Delta glutamate receptor conductance drives excitation of dorsal raphe neurons

The delta glutamate receptors, GluD1R and GluD2R, are mysterious members of the ionotropic glutamate receptor family in that they are not gated by glutamate1,2. One theory is that they are scaffolding proteins or synaptic organizers strictly, rather than ion conducting channels. Although mutant forms and wild type channels have been reported to conduct3,4,5, conduction, gating, and biophysical properties of native GluD1R remain unexplored. Here we show that the inward current induced by activation of α1-adrenergic receptors (α1-ARs) in the dorsal raphe nucleus (DR) is mediated by GluD1R. Native GluD1R channels are functional ion channels that are constitutively active under basal conditions and α1-ARs increase the tonic current. This inward current is responsible for the α1-AR-dependent induction of persistent pacemaker-type firing of neurons in the DR. Given the extensive distribution of these receptors, the ionotropic nature of GluDR is proposed to be widespread in the nervous system.

The delta glutamate receptors, GluD1R and GluD2R, are mysterious members of the ionotropic glutamate receptor family in that they are not gated by glutamate 1,2 . One theory is that they are scaffolding proteins or synaptic organizers strictly, rather than ion conducting channels. Although mutant forms and wild type channels have been reported to conduct 3,4,5 , conduction, gating, and biophysical properties of native GluD1R remain unexplored. Here we show that the inward current induced by activation of a1-adrenergic receptors (a1-ARs) in the dorsal raphe nucleus (DR) is mediated by GluD1R. Native GluD1R channels are functional ion channels that are constitutively active under basal conditions and a1-A Rs increase the tonic current. This inward current is responsible for the a1-A R-dependent induction of persistent pacemakertype firing of neurons in the DR. Given the extensive distribution of these receptors, the ionotropic nature of GluDR is proposed to be widespread in the nervous system.
The dorsal raphe nucleus (DR) is the largest serotonergic nucleus in the brain and the predominant source of central serotonin . Activation of Gaq/11 protein-coupled a1-adrenergic receptors (a1-ARs) in the DR produces a slow and long-lasting membrane depolarization 6 . In vivo, tonic noradrenergic input that activates a1-ARs is required for 5-HT neurons to fire action potentials 7,8 . Despite having a crucial role in regulating 5-HT neuron excitability, the ion channels responsible for the depolarization remain unknown.

Activation of a1-A R produces an EPSC
Electrophysiological recordings were made from DR neurons in acute brain slices from wild type mice at 35° C in the presence of NMDAR, AMPAR, KainateR, GABA-AR, and 5-HT1AR antagonists. With cell-attached recordings, a train of 5 electrical stimuli (60 Hz) produced firing in previously quiescent neurons, which was blocked by application of the a1-AR antagonist, prazosin (100 nM, Fig. 1a). The excitation produced 20±5 action potentials (APs) that lasted 9.0±3.0 s, with a latency of 650.6±0.1 ms from onset of stimulation to the first AP ( Fig. 1b-e). In whole-cell recording using a potassium-based internal solution, the same train of electrical stimuli produced prolonged AP firing (Fig. 1f). In voltage-clamp mode (Vhold -65 mV), the same stimulation produced a slow and longlasting (27.4±2.3 s, n=10) excitatory postsynaptic current (EPSC, Fig. 1f) that was eliminated by application of prazosin (Fig. 1g). Prazosin had no effect on basal whole-cell current (-3.8±3.4 pA, p=0.232, n=10, not shown) indicating a lack of persistent inward current due to noradrenaline (NA) tone. Application of tetrodotoxin (1 µM) reversibly abolished the a1-AR-EPSC, demonstrating a dependence on presynaptic action potentials (Fig. 1h).
Disruption of the vesicular monoamine transporter with reserpine (1 µM) or removal of external Ca 2+ also eliminated the a1-AR-EPSC, indicating NA release is vesicular (Fig. 1i,j). To test whether a1-AR-EPSCs were dependent on G protein-signaling, recordings were made with an internal solution containing GDP-bS-Li3 (1.8 mM) instead of GTP. Disruption of G protein signaling with intracellular dialysis of GDP-bS-Li3 eliminated the a1-AR-EPSC within 5-20 min post-break-in (p=0.004, n=9), whereas dialysis with LiCl alone had no effect on the a1-AR-EPSC amplitude (p=0.625, n=4, Fig. S1a). These findings demonstrate a cell-autonomous requirement of G protein signaling in the generation of the a1-AR-EPSC.
Removal of external CaCl2 also had no significant effect on Erev, but augmented inward INA, (Fig. S4a,b). Taken together, the data suggest that a1-AR-dependent current, whether produced by vesicular NA release or exogenous NA is carried through a mixed cation channel, with inward current carried predominantly by Na + entry.
Measurements of Erev assume voltage-independence of the channel and the signaling mechanism between a1-AR and the channel. To test for voltage-dependence, we employed a two-pulse voltage-step protocol. Current was measured at Vhold -120 mV following a conditioning pre-pulse (-120 to 30 mV, 150 ms) before and after application of NA (Fig. S2f,g). INA was isolated by subtracting the current under basal conditions from the current during NA.
Conductance (GNA) was calculated, using an Erev of -25.1 mV. Conditioning depolarizing pre-pulses incrementally reduced GNA and the increase in membrane noise induced by NA measured at Vhold -120 mV (Fig. S2h-i), demonstrating voltage-dependence of inward INA, such that prior depolarization reduced conductance.

