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. 2023 Mar 17;12:e83760. doi: 10.7554/eLife.83760

Netrin-1 regulates the balance of synaptic glutamate signaling in the adult ventral tegmental area

Marcella M Cline 1,2, Barbara Juarez 1,3, Avery Hunker 3,†, Ernesto G Regiarto 3, Bryan Hariadi 3, Marta E Soden 3, Larry S Zweifel 1,3,✉
Editors: Kate M Wassum4, Kate M Wassum5
PMCID: PMC10023152  PMID: 36927614

Abstract

The axonal guidance cue netrin-1 serves a critical role in neural circuit development by promoting growth cone motility, axonal branching, and synaptogenesis. Within the adult mouse brain, expression of the gene encoding (Ntn1) is highly enriched in the ventral midbrain where it is expressed in both GABAergic and dopaminergic neurons, but its function in these cell types in the adult system remains largely unknown. To address this, we performed viral-mediated, cell-type specific CRISPR-Cas9 mutagenesis of Ntn1 in the ventral tegmental area (VTA) of adult mice. Ntn1 loss-of-function in either cell type resulted in a significant reduction in excitatory postsynaptic connectivity. In dopamine neurons, the reduced excitatory tone had a minimal phenotypic behavioral outcome; however, reduced glutamatergic tone on VTA GABA neurons induced behaviors associated with a hyperdopaminergic phenotype. Simultaneous loss of Ntn1 function in both cell types largely rescued the phenotype observed in the GABA-only mutagenesis. These findings demonstrate an important role for Ntn1 in maintaining excitatory connectivity in the adult midbrain and that a balance in this connectivity within two of the major cell types of the VTA is critical for the proper functioning of the mesolimbic system.

Research organism: Mouse

Introduction

Proper regulation of the midbrain dopamine system is essential for numerous brain functions and behavior (Bissonette and Roesch, 2016). Disruption in the balance of midbrain dopamine neuron activity has been linked to several neurological and psychiatric conditions, including autism (Pavăl, 2017), schizophrenia (Hietala and Syvälahti, 1996), and substance use disorders (Ostroumov and Dani, 2018). Within the VTA, the activity of dopamine neurons is regulated in part by inhibitory (GABAergic) and excitatory (glutamatergic) synaptic input. The molecular mechanisms that maintain the balance of inhibitory and excitatory connectivity in the adult midbrain, however, remain poorly resolved.

Genome-wide association studies and analysis of de novo mutations have strongly implicated genes regulating neuronal axon guidance in neurodevelopmental disorders (Gilman et al., 2012; Gulsuner et al., 2013). Although the impact of mutations in these genes early in development is likely critical for their role in neurodevelopmental disorders, many of the genes maintain high levels of expression in the adult brain, and their functions in this context are less understood. We previously demonstrated that the axonal guidance receptor Robo2 is necessary for the maintenance of inhibitory synaptic connectivity in the adult VTA (Gore et al., 2017), suggesting that axonal guidance proteins have a critical function in maintaining synaptic connectivity in the adult midbrain.

Netrin-1 is predominately recognized for its role in neurodevelopmental processes (Gore et al., 2017; Manitt et al., 2010; Winberg et al., 1998; Glasgow et al., 2018; Yetnikoff et al., 2010). During development, the gene encoding netrin-1 (Ntn1) is highly expressed throughout the central nervous system (CNS). Following this critical period global expression decreases (Manitt et al., 2010), but expression within the limbic system, particularly in the ventral midbrain, persists. Consistent with the continued function of Ntn1 following early development, genetic inactivation of either Ntn1 (Winberg et al., 1998) or its receptor Dcc Glasgow et al., 2018 from forebrain glutamatergic neurons in late postnatal development results in significantly impaired spatial memory in adult mice that corresponds to a loss of hippocampal plasticity. Within the VTA, Dcc expression levels in adult mice are significantly upregulated following amphetamine exposure (Yetnikoff et al., 2010), and Dcc haploinsufficient mice display blunted locomotor response to amphetamine (Flores et al., 2005), consistent with increased excitatory synaptic strength in the VTA following amphetamine treatment (Saal et al., 2003). These results suggest a potential role for Ntn1 signaling through Dcc in regulating excitatory tone in the adult dopamine system.

To determine whether Ntn1 regulates excitatory synaptic connectivity in the VTA of adult mice, we used viral-mediated, Cre-inducible CRISPR/Cas9 (Hunker et al., 2020) to selectively mutate Ntn1 in midbrain dopamine and GABA neurons. We find that Ntn1 loss of function significantly reduces postsynaptic glutamate receptor-mediated currents in a cell-autonomous manner similar to what has been reported previously in the adult hippocampus (Glasgow et al., 2018). We further show that Ntn1 loss of function in VTA GABA neurons has a more profound effect on behavior than the loss of function in VTA dopamine neurons. Intriguingly, the simultaneous loss of function of Ntn1 in both cell types of the VTA largely rescues the behavioral phenotypes observed following mutagenesis in VTA GABA neurons alone. These data support a model in which the balance of excitatory synaptic connectivity between dopamine and GABA neurons within the VTA is maintained by the persistent expression of the developmental gene Ntn1. This continued function of Ntn1 in adulthood sustains the excitatory/inhibitory equilibrium onto dopamine neurons that is critical to the function of the mesolimbic dopamine system.

Results

Ntn1 expression and mutagenesis in the VTA

In situ hybridization analysis of Ntn1 from the Allen Institute mouse brain expression atlas (Lein et al., 2007) shows diffuse and low levels of expression throughout the adult mouse brain, with moderate expression levels in the cerebellum and hippocampus (Figure 1A), and the highest level of expression in the ventral midbrain (substantia nigra and ventral tegmental area). The VTA is comprised of multiple cell types Morales and Margolis, 2017; to determine the cell type-specific expression of Ntn1 within the heterogeneous VTA, we performed RNAscope in situ hybridization on midbrain slices from adult wild-type mice (>8 weeks of age) and probed for Ntn1, Th (tyrosine hydroxylase, a marker of dopamine neurons), and Slc32a1 (vesicular GABA transporter [Vgat], a marker of GABA neurons). We found Ntn1 expression to be present throughout the VTA, largely localized to Th-positive neurons but also present in GABA neurons (Figure 1C–F). Of the identified Ntn1 positive cells, Ntn1 expression co-localized with Th expression (dopamine producing neurons; 72.2% co-localization) and Slc32a1-expressing GABA neurons (18.1% co-localization) (Figure 1E). The remaining Ntn1 expressing cells that do not co-localize with Th or Slc32a1 are likely glutamatergic neurons (Morales and Margolis, 2017), or possibly glial cells (Phillips et al., 2022). Immunohistochemistry for Ntn1 and Th (Figure 1G) confirmed the presence of Ntn1 in dopamine and non-dopamine producing (Th-negative) cells.

Figure 1. Netrin-1 is present in the adult ventral tegmental area (VTA) and expressed by both dopamine and GABA neurons.

Figure 1.

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3D display of Ntn1 (A) and Slc6a3 (B, dopamine marker) from the Allen Brain Atlas. (C–D) 20 X magnification images of in situ hybridization (RNAScope) for Ntn1 (green) and Slc32a1 (GABA marker; red, C) and Th (dopamine marker; red; D). Arrows indicate co-labeling of Ntn1 with Slc32a1 (C) or Th (D). Scale bar indicates 20 μm. (E–F) Quantification of cell type expression. Of the cells expressing Ntn1, 72.2% were dopaminergic (Th+) and 18.1% were GABAergic (Slc32a1+; E). (F) Of the total of Th+ identified cells, 64.5% co-expressed Ntn1 (35.6% did not express Ntn1), and 30.4% of Slc32a1 identified cells co-expressed Ntn1 (69.5% did not express Ntn1). (G) Immunohistochemistry confirms the presence of Ntn1 protein (red) in both Th+ (cyan) and non-dopamine cells (Th- cells, indicated by yellow arrows).

Figure 1—source data 1. Cell counts.

To selectively mutate Ntn1 in specific cell types in the VTA, we designed a single guide RNA (sgRNA) targeting exon 2 in mice (sgNtn1; Figure 2A) and cloned it into an AAV packaging plasmid containing a Cre-recombinase dependent expression cassette for SaCas9 (Hunker et al., 2020). To determine the efficiency of Ntn1 mutagenesis, we injected DAT-Cre (Slc6a3Cre/+) mice (aged 8–10 weeks) bilaterally into the VTA with either AAV-FLEX-SaCas9-HA-sgNtn1 and AAV-FLEX-YFP (DAT-Cre Ntn1-cKO mice) or AAV-FLEX-SaCas9-sgRosa26 (a gene locus with no known function; control mice). Four to five weeks following injection, we performed immunohistochemistry for Ntn1 and Th. Ntn1 conditional knockout (cKO) resulted in a significant reduction in the proportion of VTA Th-positive cells co-labeled with Ntn1 in DAT-Cre Ntn1 cKO mice compared to controls (Figure 2D–E). In contrast to previous findings following Ntn1 deletion in the substantia nigra (Jasmin et al., 2021), the average number of Th + cells per slice was not statistically different in DAT-Cre Ntn1 cKO mice compared to controls (control: 178.2 ± 12.68 and Ntn1 cKO 173.3 ± 10.75). Although, this result is consistent with Ntn1 inactivation not compromising cell viability, without a complete stereological analysis of every neuron within the VTA, we cannot definitively conclude that some cell loss did not occur.

Figure 2. Virally delivered CRISPR-Cas9 complex targeting the Ntn1 locus results in a significant reduction in Ntn1 antibody staining.

Figure 2.

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(A–B) Schematics summarizing cell type-specific knockout procedure. (A) Adult mice were injected bilaterally into the VTA with AAV-FLEX-SaCas9-HA-sgNtn1 and AAV-FLEX-YFP. Control mice received an equivalent volume of -sgRosa26 and/or AAV-FLEX-YFP. SaCas9 is virally delivered into the genome in the inactive orientation and returned to the active orientation only in the presence of Cre recombinase, limiting Cas9 expression to target cells. (B) Schematic of the VTA (left) showing VTA GABA neurons project to and inhibit VTA dopamine neurons. By using transgenic Cre-driver mouse lines (right) viral delivery of SaCas9 results in gene disruption in specifically VTA dopamine neurons (DAT-Cre mice, top panel), or VTA GABA neurons (Vgat-Cre mice, bottom panel). (D) Example images for Th (cyan) and Ntn1 (red) immunostaining in the ventral tegmental area (VTA) of mice injected with control or sgNtn1 CRISPR virus. (E) Quantification of the percentage of Th + cells co-labled with Ntn1 (Students t-test; t=8.179, df = 10, 62.25 ± 5.796 vs 9.586 ± 2.807, ****p<0.0001).

Figure 2—source data 1. Cell counts.

