direct inhibitory feedback from the hippocampus onto septal cells, and are therefore likely to also play an important role in the circuit; these neurons fire at either low or high frequency, reflecting hippocampal network activity during theta oscillations or ripple events, respectively. Here, we optogenetically target the long-range GABAergic projection from the hippocampus to the medial septum in rats, and thereby simulate hippocampal input onto downstream septal cells in an acute slice preparation. In response to optogenetic activation of hippocamposeptal fibers at theta and ripple frequencies, we elicit postsynaptic GABAergic responses in a subset (24%) of septal cells, most predominantly in fast-spiking cells. In addition, in another subset of septal cells (19%) corresponding primarily to cholinergic cells, we observe a slow hyperpolarization of the resting membrane potential and a decrease in input resistance, particularly in response to prolonged high-frequency (ripple range) stimulation. This slow response is partially sensitive to GIRK channel and D2 dopamine receptor block. Our results suggest that two independent populations of septal cells distinctly encode hippocampal feedback, enabling the septum to monitor ongoing patterns of activity in the hippocampus.

Glutamate biosensor imaging reveals dysregulation of glutamatergic pathways in a model of developmental cortical malformation

Cortical malformations can cause intractable epilepsy, but the underlying epileptogenic mechanisms are poorly understood. We used high-speed glutamate biosensor imaging to ask how glutamatergic signaling is altered in cortical malformations induced by neonatal freeze-lesions (FL). In non-lesion neocortical slices from 2 to 8week old rats, evoked glutamate signals were symmetrical in the medio-lateral axis and monotonic, correlating with simple, brief (≈50ms) local field potentials (LFPs). By contrast, in FL cortex glutamate signals were prolonged, increased in amplitude, and polyphasic, which paralleled a prolongation of the LFP. Using glutamate biosensor imaging, we found that glutamate signals propagated throughout large areas of FL cortex and were asymmetric (skewed toward the lesion). Laminar analysis demonstrated a shift in the region of maximal glutamate release toward superficial layers in FL cortex. The ability to remove exogenous glutamate was increased within the FL itself but was decreased in immediately adjacent regions. There were corresponding alterations in astrocyte density, with an increase within the lesion and a decrease in deep cortical layers surrounding the lesion. These findings demonstrate both network connectivity and glutamate metabolism are altered in this cortical malformation model and suggests that the regional ability of astrocytes to remove released glutamate may be inversely related to local excitability.

NPY signaling through Y1 receptors modulates thalamic oscillations

Neuropeptide Y is the ligand of a family of G-protein coupled receptors (Y(1) to Y(6)). In the thalamus, exogenous and endogenously released NPY can shorten the duration of thalamic oscillations in brain slices from P13 to P15 rats, an in vitro model of absence seizures. Here, we examine which Y receptors are involved in this modulation. Application of the Y(1) receptor agonist Leu(31)Pro(34)NPY caused a reversible reduction in the duration of thalamic oscillations (-26.6+/-7.8%), while the Y(2) receptor agonist peptideYY((3-36)) and the Y(5) receptor agonist BWX-46 did not exert a significant effect. No Y receptor agonist affected oscillation period. Application of antagonists of Y(1), Y(2) and Y(5) receptors (BIBP3226, BIIE0246 and L152,806, respectively) produced results consistent with those obtained from agonists. BIBP3226 caused a reversible disinhibition, an effect that increases oscillation duration (18.2+/-9.7%) while BIIE0246 and L152,806 had no significant effect. Expression of NPY is limited to neurons in the reticular thalamic nucleus (nRt), but Y(1) receptors are expressed in both nRt and adjacent thalamic relay nuclei. Thus, intra-nRt or nRt to relay nucleus NPY release could cause Y(1) receptor mediated inhibition of thalamic oscillations.

LSPS/Optogenetics to Improve Synaptic Connectivity Mapping: Unmasking the Role of Basket Cell-Mediated Feedforward Inhibition.

Abstract Neocortical pyramidal cells (PYRs) receive synaptic inputs from many types of GABAergic interneurons. Connections between parvalbumin (PV)-positive, fast-spiking interneurons (“PV cells”) and PYRs are characterized by perisomatic synapses and high-amplitude, short-latency IPSCs. Here, we present novel methods to study the functional influence of PV cells on layer 5 PYRs using optogenetics combined with laser-scanning photostimulation (LSPS). First, we examined the strength and spatial distribution of PV-to-PYR inputs. To that end, the fast channelrhodopsin variant AAV5-EF1α-DIO-hChR2(E123T)-eYFP (ChETA) was expressed in PV cells in somatosensory cortex of mice using an adeno-associated virus-based viral construct. Focal blue illumination (100–150 µm half-width) was directed through the microscope objective to excite PV cells along a spatial grid covering layers 2–6, while IPSCs were recorded in layer 5 PYRs. The resulting optogenetic input maps showed evoked PV cell inputs originating from an ∼500-μm-diameter area surrounding the recorded PYR. Evoked IPSCs had the short-latency/high-amplitude characteristic of PV cell inputs. Second, we investigated how PV cell activity modulates PYR output in response to synaptic excitation. We expressed halorhodopsin (eNpHR3.0) in PV cells using the same strategy as for ChETA. Yellow illumination hyperpolarized eNpHR3.0-expressing PV cells, effectively preventing action potential generation and thus decreasing the inhibition of downstream targets. Synaptic input maps onto layer 5 PYRs were acquired using standard glutamate-photolysis LSPS either with or without full-field yellow illumination to silence PV cells. The resulting IPSC input maps selectively lacked short-latency perisomatic inputs, while EPSC input maps showed increased connectivity, particularly from upper layers. This indicates that glutamate uncaging LSPS-based excitatory synaptic maps will consistently underestimate connectivity.