a1-A Rs modulate tonic GluD1R current
Activation of Gaq/11 protein-coupled receptors (GqPCRs), namely metabotropic glutamate mGluRs, muscarinic acetylcholine M1 (mAChRs), or a1-ARs produces slow, noisy inward currents throughout the central and peripheral nervous system. Multiple mechanisms have been reported to underlie the inward current including: inhibition of K + current (including leak , Ca 2+ -activated, and Kv7/M-current) 9-12 , modulation of TTX-sensitive persistent Na + current 13 , and activation of transient potential receptor canonical (TRPC) 14,15 , Na + -leak (NALCN) 16 , or GluDR channels 4,5 . GluD1R-encoding (Grid1) mRNA is expressed widely throughout the brain, with notably high levels in the DR 17,18 . To assess involvement of GluD1R, we applied a channel-selective toxin. Akin to other Ca 2+ -permeable ionotropic glutamate receptors, GluDR channels are blocked by the Joro spider toxin, 1-Naphthyl acetyl spermine (NASPM) 5,19 . Application of NASPM (100 µM, 6 min) blocked the a1-AR-EPSC (96.0±12.5% reduction), which recovered to baseline after >30 min of wash ( Fig. 3a (Fig. S5a). Thus, INSP was due to block of tonic Na + -dependent inward current. INSP was not dependent on prior electrical stimulation of the brain slice, as the magnitude of INSP was similar between stimulated and unstimulated brain slices (Fig. S5b,c). Given that NASPM is an open-channel blocker 20 , we tested whether electrically evoking an a1-AR-EPSC during application of NASPM was required for block. After obtaining a steady a1-AR-EPSC baseline, NASPM was applied for 6 min without stimulating the brain slice. The a1-AR-EPSC was blocked when stimulation was reapplied (Fig. 3f), indicating that the channels underlying the a1-AR-EPSC were blocked. Thus, the a1-AR-EPSC is carried by channels that are at least transiently open at rest and may be the same channels that underlie the NASPM-sensitive tonic inward current.
GluDRs bind the amino acids D-serine and glycine which inhibit constitutively open mutant GluDR current 21,22 .
Next, a viral genetic strategy was used to functionally delete GluD1R by targeting the encoding gene, GRID1, via CRISPR/Cas9 (Fig. S7a,b). In brief, a cocktail of AAV1 viruses encoding Cas9, and mouse Grid1 guide RNA with enhanced green fluorescent protein (eGFP) reporter (GRID1) was microinjected into the DR of wild type mice. A separate cohort received microinjections of a cocktail of AAV1 viruses encoding Cas9 and eGFP reporter (control).
Brain slices were prepared after >4 weeks and the DR was microdissected and frozen on dry ice to assess the mutation of GRID1. Restriction enzyme site-digested PCR confirmed in vivo mutation of GRID1 at the expected site ( Fig. S7c). In a separate cohort, brain slices were prepared and whole-cell voltage-clamp recordings were made from infected and uninfected neurons visualized in brain slices by expression of eGFP. In eGFP + neurons from control mice, electrical stimulation produced a decrease in Rm and an a1-AR-EPSC, and application of NA caused inward INA (Fig. 4). However, in eGFP + neurons from GRID1 mice, electrical stimulation did not change Rm (Fig. 4a) and no a1-AR-EPSC was detected above baseline noise (Fig. 4b). In addition, inward INA was substantially smaller in eGFP + neurons from GRID1 mice, as compared to eGFP + neurons from control mice (Fig. 4c). In the same slices from GRID1 mice, eGFPneurons still had an a1-AR-EPSC and inward INA (Fig. 4a,c). Lastly, application of NASPM produced an apparent outward current in eGFP + neurons from control mice, but not from GRID1 mice (Fig. 4d).
Taken together, these results demonstrate that conduction through GluD1R is necessary for the a1-AR-EPSC and the NASPM-sensitive tonic inward current.