Netrin-1 regulates excitatory connectivity within the adult VTA

Previous research has shown that Ntn1 regulates excitatory synaptic connectivity in the adult hippocampus (Glasgow et al., 2018). To determine the impact of Ntn1 loss of function on synaptic connectivity, DAT-Cre or Vgat-Cre (Slc32a1Cre/+) mice were injected with AAV1-FLEX-SaCas9-U6-sgNtn1 and AAV1-FLEX–YFP (Figure 3A and E). After at least four weeks, miniature excitatory postsynaptic currents (mEPSCs) were recorded from fluorescently identified dopamine or GABA neurons of the VTA. Ntn1 mutagenesis in dopamine neurons resulted in significantly reduced mEPSC amplitude and frequency (Figure 3B–D). Similarly, Ntn1 mutagenesis in VTA GABA neurons also resulted in significantly reduced mEPSC amplitude and frequency (Figure 3F–H). We did not detect significant effects on miniature inhibitory postsynaptic currents (mIPSCs) in VTA dopamine or GABA neurons following Ntn1 mutagenesis in these cells (Figure 3—figure supplement 1), suggesting Ntn1 does not play a role in regulating inhibitory connectivity in these cells.

Figure 3. Loss of Ntn1 results in a significant reduction in excitatory postsynaptic current.

(A) Schematic of DAT-Cre dopamine specific Ntn1cKO. (B) Sample traces from control (top panel) and DAT Ntn1 cKO mice (bottom panel). (C–D) mEPSC amplitude (C) and frequency (D) measured from fluorescently identified dopamine neurons (n=35 controls, n=33 cKO, t=3.744, df = 66, ***p<0.001 and t=5.259, df = 66, ****p<0.0001). (E) Schematic of Vgat-Cre GABA specific Ntn1cKO. (F) Sample traces from control (top panel) and Vgat Ntn1 cKO mice (bottom panel). (G–H) mEPSC amplitude (G) and frequency (H) measured from fluorescently identified GABA neurons (n=30 controls, n=32 cKO, t=2.048, df = 60, *p<0.05, and t=3.966, df = 60, ***p<0.001). (I) Schematic of stimulating electrode placement in horizontal midbrain slice and example EPSCs. (J–K) Paired pulse ratio in dopamine (J, n=18 controls, n=21 cKO, t=1.271, df = 37, p>0.05), or GABA neurons (K, n=14 controls, n=21 cKO, t=1.105, df = 33, p>0.05).

Figure 3—source data 1. EPSCs and IPSCs from targeted cells.
Figure 3—source data 2. Additional EPSC and IPSC data from non-targeted cells.

Figure 3.

Figure 3—figure supplement 1. No significant differences in inhibitory synaptic connectivity associated with Ntn1 loss of function.

Figure 3—figure supplement 1.

(A) mIPSC amplitude and frequency of DAT-Cre fluorescently identified dopamine neurons (n=20 controls, n=27 cKO, t=0.8283, df = 45 p>0.05 and t=0.6668, df = 45 p>0.05). (B) mIPSC amplitude and frequency of Vgat-Cre fluorescently identified GABA neurons (n=20 controls, n=10 cKO, t=0.3183, df = 28 and t=1.106, df = 29 p>0.05).
Figure 3—figure supplement 2. No significant differences in excitatory or inhibitory synaptic connectivity in non-targeted cell types.

Figure 3—figure supplement 2.

(A) Miniature excitatory postsynaptic current (mEPSC) amplitude and frequency recorded from non-fluorescent cells in DAT-Cre mice (presumptively non-dopamine neurons) (n=16 controls, n=11 cKO, t=0.2985, df = 25, and t=1.678, df = 25 p>0.05). (B) Miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency recorded from non-fluorescent cells in DAT-Cre mice (n=10 controls, n=15 cKO, t=1.134, df = 23 and t=1.546, df = 23 p>0.05). (C) mEPSC amplitude and frequency recorded from non-fluorescent cells in Vgat-Cre mice (presumptively non-GABA neurons) (n=18 controls, n=20 cKO, t=0.2333, df = 33 and t=1.028, df = 33 p>0.05). (D) mIPSC amplitude and frequency recorded non-fluorescent cells in Vgat-Cre mice (n=27 controls, n=21 cKO, t=0.4650, df = 46 and t=0.8771, df = 46 p>0.05).
Figure 3—figure supplement 3. Loss of Netrin function results in significant decrease in AMPA response.

Figure 3—figure supplement 3.

Analysis of 1/CV2 from miniature excitatory postsynaptic current (mEPSC) data in (A) DAT-Cre mice measured from fluorescently identified dopamine neurons (n=35 controls, n=33 cKO, t=1.252, df = 66. p>0.05) and (B) Vgat-Cre mice measured from fluorescently identified GABA neurons (n=30 controls, n=32 cKO, t=0.8932, df = 60, p>0.05). (C) Bath application of 1 uM AMPA peak amplitude holding current (n=8 controls, n=17 sgNtn1, t=2.906, df = 23, p=0.008) and (D) voltage-clamped (–60 mV) holding current following 30 s bath application of 1 uM AMPA in 10 s averaged increments. (E) 50 uM NMDA evoked peak holding current (t=0.6421, df = 23, p=0.5271) and (F) voltage-clamped (+40 mV) holding current following 30 s bath application of 50 uM NMDA in 10 s averaged increments.
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Because Ntn1 is a secreted protein, it is also possible that Ntn1 loss of function in one cell type could affect synaptic connectivity in adjacent neurons in which the gene was not inactivated, inducing a non-cell autonomous effect. To address this, we recorded mEPSCs from non-YFP-expressing (presumptively non-dopamine) neurons in DAT-Cre mice injected with Ntn1 CRISPR or control virus, and from non-YFP-expressing (presumptively non-GABA) neurons in Vgat-Cre injected mice. We did not observe significant non-cell autonomous effects on mEPSCs from non-targeted cells (Figure 3—figure supplement 2). Similarly, we also did not observe non-cell autonomous effects on mIPSCs from non-targeted cells (Figure 3—figure supplement 2).

The observed reduction in mEPSC frequency suggests that loss of Ntn1 function could act presynaptically, potentially through postsynaptic Ntn1 secretion (Glasgow et al., 2018). To test potential presynaptic changes in vesicle release probability, we analyzed the paired-pulse ratio (PPR) of electrically evoked EPSCs delivered 50 ms apart. Ntn1 mutagenesis in either dopamine or GABA neurons did not result in a significant change in PPR compared to controls, suggesting no measurable change in presynaptic release (Figure 3J–K). To further resolve this question, we analyzed potential changes in quantal size by performing a 1/CV2 analysis of the coefficient of variation in the mEPSC amplitude. We did not detect a statistically significant change in 1/CV2 associated with Ntn1 loss in either dopamine or GABA cells, further suggesting netrin manipulation is altering either the number of or the function of postsynaptic AMPA receptors (Figure 3—figure supplement 3A, B).

Our data suggest that the observed changes in mEPSCs are likely a reflection of reduced AMPA-type or NMDA-type glutamate receptor levels in postsynaptic cells. To address this, fluorescently identified dopamine neurons from DAT-Cre Ntn1 cKO mice held at –60 mV, and AMPA-evoked current was measured following bath application of 1 uM AMPA. For NMDA currents, neurons were held at +40 mV and NMDA-evoked current was measured following bath application of 50 μM NMDA. Ntn1 mutagenesis in DAT-Cre mice resulted in significantly reduced AMPA-evoked current compared to controls (Figure 3—figure supplement 3C, D). In contrast, NMDA-evoked responses were similar between the groups (Figure 3—figure supplement 3E, F). These results suggest that Ntn1 regulates AMPA receptor availability in adult VTA.

Ntn1 loss of function in VTA-dopamine neurons has little effect on behavior

Dopamine producing neurons of the VTA regulate multiple aspects of locomotor activity, motivated behavior, and psychomotor activation. To determine whether conditional mutagenesis of Ntn1 in dopamine neurons, and subsequent reduction in excitatory synaptic connectivity impacts these behaviors, we injected DAT-Cre mice with AAV1-FLEX-SaCas9-sgNtn1 or AAV1-FLEX-SaCas9-sgRosa26 (control) and assayed them in multiple behavioral paradigms. First, we monitored day-night locomotion in control and AAV1-FLEX-SaCas9-sgNtn1 injected DAT-Cre mice. No significant differences were detected (Figure 4B and Figure 4—figure supplement 1).

Figure 4. Ntn1 cKO in DA neurons results in little behavioral alteration.

(A) Schematic summarizing cell type-specific knockout procedure. (B) Distance traveled in 15 min bins over the course of three nights and two days (n=21 control; n=15 cKO, Two-way ANOVA, Group F(1, 34)=1.169, p=0.2872, Time F(18.25, 620.6)=21.97 p<0.0001, Interaction F(251, 8534)=1.063 p=0.2380). (C) Earned reinforcers during three days of FR1 or FR5 operant conditioning (n=19 control; n=15, FR1; Group F(1, 96)=0.9761 p=0.3257, Time F(2, 96)=9.999 p=0.0001, Interaction F(2, 96)=0.006622 p=0.9934; FR5 Group F(1, 32)=0.6140, p=0.9808, Time F (2, 96)=2.786 p=0.0667, Interaction F(2, 96)=0.008669 p=0.9914). (D) Breakpoint (maximum presses per reinforcer) on a progressive ratio task (t=0.9434, df = 32, p=0.3525). (E) Lever presses per session during five days of extinction training (Group F(1, 32)=1.336, p=0.2562, Time F(4, 128)=87.55 p<0.0001, Interaction F(4, 128)=2.017 p=0.0959). (F) Acoustic startle response to varying intensity white noise stimuli (Group F(1, 31)=3.176 p=0.0845, Intensity F(1.737, 53.83)=37.74 p<0.0001, Interaction F(6, 186)=2.124 p=0.0525) (G) Percent inhibition of startle response following pre-pulse at indicated intensities (Group F(1, 96)=0.05032 p=0.8230, Intensity F(2, 96)=5.638 p=0.0048, Interaction F(2, 96)=0.2402 p=0.7870). (H) Time on edge or in center of an open field arena during a 10 min test session (Edge: t=2.897, df = 32, **p<0.01, Center: t=2.750, df = 32, **p<0.01).

Figure 4—source data 1. Behavioral data for Figure 4.
elife-83760-fig4-data1.xlsx (101.2KB, xlsx)

Figure 4.

Figure 4—figure supplement 1. Additional behavioral analysis of Ntn1 cKO DAT-Cre mice.

Figure 4—figure supplement 1.