Relevance of GluD1R to the dorsal raphe
In vivo, 5-HT neurons in the DR require NA release and subsequent activation of a1-ARs to maintain AP firing 8 .
Activation of a1-ARs in the DR by exogenous agonist was reported to depolarize neurons through net reduction of K + conductance and by activation of an unidentified non-K + conductance 23 . More recently, Brown et al., reported that a1-ARs activation in the DR induces Na + -dependent inward current with an Erev of -23 mV 24 , similar to our findings. Our study identifies GluD1R as the channel that carries this mixed cation current, indicating that modulation of GluD1R is a key constituent in driving persistent AP firing of the 5-HT neurons. In principle, inward GluD1R current may bring the membrane potential to threshold, but recruitment of other voltage-gated ion channels is expected to underlie the persistent pacemaker-like activity.

Ionotropic nature of GluD1R
GluDR have been characterized as synaptic organizers, regulating LTD, endocytosis and trafficking of AMPAR, formation of excitatory and inhibitory synapses, and spine density 3,25-27 , without the requirement of ion conduction. Similarly, NMDAR signal through non-ionotropic or "metabotropic" mechanisms where ion conduction is not required, to regulate LTD, AMPAR endocytosis, and spine morphology 28 . The ionotropic nature of GluDR does not conflict with its known role as a scaffold protein, but rather expands the similarities between NMDAR and GluDR.
The largest obstacle towards understanding the ionotropic nature of GluDR is the lack of known agonist and inability to gate the intact channel. The majority of studies have been performed on constitutively open mutant or chimeric channels 29,30 . Two prior studies have demonstrated that in heterologous systems and brain slices, activation of metabotropic glutamate receptors (mGluR) produces an inward current carried by GluD1R 5 or GluD2R 4 , concluding mGluR activation triggers rather than modulates gating of GluDR. The congruous explanation of our results is that, in DR neurons, GluD1R are functional and open under basal conditions, carrying subthreshold, tonic Na + current. Activation of a1-ARs, by exogenous agonist or synaptic NA release, excites DR neurons by increasing GluD1R current.
In general, the kinetics of iGluR synaptic currents are controlled by the lifetime of the receptor-agonist complex and the rates of desensitization and deactivation. In brain slices, NMDAR carry tonic inward current 31 attributed to slow desensitization and ambient levels of glutamate and glycine. What remains to be understood are the conditions that permit GluD1R opening, and why their activation has been largely elusive in heterologous expression systems. Reminiscent of times before the discovery of glycine as a necessary co-agonist at NMDAR 32,33 , it may be that an endogenous agonist needed for gating is present in brain slices. Alternatively, it is possible GluD1R are gated by an intracellular factor or require expression of accessory or interacting proteins 34 . Tonic activation of a1-ARs cannot explain the tonic inward current as a1-AR antagonism did not change basal whole-cell current.
The mechanism by which a1-ARs increase GluD1R current also remains to be described and may be distinct from tonic activation. It is well-established that GqPCRs, especially mGluR and mAChR, bidirectionally change NMDAR and AMPAR currents, producing the two major forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD), in part through a variety of postsynaptic mechanisms 35 . To our knowledge, the duration of the a1-AR-EPSC (~27 s) is exceptional for any synaptic current and more closely resembles the duration of short-term synaptic plasticity; for instance, endocannabinoid-mediated short-term depression 36 .
GqPCRs can activate phospholipase C which hydrolyzes the integral membrane lipid, phosphatidylinositol 4,5bisphosphate (PIP2). PIP2 stabilizes Kv7 channels such that PIP2 hydrolysis following mAChR activation accounts for inhibition of M-current 37 . By the same signaling cascade, GqPCRs stimulate the production of endocannabinoids that can act directly on membrane ion channels 38 . Thus, one possibility is that a1-ARs modulate GluD1R through membrane lipid signaling. Largely, it remains to be seen whether the intracellular signaling cascades known to affect NMDAR and AMPAR currents generalize to GluDR.
Using heterologous systems or constitutively open mutants, GluDR current reverses polarity around 0 mV 39 , akin to AMPAR and NMDAR, while our results show an Erev of ~ -30 mV. The difference may be due space-clamp error in brain slices, since the magnitude of subtracted current is small relative to total current at depolarized potentials. Activation of a1-ARs in the DR also inhibits A-type K + current 40 . Thus, Erev measurements may include inhibition of a voltage-gated K + conductance that is not contributing directly to the a1-AR-EPSC but is activated during voltage ramps at Vhold >-65 mV. Tail current analysis revealed voltage-dependence of INA, such that prior depolarization reduced conductance. These data may reflect block of GluD1R by endogenous intracellular polyamines, as established for calcium-permeable AMPAR and KainateR 41 . Another important consideration is that our measurements may be subject to voltage-dependence of the signaling pathway between a1-ARs and GluD1Rs.
There are reports of inward currents generated by GqPCR activation with Erevs between -40 and -23 mV 13,24,42 , measured under different recording conditions, suggesting a commonality that is unlikely to be due to space-clamp error alone. Taken together, measurements here should be considered an approximate estimate of GluD1R, and more precisely as the current-voltage relationship of the a1-AR-GluD1R signaling complex.

Summary
Native GluD1R are functional ion channels that carry ionic current. GluD1R underlie the a1-AR-mediated

Animals
All studies were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory animals with the approval of the National Institute on Drug Abuse Animal Care and Use Committee.
Wild type C57BL/6J (>3 months old) mice of either sex were used. Mice were group-housed on a 12:12 h reverse light cycle.

Brain slice preparation and electrophysiological recordings
Mice were deeply anesthetized with isoflurane and killed by decapitation. Brains were removed quickly and placed in warmed modified Krebs' buffer containing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 CaCl2, 1.2 NaH2PO4, 21.5 NaHCO3, and 11 D-glucose with 5 μM MK-801 to reduce excitotoxicity and increase viability, bubbled with 95/5% O2/CO2. In the same solution, coronal DR slices (220 μm) were obtained using a vibrating microtome (Leica 1220S) and incubated at 32 °C >30 minutes prior to recording. All experiments were conducted with NMDAR, AMPAR, KainateR, GABA-AR, and 5-HT1AR antagonists in the external solution. In addition, a a2-adrenergic receptor antagonist was added for experiments where NA was applied and a glycine receptor antagonist was added when glycine was applied. Unitary current was calculated from fluctuation analysis, as previously described 43 , assuming the macroscopic current arises from independent, identical channels with a low probability of opening, according probability theory; i = s 2 /[I(1 -p)] where i is unitary current, s 2 is the variance, I is mean current amplitude, and p is probability of opening.

Vector construction gRNA identification
CRISPR SpCas9 gRNA target sites were identified in the mouse Grid1 gene (NC_000080.6) using CRISPOR 44 . The seed sequence (GAACCCTAGCCCTGACGGCG) was chosen based on its relatively high specificity scores and the observation that it contains a Bgl I restriction enzyme site (GCCNNNN^NGGC) that overlaps with the Cas9 cleavage site.

mGrid1 genotyping
C57BL/6J mouse genomic DNA was isolated from tail biopsies or brain pieces containing microdissected DR by digestion in DNA lysis buffer (50 mM KCl, 50 mM Tris-HCl (pH 8.0), 2.5 mM EDTA, 0.45% NP-40, 0.45% Tween-20, 0.5 ug/mL proteinase K) for 3 h at 55 °C, and 1 h at 65 °C. Lysates were then used as templates to amplify a 654 basepair fragment including the 390F gRNA target site using Q5 HotStart Master mix (New England Biolabs). A portion of the finished PCR reaction was treated with Bgl I restriction enzyme (New England Biolabs) for 60 minutes and processed on an AATI fragment analyzer.

Construction and packaging of AAV vectors
The AAV vector plasmid encoding SpCas9 45 (pX551) expressed from the MeCP2 promoter was a gift from Feng Zhang (Addgene plasmid # 60957, AAV-Cas9). The AAV packaging plasmid encoding a nuclear envelope-embedded eGFP reporter (Addgene 131682) was constructed by amplifying the KASH domain from (Addgene 60231, a gift of Feng Zhang) and fusing it (in-frame) to the end of coding region for eGFP in (Addgene 60058, pOTTC407) using ligation-independent cloning (AAV-empty, Fig. S8). gRNA was cloned into a mU6 expression cassette and then moved into an AAV backbone expressing a nuclear envelope-embedded (KASH-tagged) eGFP reporter (Addgene 131683) by PCR amplification and ligation-independent cloning (AAV-GRID1). Insert-containing clones were verified by sequencing and restriction fragment analysis prior to virus production. All AAV vectors were produced using triple transfection method as previously described 46 . All vectors were produced using serotype 1 capsid proteins and titered by droplet digital PCR.