(A) Average ambulatory activity (beam breaks) averaged across three nights and two days (n=21 control; n=18 cKO, Night: t=0.3938, df = 37, p=0.6960, Day: t=1.271, df = 37, p=0.2117). (B) Distance traveled during the open field task (t=0.2275, df = 32, p>0.05). (C) Initial mouse weight prior to instrumental conditioning (n=22 control, n=22 cKO, t=2.053, df = 42, *p<0.05) (D) FR1 reinstatement (t=0.1192, df = 32 p=0.9058).
Figure 4—figure supplement 1—source data 1. Behavioral data for Figure 4—figure supplement 1.
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To determine whether appetitive conditioning behaviors are disrupted by the loss of Ntn1 function in VTA dopamine neurons, we assayed mice in a simple instrumental conditioning paradigm using a fixed-ratio 1 (FR1) followed by a fixed ratio 5 (FR5) schedule of reinforcement in which one or five lever presses are required to obtain a food reward, respectively. We did not observe significant differences in either of these behavioral tasks (Figure 4C). Next, we monitored motivated behavior using a progressive ratio schedule of reinforcement in which the number of lever presses required for reinforcement increases non-arithmetically (1, 2, 4, 7, 13, 19, 25, 34, 43, 52, 61, 73…), and again did not observe significant differences between control and experimental mice (Figure 4D). Following PR, we reinstated FR1 responding for three days followed by extinction training, and again did not detect any differences between the two groups (Figure 4E and Figure 4—figure supplement 1), indicating Ntn1 loss of function in VTA dopamine neurons did not alter appetitive conditioning behaviors. Although appetitive conditioning was not affected by Ntn1 loss of function in dopamine neurons, we did observe a slight but significant reduction in body weight in these mice relative to controls prior to calorie restriction (Figure 4—figure supplement 1).

To determine whether sensory-motor gating is altered in mice with loss of Ntn1 function in VTA dopamine neurons, we assayed them in acoustic startle and pre-pulse inhibition (PPI) paradigms. Although acoustic startle responses were reduced in AAV1-FLEX-SaCas9-sgNtn1 injected mice, this did not reach significance (Figure 4F). Moreover, we did not observe differences in PPI percentage inhibition (Figure 4G). These results indicate that loss of Ntn1 function in VTA dopamine neurons does not appear to affect psychomotor activation.

In addition to reinforcement and motivation, dopamine regulates other dimensions of affective behavior. To test whether anxiety-related behavior is affected in experimental mice relative to control mice, we assayed them in an open-field test. AAV1-FLEX-SaCas9-sgNtn1 injected DAT-Cre mice spent significantly more time on the edge of the open field arena and significantly less time in the center of the arena, consistent with an elevation in anxiety-like behavior (Figure 4H). There were no significant locomotor differences associated with the loss of Ntn1 function in the open field arena (Figure 4—figure supplement 1).

Ntn1 loss of function in VTA-GABA neurons affects multiple behaviors

To determine whether reducing excitatory synaptic connectivity onto VTA GABA neurons through the loss of Ntn1 function in these cells impacts behavior, we injected Vgat-Cre mice with AAV1-FLEX-SaCas9-sgNtn1 or AAV1-FLEX-SaCas9-sgRosa26 (control) into the VTA as described previously and tested these mice using the same behavioral paradigms described above. In contrast to Ntn1 mutagenesis in dopamine neurons, this manipulation in VTA GABA neurons resulted in a significant increase in locomotor activity (Figure 5B and Figure 5—figure supplement 1).

Figure 5. Ntn1 cKO in GABA ventral tegmental area (VTA) neurons resulted in significant behavioral alterations.

(A) Schematic summarizing cell type-specific knockout procedure. (B) Distance traveled in 15 min bins over the course of three nights and two days (n=26 controls, n=23 cKO, Two-way ANOVA Group F(1, 11797)=527.4, ****p<0.0001, Time F(250, 11797)=14.61 p<0.0001. Interaction F(250, 11797)=1.342, p=0.0003). (C) Earned reinforcers during three days of FR1 or FR5 operant conditioning (n=18 control; n=15 cKO; FR1: Group F(1, 31)=0.08647 p=0.7707, Time F(2, 62)=30.46 p<0.0001, Interaction F(2, 62)=3.186 p=0.0482; FR5: Group F(1, 31)=4.261, *p<0.05, Time F (1.992, 61.74)=0.3131 p=0.7314, Interaction F(2, 62)=1.448 p=0.2428). (D) Breakpoint (maximum presses per reinforcer) on a progressive ratio task (t=2.577, df = 31, *p<0.05). (E) Lever presses per session during five days of extinction training (Group F(1, 31)=10.23, **p<0.01, Time F(1.491, 46.23)=83.84 p<0.0001, Interaction F(4, 124)=3.546 p=0.0089). (F) Acoustic startle response to varying intensity white noise stimuli (Group F(1, 31)=7.891, **p<0.0085, Intensity F(1.790, 55.49)=24.94 p<0.0001, Interaction F(6, 186)=2.186, p=0.0462). (G) Percent inhibition of startle response following pre-pulse at indicated intensities (Group F(1, 93)=9.181, **p<0.01, Intensity F(2, 93)=5.101 p=0.0079, Interaction F(2, 93)=0.002227 p=0.9978). (H) Time on edge or in center of open field arena during a 10 min test session (edge: t=2.248, df = 31, *p<0.05, center t=1.366, df = 33, p>0.05).

Figure 5—source data 1. Behavioral data for Figure 5.

Figure 5.

Figure 5—figure supplement 1. Additional behavioral analysis of Ntn1 cKO Vgat-Cre mice.

Figure 5—figure supplement 1.

(A) Average ambulatory activity (beam breaks) averaged across three nights and two days (n=26 control; n=26 cKO, t=3.109, df = 50 **p=0.01 and t=3.227, df = 50 **p=0.01). (B) Distance traveled during open field test (t=2.064, df = 33, p<0.05*) (C) Initial mouse weight prior to instrumental conditioning (n=24 control, n=17 cKO, t=0.5342, df = 39 p>0,05). (D) FR1 reinstatement (t=0.4193, df = 31, p=0.6779). (E) Extinction Day 1 lever presses per 10 min time bins (Two-way ANOVA Group F(1, 31)=8.715 **p<0.01, Time F (1.961, 60.79)=35.65 p<0.0001, Interaction F(5, 155)=0.7968 p=0.5535). (F) Extinction Day 2 lever presses per 10 min time bins (Group F(1, 31)=6.932, *p<0.05, Time F(3.894, 120.7)=11.61 p<0.0001, Interaction F(5, 155)=0.9574 p=0.4459).
Figure 5—figure supplement 1—source data 1. Behavioral data for Figure 5—figure supplement 1.
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In the FR1 schedule of reinforcement, we did not observe a significant difference between the groups; however, we observed an increase in the number of earned reinforcements in the FR5 schedule in mice with Ntn1 loss of function in VTA GABA neurons (Figure 5C). We also observed an increase in the PR schedule of reinforcement in these mice relative to controls (Figure 5D). In contrast to DAT-Cre Ntn1 cKO mice, pre-calorie restriction body weights in Vgat-Cre Ntn1 cKO mice did not differ from controls (Figure 5—figure supplement 1).

Reinstatement of FR1 responding in Vgat-Cre Ntn1 cKO following PR was not different than controls (Figure 5—figure supplement 1). However, during extinction training, Vgat-Cre Ntn1 cKO mice displayed high extinction bursts (elevated pressing following reward omission) compared to controls that remained elevated on the second day of extinction training (Figure 5E). While these data likely reflect an altered motivational state with loss of Ntn1, it is also possible that the hyperactivity observed in Vgat-Cre Ntn1 cKO mice contributes to the elevated lever press rates during FR5, PR, and extinction.

Analysis of sensory-motor gating in these mice revealed that Vgat-Cre mice injected with AAV1-FLEX-SaCas9-sgNtn1 had a significant reduction in the acoustic startle relative to control mice (Figure 5F) that was accompanied by a reduction in PPI (Figure 5G). Similar to mutagenesis of Ntn1 in dopamine neurons, this manipulation in GABA neurons resulted in an increase in anxiety-like behavior as demonstrated by an increased time on edge; though we only observed a trend towards a reduction in time spent in the center of the open field arena (Figure 5H). The lack of observed significance in the time in center in the context of increased edge time may reflect the hyperactivity observed following Ntn1 mutagenesis in VTA GABA neurons, consistent with this possibility, we did observe increased distance traveled during the open field test in these mice relative to controls (Figure 5—figure supplement 1).

Loss of netrin-1 in dopamine neurons largely reverses the effects of Ntn1 mutagenesis in GABA neurons

A loss of Ntn1 in VTA-dopamine neurons resulted in decreased excitatory synaptic input to those cells (theoretically reducing dopamine activity) (Figure 6A), and loss of Ntn1 in VTA-GABA neurons resulted in decreased excitatory tone onto GABA neurons, which would be predicted to increase dopamine activity through disinhibition (Tan et al., 2012; Figure 6A). Based on these observations, we asked whether a loss of Ntn1 in both cell types would restore the balance of activity in the midbrain, or whether there is a hierarchical effect of Ntn1 loss of function in GABA neurons. To address this, we crossed DAT-Cre with Vgat-Cre mice to develop a DAT-Cre::Vgat-Cre transgenic line, injected these mice with AAV1-FLEX-SaCas9-sgNtn1 or AAV1-FLEX-SaCas9-sgRosa26 (control) (Figure 6B), and assayed them using the previous behavioral battery.

Figure 6. Ntn1 cKO in DATIRES::Vgat-Cre mice partially rescues behavioral phenotype.

(A) Model of Ntn1 loss of function in the ventral tegmental area (VTA) on excitatory and inhibitory balance. (B) Schematic of GABA and Dopamine Ntn1 cKO. (C) Distance traveled in 15 min bins over the course of three nights and two days (Two-Way ANOVA Group F(1, 45)=0.004273, p>0.05, Time F(17.16, 772.1)=23.36, p<0.0001, Interaction F (247, 11115)=1.492, p<0.0001). (D) Earned reinforcers during three days of FR1 or FR5 operant conditioning (n=21 controls, n=20 Ntn1 cKO, FR1: Group F(1, 40)=0.04247 p=0.8378, Time F(2, 80)=25.70 p<0.0001, Interaction F(2, 80)=1.402 p=0.2522; FR5: Group F(1, 40)=0.2244 p=0.6383, Time F(1.499, 59.95)=2.226 Pp0.1295, Interaction F(2, 80)=0.1385 p=0.8708) (E) Breakpoint (maximum presses per reinforcer) on a progressive ratio task (t=2.502, df = 39, *p<0.05) (F) Lever presses per session during five days of extinction training (Group F(1, 39)=6.990, *p=0.0117, Time F(2.381, 92.87)=42.95 p<0.0001, Interaction F(4, 156)=0.1470 p=0.9641). (G) Acoustic startle response to varying intensity white noise stimuli (Group F(1, 40)=0.1207 p=0.7301, Intensity F(1.775, 70.99)=36.77 p<0.0001, Interaction F(6, 240)=0.6127 p=0.7201). (G) Percent inhibition of startle response following pre-pulse at indicated intensities (Group F(1, 120)=0.9661 p=0.3276, Intensity F(2, 120)=7.067 p=0.0013, Interaction F(2, 120)=0.8861 p=0.4150). (H) Time on edge or in center of open field arena during a 10 min test session (edge: t=0.3584, df = 45 p>0,05, center: t=0.4233, df = 45, p>0.05).

Figure 6—source data 1. Behavioral data for Figure 6.
elife-83760-fig6-data1.xlsx (109.2KB, xlsx)

Figure 6.

Figure 6—figure supplement 1. Average day and night locomotion in DAT-Cre::Vgat-Cre mice.

Figure 6—figure supplement 1.

(A) Ambulatory activity (beam breaks) averaged across three nights and two days (n=26 controls n=21 cKO, Night: t=1.010, df = 45, p=0.3178 and Day: t=2.091, df = 45 *p<0.05). (B) Distance traveled during open field test (t=1.414, df = 45, p>0.05). (C) Initial mouse weight prior to instrumental conditioning (n=10 control, n=13 cKO, t=0.08465, df = 21 p>0.05). (D) FR1 reinstatement (t=1.127, df = 39 p>0.05) (E) Extinction Day 4 lever presses per 10 min time bins (Two-way ANOVA Group F (1, 39)=5.428 *p<0.0.5, Time F (3.691, 144.0)=4.539 p=0.0023, Interaction F (5, 195)=0.6146 9=0.6888) (F) Extinction Day 5 lever presses per 10 min time bins (Group F (1, 39)=5.232 *p<0.05, Time F F (3.524, 137.4)=3.115 p=0.0218, Interaction F (5, 195)=2.802 p=0.0181).
Figure 6—figure supplement 1—source data 1. Behavioral data for Figure 6—figure supplement 1.
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Simultaneous Ntn1 loss of function in VTA GABA and dopamine neurons largely reversed the hyperlocomotor phenotype (Figure 6C) observed with Ntn1 mutagenesis in VTA GABA neurons alone, though a modest, increase in daytime locomotion remained (Figure 6—figure supplement 1). Similarly, loss of Ntn1 in both VTA GABA and dopamine neurons resulted in operant responding during FR1 and FR5 that was similar to controls (Figure 6D) and pre-calorie restriction body weights did not differ between the groups. Motivation, as measured in the PR task, was elevated in the double transgenic Cre line following Ntn1 mutagenesis (Figure 6E) and extinction was impaired (Figure 6F), though these phenotypes were less robust than those observed in the VTA GABA-only mice. Further analysis of extinction training days four and five revealed significant differences in both the number of lever presses and the rate of lever presses between groups (Figure 6—figure supplement 1). Finally, loss of Ntn1 in both cell types resulted in acoustic startle and PPI responses (Figure 6G–H), and open field activity (Figure 6I) that was similar to control mice.

Discussion

Here, we show that Ntn1 is present in both dopamine and GABA-producing neurons of the adult VTA, and loss of Ntn1 function via genetic inactivation in either cell type results in a significant disruption of excitatory synaptic connectivity. The exact mechanisms by which Ntn1 regulates glutamatergic connectivity remain to be resolved. Likely mechanisms include Ntn1 regulation of the actin cytoskeleton and receptor transport vesicles through its activation of the cognate receptor DCC (Yetnikoff et al., 2010; Rajasekharan and Kennedy, 2009). The latter is consistent with our observed decrease in the amplitude of mEPSCs and reduced AMPA-evoked currents and with previous reports of Ntn1 regulating the delivery of GluA1-containing AMPA receptors to the postsynaptic density (Jasmin et al., 2021). Our finding that mEPSC frequency, but not paired-pulse ratio, was affected by Ntn1 loss further suggests netrin’s role in modulating excitatory synaptic connectivity is likely confined to postsynaptic mechanisms.

Loss of Ntn1 in dopamine neurons had little effect on behavior; however, we did observe an increase in anxiety-like behavior as measured by the open field assay consistent with the proposed role of dopamine in the modulation of anxiety-related behavior (Zarrindast and Khakpai, 2015). The general lack of effect of reduced glutamatergic synaptic connectivity on appetitive behavior, locomotion, and sensory-motor gating is consistent with previous observations that reduced glutamatergic signaling in dopamine neurons largely does not affect these behaviors (Zweifel et al., 2009; Hutchison et al., 2018). However, this does not discount the importance of glutamatergic inputs to the VTA (see additional discussion below). In contrast, loss of Ntn1 in VTA GABA neurons had a significant effect on multiple behaviors including locomotion, motivation, and acoustic pre-pulse inhibition, all of which are consistent with a hyperdopaminergic phenotype and with previous reports that disrupting GABA neuron function in the VTA induces similar phenotypes (Gore et al., 2017; Soden et al., 2020).

Given the robust nature of the behavioral effects observed following Ntn1 mutagenesis in VTA GABA neurons, we were initially surprised that simultaneous loss of Ntn1 in both GABA and dopamine neurons largely rescued the observed hyperdopaminergic phenotype. These results suggest that a balance of glutamatergic signaling in these two cell types is essential for the normal functioning of the mesolimbic dopamine system (Figure 6A). This finding is similar to what has been reported previously in the striatum.(Beutler et al., 2011) , demonstrated that loss of NMDA receptor signaling in dopamine D1 receptor-expressing neurons prevented the development of amphetamine sensitization; however, inactivation of NMDA receptors in D1R and D2R-expressing medium spiny neurons reversed this phenotype (Beutler et al., 2011). We have previously shown that blocking all synaptic transmission from GABA neurons in the VTA (Gore et al., 2017), or blocking selectively GABA release from VTA GABA neurons (Hutchison et al., 2018) results in hyperactivity and increased operant responding, though to a much greater degree than the effects observed here. We and others have also shown that loss of glutamate signaling in dopamine neurons has only a modest behavioral effect (Zweifel et al., 2009; Hutchison et al., 2018) so it is not surprising that reducing mEPSCs onto VTA dopamine neurons has little effect. The partial rescue of the GABA-Ntn1 knockout experiment suggests that reducing glutamatergic input onto dopamine neurons can abrogate effects associated with reduced inhibitory tone; thus, restoring the excitatory and inhibitory balance. Future research directly measuring the amount of inhibitory and excitatory current from the same cell (for example, by recording mIPSC at the reversal potential for EPSCs and mEPSCs at the reversal potential for mIPSCs) would be helpful in supporting this hypothesis, but was not performed in the current paper.

There are numerous disinhibitory projections to the VTA (Soden et al., 2020), and it is hypothesized that activation of GABAergic inputs onto VTA GABA neurons during behavior suppresses the activity of these neurons releasing the inhibitory brake onto dopamine neurons. Simultaneously, excitatory inputs onto VTA dopamine neurons, such as those from the pedunculopontine tegmental nucleus (Soden et al., 2020; Lodge and Grace, 2006), or dorsal raphe nucleus (Qi et al., 2014) would drive the glutamate-dependent burst activation of dopamine neurons (Zweifel et al., 2009; Lodge and Grace, 2006). Loss of excitatory drive onto dopamine neurons can likely be compensated for through the many disinhibitory circuits of the VTA. Indeed, inhibitory synaptic connectivity is not altered by the loss of Ntn1, and suppression of inhibition onto dopamine neurons is a potent means to drive the burst activation of these cells (Paladini and Tepper, 1999). The loss of glutamatergic inputs onto VTA GABA neurons is less likely to be bypassed at the circuit level, and we do not observe compensatory changes in excitatory or inhibitory synapses onto dopamine neurons following Ntn1 mutagenesis in VTA GABA neurons or compensatory changes in inhibitory synaptic connectivity onto VTA GABA neurons. The effects of Ntn1 loss of function in VTA GABA neurons can be abrogated if the excitatory drive onto dopamine neurons is also reduced by Ntn1 mutagenesis. Excitatory inputs onto GABA neurons within the VTA proper, as well as the caudal tail of the VTA, or RMTg, arise from multiple locations including the lateral habenula and ventral pallidum (Omelchenko et al., 2009; Brinschwitz et al., 2010; Tooley et al., 2018). Activation of these circuits is aversive, likely through the activation of GABAergic inputs onto dopamine neurons (Tooley et al., 2018; Wulff et al., 2019; Faget et al., 2018; Stamatakis and Stuber, 2012). Thus, reduced glutamatergic inputs onto VTA GABA neurons would suppress the inhibition of dopamine neurons allowing excitatory drive onto these cells to go unchecked, resulting in the phenotypes observed here. When excitatory inputs onto dopamine neurons are also reduced the combined driving forces of disinhibition and excitation of the dopamine neurons are re-equilibrated, restoring balance to the system.

While our findings shed light on the role of Ntn1 in adult VTA neurons, the question remains as to which netrin-1 receptors may be involved. Indeed, though Dcc is considered to be its canonical receptor, Ntn1 is a known ligand for several additional receptors, including DSCAM, Neogenin, and Unc5 homologs A-D (Rajasekharan and Kennedy, 2009; Lai Wing Sun et al., 2011). Previous work has identified the presence of both Dcc and Unc5c receptors in the adult VTA (often in the same cells) (Manitt et al., 2010). It is also interesting to note that during the development of the spinal cord, Ntn1 expression in the floor plate attracts commissural axons to the midline, but following the arrival of these axons at the floor plate, Unc5 expression increases to suppress the attractive actions of DCC signaling (Lai Wing Sun et al., 2011). Whether a similar relationship exists for the formation of nascent synapses and the maintenance of excitatory synapses occurs in the VTA will be important to resolve. Of further note, in addition to the role of Ntn1/Dcc/Unc5 signaling in the regulation of commissural axons crossing the midline, Slit/Robo signaling repels axons away from the floor plate (Kidd et al., 1998; Kidd et al., 1999) setting up a push-pull relationship between these pathways. We previously demonstrated that Robo2 maintains inhibitory synaptic connectivity in the adult VTA (Gore et al., 2017), suggesting the existence of another ‘push/pull’ relationship between these two pathways in which netrin/Dcc/Unc5 regulates excitation and Slit/Robo signaling regulates inhibition.

We find that Ntn1 is expressed in both Vgat- and Th-expressing neurons which is consistent with single nuclear RNA sequencing data recently obtained in rats from Phillips et al., 2022, though the levels of nuclear Ntn1 mRNA levels in this study were relatively low. This may reflect differences between rat and mouse, or the increased sensitivity of in situ hybridization in detecting cytosolic mRNA compared to nuclear mRNA levels observed with snRNA seq. Interestingly, (Phillips et al., 2022) also observed the expression of Ntn1 in glial cells. Thus, the identity and potential role of the roughly 10% of Ntn1 expressing neurons that do not co-label with markers of dopamine or GABA neurons in the VTA, potentially glutamatergic neurons or glial cells, warrants further investigation.

In our proposed model, reduced excitatory input onto GABA neurons results in a reduced inhibition of dopamine neurons causing the observed phenotypes, consistent with previous observations that reducing VTA GABA neuron function causes hyperactivity (Gore et al., 2017; Soden et al., 2020). However, it is also likely that GABA neurons projecting outside the VTA (for example, to the NAc Brown et al., 2012) are also affected and could contribute to the observed behavioral phenotypes.

Mutations in NTN1 (netrin-1) and DCC in humans have been associated with several dopamine-associated psychiatric conditions, including neurodevelopmental disorders such as schizophrenia (Wang et al., 2018; Tang et al., 2019; Grant et al., 2012) and major depressive disorder (Tang et al., 2019; Zeng et al., 2017; Vosberg et al., 2020; Torres-Berrío et al., 2020), as well as multiple neurodegenerative disorders (Lesnick et al., 2007; Lesnick et al., 2008; Lin et al., 2009). Our findings that Ntn1 plays a key role in maintaining excitatory connectivity in the adult midbrain and controlling the inhibitory/excitatory balance in this region highlights the importance of understanding these critical developmental signaling pathways in the adult nervous system that are likely important for therapeutic considerations in targeting these pathways.