Stereotaxic intracranial microinjections
Mice were anesthetized with a cocktail of ketamine/xylazine, immobilized in a stereotaxic frame (David Kopf Instruments), and received one midline injection of a 1:1 (v/v) cocktail of viruses AAV-Cas9 and AAV-empty or AAV-GRID1 for total volume 400 nL delivered over 4 minutes. The coordinates for injection were AP -4.4; ML 1.19, 20° angle; DV -3.62 mm, with respect to bregma. Prior to surgery, mice were injected subcutaneously with warm saline (0.5 mL) to replace fluid lost during surgery and given carprofen (5 mg/kg) post-surgery for pain relief. Mice recovered for >4 weeks to allow expression. Infected neurons were identified in the slice by visualization of eGFP.

Data analysis and visualization
Data were analyzed using Clampfit 10.7. Data are presented as representative traces, or in scatter plots where each point is an individual cell, and bar graphs with means ± SEM. Unless otherwise noted, n = number of cells. Erevs were determined by linear regression for each cell. Recordings in which current did not cross 0 pA were omitted from analysis. To minimize space-clamp errors, analysis of current during voltage ramps was limited to -10 mV where the currents were typically less than 500 pA. Ramp currents were averaged in 2 mV bins (20 ms). Data sets with n>30 were tested for normality with a Shapiro-Wilk test. When possible (within-group comparisons), significant differences were determined for two group comparisons by paired t-tests, Wilcoxon matched-pairs signed rank test, and in more than two group comparisons by nonparametric repeated-measures ANOVA (Friedman test). Significant mean differences in between-group comparisons were determined for two group comparisons by Mann Whitney tests, and in more than two group comparisons by Kruskal-Wallis tests. ANOVAs were followed, when p<0.05 by Dunn's multiple comparisons post hoc test. Linear trends were analyzed using a

COMPETING INTEREST DECLARATION
The authors declare no competing interests.

CORRESPONDING AUTHOR
Stephanie C. Gantz email: stephanie.gantz@nih.gov         (NSP, 100 µM). Replacing 126 mM NaCl with NMDG eliminated the apparent outward current, suggesting a block of a tonic inward Na + current. b, Representative trace of whole-cell voltage-clamp recording of the apparent outward current induced by application of NASPM in an unstimulated brain slice. Dashed line is at 0 pA (Vhold -65 mV). c, Plot of amplitude of NASPM-induced apparent outward current in stimulated and unstimulated brain slices demonstrating no effect of prior electrical stimulation (p=0.850, n=21&5). Line and error bars/shaded area represent mean±SEM, ns indicates not significant.   (Table S1). b, A nucleotide representation of gRNA target site with the translated peptide sequence underneath. The BglI recognition site (GCCNNNN^NGGC) is contained by the grey box and overlaps with the predicted Cas9 cleavage site (|). c, Fragment analysis on BglI-digested Grid1 PCR products amplified from the DR of mice injected with (AAV-Cas9 and AAV-empty) or (AAV-Cas9 and AAV-GRID1). "% PCR undigested" refers to the amount of the undigested PCR product relative to the total PCR product as determined by AATI fragment analysis. Figure 4 | The a1-A R-EPSC is eliminated by targeting of GluD1 receptors via CRISPR/Cas9. a, Membrane resistance (Rm, DV -65 to -120 mV) decreased after stimulation in infected neurons from mice injected with AAV-Cas9 and AAV-empty (control, p=0.0002, n=13), but not in infected neurons from mice injected with AAV-Cas9 and AAV-GRID1 (GRID1, p=0.562, n=16). b, Representative traces (left) and grouped data (right, p<0.0001, n=16&16) demonstrating the presence of an a1-AR-EPSC in infected neurons from control mice, but not from GRID1 mice. Neighboring uninfected neurons from GRID1 mice (uninf.) had an a1-AR-EPSC that was indistinguishable from infected neurons from control mice (p>0.999, n=7). c, Targeting GluD1R reduced the inward current to NA (INA, 30 µM) as compared to infected neurons from control mice, as shown in representative traces (left) and grouped data (right, p=0.009, n=16&16). Capacitive transients and ramp-induced currents have been truncated for clarity. d, Targeting GluD1R reduced the tonic inward current revealed by application of NASPM (100 µM, INSP) as compared to infected neurons in control mice (p=0.009, n=5&11). Line and error bars represent mean±SEM, * denotes statistical significance, ns denotes not significant. Table S1 | Primer sequences used in PCR amplification (amp) and sequencing (seq).

Primer name Primer sequence (linkers in lowercase)
Forward amp primer tgattacgccaagctt GGTGGAGCTGTGTGGATGAAGC Forward seq primer CCAGCCTGTGACCTCATGACC Reverse amp primer gacggccagtgaattc CTTCAGCTGTCATGATAAGGTGATGTTG