Methods

Mice

All procedures were approved and conducted in accordance with the guidelines of the University of Washington’s Institutional Animal Care and Use Committee (protocol number: 4249–01). Mice were housed on a 12:12 light:dark cycle with ad libitium access to food and water, except when undergoing food restriction for operant behavioral conditioning. Approximately equal numbers of male and female mice were used for each experiment. Post-hoc analysis to test for sex-specific differences was performed for each experiment and no differences were observed so mice from each sex were pooled within experimental and control groups. Mice were group housed (2–5 mice per cage, separated based on sex at the time of weaning). Mice injected with CRISPR/YFP were allowed 4–5 weeks of recovery after surgery to allow for viral expression, mutagenesis, and protein turnover before any testing. Dat-Cre (Slc6a3Cre/+) mice and Vgat-Cre (Slc32a1Cre/+) mice were obtained from Jackson Laboratories (Strain # 006660 and 028862).

Viruses

All adeno-associated viruses (AAV) for CRISPR/SaCas9 mutagenesis were produced in-house, as previously described (Hunker et al., 2020). CRISPR viruses employed for this research: AAV1-FLEX-SaCas9-U6-sgNtn1 (Addgene: #159907) and AAV1-FLEX-SaCas9-U6-sgRosa26 (Addgene: #159914) are available through Addgene or upon request to the corresponding author.

Surgeries

All mice used were 8–10 weeks of age at the time of surgery. Mice were inducted using isoflurane at 5.0% and held at 2% throughout the procedure. Mice were stereotaxically injected bilaterally into the VTA using the following coordinates in mm, relative to bregma: A/P: –3.25; M/L±0.5; D/V: (–4.9) – (–4.4), total volume 0.5 µL into each side. A/P coordinates were adjusted for Bregma/Lambda distances using a correction factor of 4.2 mm.

In situ hybridization

Male and female mice (n=2 each sex, 8–12 weeks old) were used to verify mRNA expression in the VTA using RNAscope. Brains were flash-frozen in 2-methylbutane and representative coronal sections that spanned the VTA were sliced at 20 µm and slide mounted for hybridization. Sections were prepared for hybridization per the manufacturer’s (Advanced Cell Diagnostics, Inc) instructions using probes for Th (Mm-Th), Ntn1 (Mm-Ntn1-C2), and Slc32a1 (Vgat; Mm-Slc32a1-C3). Slides were coverslipped with Fluoromount with DAPI (Southern Biotech) and imaged using a confocal fluorescent microscope (the University of Washington Keck Center Leica SP8X confocal) and Keyence Fluorescence Microscope (Keyence). Quantification of co-labeled cells was performed using CellProfiler, with thresholding and cell identification/overlap for each channel verified for each image manually prior to quantification.

Immunohistochemistry

Mice were anesthetized with pentobarbital and transcardially perfused with PBS followed by 4% PFA. Brains were post-fixed for 24 hr in PFA at 4°C, followed by 48 hr in 30% sucrose. The VTA was coronally sectioned at 30 µm. Sections were kept in PBS with 0.3% Sodium Azide. Free-floating sections were treated with 0.3% TBS-Triton-X 100 3x10 min, blocked in 3% Normal Donkey Serum for 1 hr and treated overnight in primary antibody. Following 1–3 hr in secondary antibody (JacksonImmuno), sections were slide mounted and coverslipped with Fluoromount with DAPI. Images were collected on a Keyence Fluorescence Microscope (Keyence). For CRISPR validation, male and female DAT-Cre mice (8–12 weeks old) received AAV1-FLEX-SaCas9-U6-sgNtn1/AAV1-FLEX-YFP (Ntn1-cKO) or AAV1-FLEX-SaCas9-U6-sgROSA26/AAV1-FLEX-YFP (controls) injections as described above, and quantification of co-labeled cells for immunohistochemistry were performed using ImageJ 1.53 Cell Counter/Multi-point tool. Total Th-positive cells were recorded for all images and averaged across all slices for each mouse to give a total number of Th-positive cells. Primary antibodies used: mouse anti-TH (1:1500, Millipore), chicken anti-Netrin-1 (1:1000, Abcam), and rabbit anti-HA (1:1500, Sigma).

Slice electrophysiology

Mice injected with CRISPR/YFP were allowed 4–5 weeks of recovery after surgery to allow for viral expression, mutagenesis, and protein turnover. All solutions were continuously bubbled with O2/CO2. Horizontal (200 µm) brain slices were prepared from 12 to 20 week-old mice in a slush NMDG cutting solution (Lin et al., 2009) (in mM: 92 NMDG, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2, 10 MgSO4, pH 7.3–7.4). Slices recovered for ~12 min in the same solution warmed in a 32 °C water bath, then transferred to room temperature HEPES-aCSF solution (in mM: 92 NaCl, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiouria, 5 Na-ascorbate, 3 Na-pyruvate, 2 CaCl2, 2 MgSO4). Slices recovered for an additional 30–60 min in HEPES solution at room temp. Whole-cell patch-clamp recordings were made using an Axopatch 700B amplifier (Molecular Devices) using 3–5 MΩ electrodes. Recordings were made in aCSF (in mM: 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 11 D-glucose, 18 NaHCO3, 2.4 CaCl2) at 32 °C continually perfused over slices at a rate of ~1 ml/min. VTA dopamine and non-dopamine neurons were identified by fluorescence.

mE/IPSC

For miniature excitatory postsynaptic currents (mEPSCs), the internal solution contained: 130 mM K-gluconate, 10 mM HEPES, 5 mM NaCl, 1 mM EGTA, 5 mM Mg-ATP, 0.5 mM Na-GTP. Picrotoxin (200 μM) was added to ACSF to block GABAA receptor-mediated events. For miniature inhibitory postsynaptic currents (mIPSCs), the internal solution contained: 135 mM KCl, 12 mM NaCl, 0.05 mM EGTA, 100 mM HEPES, 0.2 mM Mg-ATP, 0.02, and Na-GTP mM. To block glutamatergic events, 2 mM kynurenic acid was bath applied in the ACSF. All mIPSCs and mEPSCs cells were recorded in the presence of 1 mM tetrodotoxin (TTX) to block action potentials. Cells were held at −60 mV for a minimum of 5 minprior to data acquisition. Data were analyzed using Clampfit 10.3 (pCLAMP 11 Software Suite, Molecular Instruments).

Paired pulse ratio

For PPR, the internal solution contained: 130 mM K-gluconate, 10 mM HEPES, 5 mM NaCl, 1 mM EGTA, 5 mM Mg-ATP, 0.5 mM Na-GTP. Picrotoxin (200 μM) was added to ACSF to block GABAA receptor-mediated events. Electrical stimulation was delivered using a concentric bipolar electrode placed rostral to the VTA. Data were analyzed using Clampfit 10.3 (pCLAMP 11 Software Suite, Molecular Instruments).

Bath AMPA and NMDA application

For AMPA currents, neurons were held at −60 mV and 50 μM cyclothiazide was perfused onto the slice for 30 s, followed by 1 μM AMPA (with cyclothiazide) for 30 s. For NMDA currents, neurons were held at + 40 mV and 50 μM NMDA was perfused onto the slice for 30 s. Picrotoxin (100 μM) and tetrodotoxin (500 nM) were included in the bath.

Behavior

Locomotor activity

Four weeks after surgery, baseline locomotion was measured using locomotion chambers (Columbus instruments) that use infrared beam breaks to calculate ambulatory activity. Mice were singly housed in Allentown cages with reduced corncob bedding and provided with ad libitum access to food and water. Locomotion was monitored continuously for three nights two days.

Open field testing

Mice were placed in a large circular arena (120 cm diameter) and activity was recorded for a period of 10 min using Ethovision software. Zones signifying arena edge and center were generated for each video using Ethovision and kept consistent in size and placement across all trials. Time in center, time on edge, and total distance were calculated. Thigmotaxis, the tendency of mice to remain in close proximity to the walls of an enclosure, has been validated as a measure of anxiogenic behavior (Simon et al., 1994; Seibenhener and Wooten, 2015), with increased time on edge signifying increased anxiety-like behavior.

Operant conditioning

Prior to testing, all mice were food restricted to 85% body weight and maintained at this level throughout operant conditioning. Mouse weights are included in Figure 4—figure supplement 1, Figure 5—figure supplement 1, Figure 6—figure supplement 1. Mice were tested on an operant conditioning paradigm in Med Associates boxes in the following order: FR1, FR5, Progressive Ratio, Reinstatement, and Extinction. Each fixed ratio 1 (FR1) session lasted for 60 min. Levers were extended and remained extended until a lever press. Upon a lever press, levers were retracted and a sucrose pellet was immediately delivered into the food hopper. The levers did not extend again until the mouse made a head entry into the food hopper to retrieve the pellet. Reinforced FR1 sessions lasted for three days, followed by three days of FR5 (five lever presses required to obtain sucrose pellet), and a single day of a progressive ratio where the number of lever presses necessary for sucrose pellet delivery increases non-arithmetically (i.e. 1, 2, 4, 6, 9, 13…) over the course of the session. The progressive ratio session ended after three consecutive min of no lever presses or after 3 hr. After progressive ratio, mice again underwent FR1 reinforced training, followed by extinction for 60 min each session for five days. Here, levers extend and retract similarly to the FR1 reinforced paradigm, yet a sucrose pellet reward is omitted.

Acoustic startle and prepulse inhibition

Acoustic startle responses were measured using acoustic startle chambers (San Diego Instruments). Prior to testing mice received a 10 min habituation period. Background noise was maintained at 65 dB throughout testing. After habituation, mice were presented with 5, 40 ms duration 120 dB, pulse-alone trials to obtain baseline startle responses, followed by 50 trials of either a startle pulse-alone, 1 of 3 prepulse trials, or a null trial, in which no acoustic stimulus is presented. Startle trials consisted of a 40 ms, 120 dB pulse of white noise. The three prepulse trials consisted of a 20 ms prepulse of 70-, 75-, or 80 dB intensity (5, 10, and 15 dB above background) that preceded 120 dB startle pulse by 100 ms. Peak amplitude of the startle response (65 ms after pulse onset) was used as the measure of startle response magnitude.

Sex differences

No sex differences were observed in any of the behavioral or electrophysiological results.

Statistics

Data were analyzed for statistical significance using GraphPad Prism. All statistical tests were two-sided and corrected for multiple comparisons where appropriate. All experiments were repeated at least twice using a double-blind design.

Materials availability

All newly created reagents are freely available upon request to the corresponding author.

Acknowledgements

We would like to thank the staff of the University of Washington’s Comparative Medicine Animal Facilities, the University of Washington’s Keck Imaging Center, and the administrative staff of the Molecular and Cellular Biology Graduate Program.

This study was supported by grants from the National Institutes of Health T32GM007270 (MC), 1F31MH126489-01A1 (MC), T32DA727825 (B.J.), K99DA054265 (B.J.), R01MH104450 (LSZ), and. R01DA044315 (LSZ). B.J., Ph.D., holds a Postdoctoral Enrichment Program Award from the Burroughs Wellcome Fund. We would also like to acknowledge support from the University Of Washington Center Of Excellence in Opioid Addiction Research/ Molecular Genetics Resource Core (P30DA048736). The authors declare no conflicting interests.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Larry S Zweifel, Email: larryz@u.washington.edu.

Kate M Wassum, University of California, Los Angeles, United States.

Kate M Wassum, University of California, Los Angeles, United States.

Funding Information

This paper was supported by the following grants:

  • National Institutes of Health R01DA044315 to Larry S Zweifel.

  • National Institutes of Health R01MH104450 to Larry S Zweifel.

  • National Institutes of Health 1F31MH126489 to Marcella M Cline.

  • National Institutes of Health K99DA054265 to Barbara Juarez.

Additional information

Competing interests

No competing interests declared.

No competing interests declared.

Author contributions

Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Writing – review and editing.

Investigation, Methodology.

Formal analysis, Methodology.

Investigation.

Investigation.

Data curation, Formal analysis, Methodology.

Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing.

Ethics

All procedures were approved and conduced in accordance with the guidelines of the University of Washington's Institutional Animal Care and Use Committee, protocol number 4249-01.

Additional files

MDAR checklist

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file.

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Editor's evaluation

Kate M Wassum 1

This manuscript reports an important, previously unappreciated, non-developmental role for the guidance cue netrin-1 in midbrain physiology and related behavior in adult animals. Using multiple experimental tools in adult mice, the study convincingly shows that netrin-1 within midbrain dopamine and GABA neurons is necessary to maintain dopamine excitatory tone and plays a role in motivated and anxiety-like behavior. This paper will be of interest to neuroscientists studying dopamine function and/or motivated behavior and those interested in ways that neurodevelopmental genes can continue to play a role in neuronal function and behavior into adulthood.

Decision letter

Editor: Kate M Wassum1

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting the paper "Netrin-1 regulates the balance of glutamatergic connectivity in the adult ventral tegmental area" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers opted to remain anonymous.

Comments to the Authors:

We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered further for publication by eLife.

We agreed that the work addresses an interesting question and that the manipulation to knockdown netrin-1 in adult VTA was elegant. However, we noted several concerns. These include unaddressed issues related to the precise role of netrin function in DA neurons leading to challenges in interpreting the behavioral results, the lack of mechanistic insight into the observed effects of glutamate, and contextualization in prior literature examining netrin's effects in the VTA. Addressing the concerns is likely to be well beyond the scope of an eLife revision (which under normal circumstances is limited to 2 months).

Reviewer #1 (Recommendations for the authors):

The study seeks to investigate whether the guidance cue netrin-1 in the VTA is involved in the regulation of glutamatergic synaptic connectivity of dopamine and GABA neurons and influences behaviors known to be mediated by mesolimbic dopamine systems.

The study involves multiple approaches: viral mediated inactivation of NTn1 in VTA GABA or dopamine neurons, using Cre inducible CRISPR/Cas9; in situ hybridization to confirm colocalization of Ntn1 and tyrosine hydroxylase or Ntn1 and SlC31a1; electrophysiological recordings to evaluate changes in neural excitability of dopamine or GABA neurons, a battery of behavioral tests to evaluate whether loss of Ntn1 in either cell type alters sensorimotor gating function and motivated and anxiety-like behaviors.

Major strengths of the paper include (i) the novelty of the question addressed and the multidisciplinary approach, (ii) most of the experiments address functional questions, (iii) the careful characterization of functional and behavioral phenotypes for the majority of the experiments, (iv) the generation of a mechanistic model potentially explaining how netrin-1 in the adult VTA, by regulating the excitatory tone of dopamine and GABA neurons, regulates overall dopamine excitability and behavior.

Weaknesses of the study are mainly related to the interpretation of their results and the conclusions made: (i) Regarding the results from the in situ hybridization experiment, the authors do not compare their findings with results from a recent single-cell RNA seq study (PMID: 35385745). There is a discrepancy between the findings, particularly in the proportion of GABA neurons expressing NTn1 (as well as in the expression of NTn1 in other cell types, such as astrocytes). The authors do not mention this issue in their results or discussion. (ii) The authors conclude that their findings demonstrate that netrin-1 regulates the balance of glutamatergic connectivity in the VTA (title, last paragraph introduction, results, and conclusion). Indeed they find that downregulation of Ntn1 in VTA dopamine or GABA cells reduces the frequency and amplitude of their miniature excitatory postsynaptic currents (mEPSCs), without altering miniature inhibitory postsynaptic currents (mIPSCs) or leading to statistically reliable changes in frequency or amplitude in other (non-specified) cell types. They also show that reduced mEPSCs is not mediated by changes in presynaptic release, as revealed by paired-pulse ratio measures. However, these findings are not directly linking electrophysiological changes to alterations in glutamatergic synaptic connectivity (iii) Whether NTn1 deletion leads to dopamine or GABA neuronal loss remains unknown. This is important considering previous studies linking (or not) changes in the netrin-1 system in VTA neurons and cell loss. (iv) The study shows that downregulation of Ntn1 in dopamine neurons has no significant effect on reward but influences anxiety-like behaviors. In contrast, downregulation of Ntn1 in GABA neurons produces changes in most of the behaviors tested. It is not clear, however, whether increased locomotor activity in mice with Ntn1 deletion in GABA neurons could influence changes in lever pressing in the operant behaviors. (v) Regarding the changes in extinction training in the mice with reduced Ntn1 in GABA neurons, it seems that they are lever pressing at a higher rate from the beginning but they extinguish their behavior at a similar rate (similar for the animals with reduced Ntn1 in GABA and in dopamine neurons). (vi) the model and hypothesis put forward and tested in the experiments shown in Figure 6 are very interesting. However, the results obtained do not justify the conclusion that Loss of Ntn1 function in both cell types simultaneously largely rescues the consequences induced by GABA- only Ntn1 deletion.

To be able to link the electrophysiological changes to glutamatergic synaptic connectivity, other experiments are required, including assessing structural changes (e.g. PMID: 24174661) in dopamine and GABA neurons as well as the proportion of AMPA/NMDA receptors and AMPA/NMDA ratios. In this regard, there is a previous study relating the netrin-1 guidance cue system in adult VTA synaptic plasticity (PMID: 20345916).

Whether NTn1 deletion leads to dopamine or GABA neuronal loss could be addressed using stereology.

To be able to conclude that the loss of Ntn1 function in both cell types simultaneously largely rescues the consequences induced by GABA- only Ntn1 deletion, the electrophysiological properties of dopamine (and also GABA) neurons could be assessed. This experiment will also test more directly the model the authors are proposing.

In the discussion, evidence showing the role of glutamatergic inputs of VTA dopamine neurons on behavior needs to be revised more carefully (e.g. PMID 25388237, PMID 26631475, and PMID 30699344).

Reviewer #2 (Recommendations for the authors):

This manuscript by Cline et al. sought to define the role of the axonal guidance cue netrin-1 in synaptic signaling in the ventral tegmental area (VTA). The authors used CRISPR-Cas9 mutagenesis to reduce netrin-1 expression/function in the two predominant neuronal types in the VTA, dopaminergic and GABAergic neurons. This work builds on previous work from this group showing a role for the axon guidance receptor ROBO2 in inhibitory connectivity in adult VTA. Netrin-1 is examined here because of its persistent expression in the VTA into adulthood, despite its established role as a developmental protein. A strong combination of techniques is used, including selective knockdown of the netrin-1 gene in multiple neuron types in the VTA, patch clamp electrophysiology, and several behavioral assays. The results clearly indicate that knockdown of netrin-1 specifically in either GABA or dopamine neurons reduces miniature glutamatergic synaptic currents specifically in that cell type. Interestingly, inhibitory input was not significantly affected. This is consistent with previous reports of effects on excitatory synapses in the hippocampus. Robust behavioral consequences were only reported in the GABA neuron knockdown and were not evident when netrin-1 was knocked down in both cell types. The authors conclude that netrin-1 is important for maintaining the balance of excitatory input onto the two main neuronal subtypes in the VTA. This conclusion is largely supported by the results from the experiments, which were performed rigorously. The manuscript itself was easy to follow and well written, save for some minor omissions.

Operant responding for food was used as one of the dependent measures in Figures 4 and 5, with differences observed in the GABA neuron knockdown, however, one omission of the study was that body weights of the mice before and especially after treatment were not reported. It may be that viral knockdown of netrin-1 in one cell type has effects for instance on satiety, producing effects on behavior along with energy balance. The addition of body weight data would help round out the data set and, if different, might help with interpretation. Details were also not provided for the food restriction procedure that was used during operant conditioning for food pellet responding, which could have further interacted with the netrin-1 manipulation in the mouse lines to affect physiology, behavior, or both.

As discussed above, most of this study is strong, with interpretations supported by largely convincing results. However, the manuscript could be improved with additions and clarifications.

While both cell types showed increased mEPSC frequency and amplitude after netrin-1 knockdown, only the GABA neuron knockdowns showed robust behavioral effects, including increased locomotion (day and night), and increased operant responding for food, and decreased acoustic startle and pre-pulse inhibition. As some of the behavioral tasks involve responding for food pellets, interpretation of the results would benefit from reporting the weights of the mice before and after treatment. Additionally, details about the food restriction procedure that was used for operant conditioning should be provided. Several reports have identified the effects of the feeding state on dopamine neuron excitability and synaptic input to the area. Was the food restriction controlled to a percent loss of body weight, applied acutely or chronically, done throughout the operant study or just during FR1, etc.? Did the mice always eat the pellets when performing the operant task? It's interesting that responding in all of the dopamine mice stayed at the same values when they switched from FR1 to FR5, but in the GABA mice, the control animals fell while the netrin-1 knockdowns stayed the same. Full disclosure of these details would help the reader interpret small observations in the data and later assist in reproducing the results, should they wish to do so.

The evidence for the anxiety behavioral phenotype in the dopamine neuron-specific netrin-1 knockdown is pretty thin, as a time-in-center measure in an open field could be affected by other factors, and other supporting data for instance from elevated mazes were not used. Admittedly, anxiety can be difficult to show in mice. However, Reference 20 which was used to support the "proposed role of dopamine in the modulation of anxiety-related behavior" was a dead-end non-citation. If the authors wish to keep this part of their interpretation they should bolster the explanation of the link between dopamine and anxiety with further evidence (experimental or literature). Also, details about the meaning of "time in center" and "time on edge" should be provided in the Methods.

The extinction result in the GABA mice is presented as "a significant delay in the rate of extinction following reinstatement of FR1." This is hard to interpret because responding immediately before extinction is not given and the data are presented only as raw numbers instead of percent of baseline. If the netrin-1 knockdowns were responding more at FR1 when they were returned to that condition, the shape of the curve would actually indicate no difference in extinction. The same could be true in Figure 6 with the double cell knockdowns. Depending on what the data look like, these graphs may be more accurately presented as normalized numbers. This is a minor issue in the scheme of this paper but it should nonetheless be clarified.

The schematics in Figure 6A show that the effects on the GABA neurons proceed through the dopamine neurons. While this is entirely plausible, the authors never actually show this experimentally, for instance by locally manipulating GABA input in virus-injected mice. It may be at least as likely that the important interactions occur in the nucleus accumbens, or some other area to which both cell types project. As a minimum, the authors should point out this caveat in the Discussion, or point out any other possibility that could also explain the somewhat surprising data in Figure 6.

There's some confusion about the Ntn1 co-localization in Figure 1. The language in the caption and Figure 1 itself seem clear. However, the text on page 3 seems to contradict the language in the figure caption. The way it is worded, instead of 64, 30, and 6% shouldn't these numbers be 72, 18, and 10%? Please clarify with the correct information, or point out where the confusion lies.

In the abstract, "simultaneously" is placed confusingly in the sentence about rescuing the GABA phenotype. This would be more clear if the sentence started "Simultaneous loss… in both cell types."

Figure 1D has two (identical) scale bars.

Reviewer #3 (Recommendations for the authors):

In this study, they used genetic strategies to decrease Netrin-1 expression in either dopamine or GABA neurons of the VTA. Reduction of Netrin-1 expression in VTA dopamine neurons decreased excitatory, but not inhibitory postsynaptic currents onto TH+ or GABA VTA neurons. While the loss of netrin-1 in dopamine neurons did not significantly influence behaviour, loss of netrin-1 in GABAergic neurons increased locomotor activity, effort for rewards, extinction delay, and decreased prepulse inhibition. Finally, effects on locomotor activity, reward seeking, and prepulse inhibition observed in GABA-netrin mice were not present when there was the loss of netrin-1 in both dopamine and GABA neurons, suggesting that loss of netrin-1 in both could restore the behavioural effects of loss of netrin-1 in GABA neurons.

Major strengths:

This is a nicely written, clearly illustrated study describing the loss of netrin-1 in VTA dopamine neurons and GABA neurons. The authors use an elegant genetic methodology to knock down netrin-1 in select populations of neurons within the VTA. They use a battery of behavioural assays to examine the effects of this knockdown.

Major weaknesses:

While this study provides a potential physiological mechanism underlying the changed behavioural effects, it doesn't connect these changes to the behaviour or identify the mechanism by which adult netrin-1 influences these changes in synaptic transmission. While netrin-1 is involved in synapse formation during development, it is not clear how netrin-1 is influencing synapses in adulthood. For example, is it necessary for the stabilization of synapses?

Author suggestions:

The authors propose that loss of netrin-1 in dopamine neurons leads to enhanced excitation and decreased inhibition, whereas loss in GABA neurons would lead to enhanced inhibition and decreased excitation, and the E:I ratio would be balanced when netrin is lost from both cell population. However, they do not test this assertion by measuring excitatory:inhibitory ratio in each model. This would support their hypotheses in figure 6.

Some of the discussion focuses on the role of netrin-1 in development. However, the manipulations done in this paper were to remove netrin-1 in adulthood after axon migration and synapse formation occur. They do not discuss what the 'adult function' of netrin-1 is. While it seems to play a role in excitatory synaptic transmission, given its effects on mEPSCs, the authors did not provide sufficient information to conclude if this was a reduction in the number of synapses, a silencing of synapses, or a decrease in release probability with compensatory postsynaptic changes. Experiments addressing how adult netrin-1 signaling in the VTA specifically influences synaptic transmission onto dopamine or GABA neurons of the VTA may highlight how netrin-1 might be contributing to associated behavioral changes.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Netrin-1 regulates the balance of glutamatergic connectivity in the adult ventral tegmental area" for further consideration by eLife. Your revised article has been evaluated by Kate Wassum (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

Essential revisions:

We have a few remaining concerns that can be addressed with an additional analysis of existing data (see R3 point #1) and edits to the text to add clarity, change language, temper conclusions, discuss alternatives, etc.

Please provide a point x point response to each reviewer point along with your revision.

Reviewer #1 (Recommendations for the authors):

This resubmitted manuscript is substantially improved from the previous version. Additions and clarification to methods and interpretations have largely addressed my previous concerns, which were minor. Additional discussion has bolstered the notion that netrin-1 is impotant for maintaining the balance of excitatory and inhibitory inputs to dopamine neurons. The addition of maze data would have bolstered conclusions about the anxiety phenotype, but "time on edge" and locomotor data was added and this was always a minor point that did not detract substantially from the rest of the manuscript.

One item does need to be clarified. The experiment represented in Figure 3 Supplement 3 was performed to address a concern of Reviewer 1, however the text describing this is very confusing. The Results describe these data as evoked EPSCs following bath perfusion of AMPA or NMDA. The data themselves seem to be showing changes in holding current in a voltage clamp experiment, although the axis labels and the lack of sample traces leave this in doubt. If it is holding current, more details should to be provided in the figure (including better axis labels and a description of how long the drug went on and the holding voltage, both of which are currently only in the Methods). If instead it is evoked EPSCs in the presence of bath perfused agonists, then other details also need to be provided to make this result make sense (such as sample traces and an explanation of what they were after).

The addition of body weight data and food restriction information is appreciated. However in the Response to Reviewers the level of restriction was listed as 85% of initial body weight, whereas in the manuscript this is given as 80%. Please make sure the correct number appears in the manuscript.

Reviewer #2 (Recommendations for the authors):

The revised version improved the interpretation of the results, and I am pleased that the authors replied to most of my comments on the previous version. They added needed citations, clarification on their manipulation, and additional experiments to understand the changes in synaptic connectivity better and clarify the electrophysiological changes that occur with Ntn1 mutagenesis, as well as their proposed model.

The following issues remain unresolved:

– Since the study does not assess alterations in neuronal structure and connectivity, we suggest the word "connectivity" to be dropped or modified.

– It appears that stereology was not performed to calculate the number of neurons expressing netrin-1 in TH+ or GABA+ cells in the mice with conditional netrin-1 KO. The statement "Total Th-positive cells were recorded for all images and averaged across all slices for each mouse to give a total number of Th-positive cells" needs to be revised, because total number of cells can only be assessed using stereological analysis. It remains unknown whether NTn1 deletion leads to dopamine or GABA neuronal loss. This is an important issue that needs to be acknowledged in the manuscript. Please see

https://www.embopress.org/doi/full/10.15252/embj.2020105537

– Regarding the increase in locomotor activity observed after the downregulation of Ntn1 in GABA neurons, the authors argue that the level of responding in the FR1 task is not altered in GABA-Ntn1 mutant mice, suggesting that elevated responding in the FR5 and PR assays is not likely a reflection of hyperactivity. Yet, increasing the response rate in the FR1 protocol is no possible. Once the animals respond to the reward in the FR1, the lever retracts, and no more lever-pressing is possible until a reward is collected. The statement added by the authors "however, we did not observe differences in operant responding during FR1 training or reinstatement indicating that the observed effects are likely a reflection of altered motivational states rather than general hyperactivity" needs to be modified because hyperactivity may be influencing the findings reported

– Regarding the lever pressing extinction data, (1) the authors acknowledge that mice with Ntn1 cKO in GABA VTA neurons have similar extinction rates than WT. They now need to mention that this finding suggests that the increase in the number of lever-pressing during the extinction phase in Ntn1 cKO in GABA mice is most likely associated with an increase in baseline responding rather than with extinction deficits. This same point needs to be highlighted for the Ntn1 cKO in DATIRES::Vgat-Cre mice too. (2) the rate of extinction is not analyzed nor discussed. (3) it is unclear how plotting the extinction bin data for day1 and day 2 and for day 4 and day 5, for suppl Figures5 and 6 respectively, addresses this issue. The authors need to consider removing these data because they do not seem to provide relevant information.

Reviewer #3 (Recommendations for the authors):

I appreciate the addition of the bath application of AMPA or NMDA experiments. While these add support to the effect of netrin being postsynaptic, it should be indicated that these experiments do not distinguish between synaptic responses and extrasynaptic responses. Further a change in holding current after bath application of AMPA could be due to reduced synapse number that is associated with a reduced postsynaptic AMPA receptor complement. Further, while they show no change in PPR consistent with no effect on release probability (Pr), their decrease in mEPSC amplitude and frequency could be consistent with a reduction in synapse number (number of release sites N) or a change in number or function of postsynaptic AMPA receptors. To distinguish this, you can measure 1/CV2 which depends on N and Pr but is independent of quantal size. Given that there is no change in Pr, if you see a change in 1/CV2 it might be consistent with a decrease in release sites as opposed to a change in postsynaptic receptor number or function. Alternatively, if there is no change in 1/CV2, you can make a stronger conclusion that the netrin manipulation is altering number or function of postsynaptic AMPA receptors.

Regarding the shift in the E:I balance, while I do not disagree with their model, I was proposing that they could test their model directly by recording mIPSCs at the reversal potential for EPSCs (0 to 10mV) and mEPSCs at the reversal potential for GABA mIPSCs (~-60 to -70mV) and converting the average peak current amplitude into conductance. This way, you can directly measure the amount of inhibitory current and amount of excitatory current the same cell is receiving. Perhaps this experiment is beyond the scope of the current manuscript, but when discussing their model, they could indicate that they could test the validity of this hypothesis in future experiments.

eLife. 2023 Mar 17;12:e83760. doi: 10.7554/eLife.83760.sa2

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1 (Recommendations for the authors):

The study seeks to investigate whether the guidance cue netrin-1 in the VTA is involved in the regulation of glutamatergic synaptic connectivity of dopamine and GABA neurons and influences behaviors known to be mediated by mesolimbic dopamine systems.

The study involves multiple approaches: viral mediated inactivation of NTn1 in VTA GABA or dopamine neurons, using Cre inducible CRISPR/Cas9; in situ hybridization to confirm colocalization of Ntn1 and tyrosine hydroxylase or Ntn1 and SlC31a1; electrophysiological recordings to evaluate changes in neural excitability of dopamine or GABA neurons, a battery of behavioral tests to evaluate whether loss of Ntn1 in either cell type alters sensorimotor gating function and motivated and anxiety-like behaviors.

Major strengths of the paper include (i) the novelty of the question addressed and the multidisciplinary approach, (ii) most of the experiments address functional questions, (iii) the careful characterization of functional and behavioral phenotypes for the majority of the experiments, (iv) the generation of a mechanistic model potentially explaining how netrin-1 in the adult VTA, by regulating the excitatory tone of dopamine and GABA neurons, regulates overall dopamine excitability and behavior.

Weaknesses of the study are mainly related to the interpretation of their results and the conclusions made: (i) Regarding the results from the in situ hybridization experiment, the authors do not compare their findings with results from a recent single-cell RNA seq study (PMID: 35385745). There is a discrepancy between the findings, particularly in the proportion of GABA neurons expressing NTn1 (as well as in the expression of NTn1 in other cell types, such as astrocytes). The authors do not mention this issue in their results or discussion.

We find that approximately 30% of Vgat-expressing neurons also express Ntn1. Analysis of data from PMID: 35385745 confirms that a subset of neurons in their population designated GABA1, express Ntn1. We have also performed snRNA seq in mouse VTA and find that netrin 1 displays partially enriched expression in one of 3 of the major GABA populations that we identified (unpublished data). Differences in the proportion of GABA neurons we identified that express Ntn1 compared to the data in PMID: 35385745 may reflect differences between rat and mouse, or increased sensitivity of in situ hybridization compared to snRNA seq. We have added discussion of this point to the updated manuscript. The role of Ntn1 expression in glial cells is interesting, and we have added this point to our revised discussion.

(ii) The authors conclude that their findings demonstrate that netrin-1 regulates the balance of glutamatergic connectivity in the VTA (title, last paragraph introduction, results, and conclusion). Indeed they find that downregulation of Ntn1 in VTA dopamine or GABA cells reduces the frequency and amplitude of their miniature excitatory postsynaptic currents (mEPSCs), without altering miniature inhibitory postsynaptic currents (mIPSCs) or leading to statistically reliable changes in frequency or amplitude in other (non-specified) cell types. They also show that reduced mEPSCs is not mediated by changes in presynaptic release, as revealed by paired-pulse ratio measures. However, these findings are not directly linking electrophysiological changes to alterations in glutamatergic synaptic connectivity.

We apologize for our confusion, but we are not quite clear what the reviewers means when they state the mEPSCs are “not directly linking electrophysiological changes to alterations in glutamatergic synaptic connectivity”. mEPSC and mIPSP frequency and amplitude have been the gold standard in establishing excitatory synaptic connectivity since the original description of quantal synaptic transmission at the motor endplate (Fatt and Katz, 1952 and Del Castillo and Katz, 1954), the introduction of TTX to block action potential firing (Colomo and Erulkar, 1968) and the isolation of miniature inhibitory synaptic currents (Takahashi et al., 1983). A reduction in mEPSC frequency with no change in paired pulse ratio is commonly interpreted as a loss of functional synapses. Additional measures of synaptic connectivity, such as ultrastructural analysis can be performed, but dopamine neurons of the ventral midbrain have long been viewed as generally aspiny neurons (Juraska et al., 1977 J. Comp Neurol, see also Henny et al., 2012 Nat. Neurosci), though there is certainly no clear consensus on this (e.g. Jang et al., 2015 Scientific Reports). To better resolve the observed changes in synaptic connectivity in mEPSCs, we included bath application of AMPA and NMDA in the updated manuscript to quantify the degree of ‘total’ receptors present on surface of these cells with or without Ntn1 mutagenesis (Figure 3 supplement 3). We believe this addition clarifies the electrophysiological changes that occur with Ntn1 mutagenesis.

(iii) Whether NTn1 deletion leads to dopamine or GABA neuronal loss remains unknown. This is important considering previous studies linking (or not) changes in the netrin-1 system in VTA neurons and cell loss.

We agree that determining if Ntn1 deletion leads to neuronal loss is an important question. To address this, we have counted TH-positive cells in the VTA of mice with mutagenesis of Ntn1, which are more easily assessed by IHC, and determined that loss of Ntn1 function does not appear to result in the loss of dopamine neurons within the VTA.

(iv) The study shows that downregulation of Ntn1 in dopamine neurons has no significant effect on reward but influences anxiety-like behaviors. In contrast, downregulation of Ntn1 in GABA neurons produces changes in most of the behaviors tested. It is not clear, however, whether increased locomotor activity in mice with Ntn1 deletion in GABA neurons could influence changes in lever pressing in the operant behaviors.

We agree that locomotor effects can confound interpretations in behavioral assays. It is important to note that the level of responding in the FR1 task was not altered in GABA-Ntn1 mutant mice, suggesting that elevated responding in the FR5 and PR assays are not likely a simple reflection of hyperactivity. We have more explicitly addressed this in the revised manuscript.

(v) Regarding the changes in extinction training in the mice with reduced Ntn1 in GABA neurons, it seems that they are lever pressing at a higher rate from the beginning but they extinguish their behavior at a similar rate (similar for the animals with reduced Ntn1 in GABA and in dopamine neurons).

The reviewer makes an interesting point. The extinction burst observed in controls is part of the extinction process that reflects the invigoration of the behavioral response when the outcome does not match expectation at the beginning of the extinction process. We have altered the manuscript to be more specific on what we are referring to regarding the deficits in extinction behavior and have provided additional analysis of the rate of responding on the days of extinction training where differences were observed in the overall numbers of lever presses but not the slopes of the curves.

(vi) the model and hypothesis put forward and tested in the experiments shown in Figure 6 are very interesting. However, the results obtained do not justify the conclusion that Loss of Ntn1 function in both cell types simultaneously largely rescues the consequences induced by GABA- only Ntn1 deletion.

We apologize for the lack of clarity. We have altered the discussion to more explicitly provide an explanation of how mutagenesis in both cell types can result in a diminution of the observed phenotype in the GABA-Ntn1 knockout mice. In the model proposed, reduced excitatory input onto GABA neurons results in a reduced inhibition of dopamine neurons causing the observed phenotypes. For reference, we have previously shown that blocking all synaptic transmission from GABA neurons in the VTA (Gore et al., 2017 ELife), or blocking selectively GABA release from VTA GABA neurons (Soden et al., 2020, Nat. Neurosci.) results in hyperactivity and increased operant responding, though to a much greater degree than the effects observed here. We, and others have also shown that loss of glutamate signaling in dopamine neurons has only a modest behavioral effect (Zweifel et al., 2009 PNAS, and Hutchinson et al., 2017 Mol Pscyh.), so it is not surprising that reducing mEPSCs onto VTA dopamine neurons has little effect in this context. The partial rescue of the GABA-Ntn1 knockout experiment suggests that reducing glutamatergic input onto dopamine neurons can abrogate effects associated with reduced inhibitory tone, thus restoring the excitatory and inhibitory balance. In the revised discussion we outline a detailed mechanism that we propose explains this observation.

To be able to link the electrophysiological changes to glutamatergic synaptic connectivity, other experiments are required, including assessing structural changes (e.g. PMID: 24174661) in dopamine and GABA neurons as well as the proportion of AMPA/NMDA receptors and AMPA/NMDA ratios. In this regard, there is a previous study relating the netrin-1 guidance cue system in adult VTA synaptic plasticity (PMID: 20345916).

An initial address of this point was provided above. Assessing AMAPA/NMDA ratio is another measure of synaptic strength; however, this assumes that NMDA receptor levels remain unchanged, which we know from previous studies is not always true in the VTA (Mameli, M., Bellone, C., Brown, M. T. & Lüscher, C 2011 Nature). To address this, we recorded both AMPA and NMDA-evoked current in control and Ntn1 mutant dopamine neurons. We have also made sure that we reference the above highlighted paper which describes the impact of Dcc heterozygous loss of function on amphetamine sensitization.

Whether NTn1 deletion leads to dopamine or GABA neuronal loss could be addressed using stereology.

We have included a count of the number of virally labelled dopamine neurons to address this point and show that Ntn1 deletion did not result in a loss of dopamine neurons.

To be able to conclude that the loss of Ntn1 function in both cell types simultaneously largely rescues the consequences induced by GABA- only Ntn1 deletion, the electrophysiological properties of dopamine (and also GABA) neurons could be assessed. This experiment will also test more directly the model the authors are proposing.

This experiment is more difficult to address than the reviewer may appreciate. Both dopamine and GABA neurons express Cre, so fluorescent isolation of these cells will not be possible. Ih currents can be used, but not all dopamine neurons are Ih positive. This is particularly problematic as the number of excitatory synapses onto GABA neurons is higher than onto dopamine neurons, thus there will be a large spread in the distribution of responses making statistical analysis difficult to achieve. To address this, we have been more explicit in our discussion of what we hypothesize the double mutagenesis is achieving and why this is important for considerations of the cell-type and circuit-specific impacts of Ntn1 loss of function are and how they are consistent with what is known about the organization of the VTA cell types.

In the discussion, evidence showing the role of glutamatergic inputs of VTA dopamine neurons on behavior needs to be revised more carefully (e.g. PMID 25388237, PMID 26631475, and PMID 30699344).

We have expanded this area of the discussion to address this point.

Reviewer #2 (Recommendations for the authors):

This manuscript by Cline et al. sought to define the role of the axonal guidance cue netrin-1 in synaptic signaling in the ventral tegmental area (VTA). The authors used CRISPR-Cas9 mutagenesis to reduce netrin-1 expression/function in the two predominant neuronal types in the VTA, dopaminergic and GABAergic neurons. This work builds on previous work from this group showing a role for the axon guidance receptor ROBO2 in inhibitory connectivity in adult VTA. Netrin-1 is examined here because of its persistent expression in the VTA into adulthood, despite its established role as a developmental protein. A strong combination of techniques is used, including selective knockdown of the netrin-1 gene in multiple neuron types in the VTA, patch clamp electrophysiology, and several behavioral assays. The results clearly indicate that knockdown of netrin-1 specifically in either GABA or dopamine neurons reduces miniature glutamatergic synaptic currents specifically in that cell type. Interestingly, inhibitory input was not significantly affected. This is consistent with previous reports of effects on excitatory synapses in the hippocampus. Robust behavioral consequences were only reported in the GABA neuron knockdown and were not evident when netrin-1 was knocked down in both cell types. The authors conclude that netrin-1 is important for maintaining the balance of excitatory input onto the two main neuronal subtypes in the VTA. This conclusion is largely supported by the results from the experiments, which were performed rigorously. The manuscript itself was easy to follow and well written, save for some minor omissions.

Operant responding for food was used as one of the dependent measures in Figures 4 and 5, with differences observed in the GABA neuron knockdown, however, one omission of the study was that body weights of the mice before and especially after treatment were not reported. It may be that viral knockdown of netrin-1 in one cell type has effects for instance on satiety, producing effects on behavior along with energy balance. The addition of body weight data would help round out the data set and, if different, might help with interpretation.

This is an excellent point. All mice were food restricted to 85% of their body weight, and the methods have been changed to reflect that omission. Initial bodyweights at the start of calorie restriction have also been included in the supplemental data.

Details were also not provided for the food restriction procedure that was used during operant conditioning for food pellet responding, which could have further interacted with the netrin-1 manipulation in the mouse lines to affect physiology, behavior, or both.

We apologize for the oversight and have corrected the methods to reflect the calorie restriction used.

As discussed above, most of this study is strong, with interpretations supported by largely convincing results. However, the manuscript could be improved with additions and clarifications.

While both cell types showed increased mEPSC frequency and amplitude after netrin-1 knockdown, only the GABA neuron knockdowns showed robust behavioral effects, including increased locomotion (day and night), and increased operant responding for food, and decreased acoustic startle and pre-pulse inhibition. As some of the behavioral tasks involve responding for food pellets, interpretation of the results would benefit from reporting the weights of the mice before and after treatment. Additionally, details about the food restriction procedure that was used for operant conditioning should be provided. Several reports have identified the effects of the feeding state on dopamine neuron excitability and synaptic input to the area. Was the food restriction controlled to a percent loss of body weight, applied acutely or chronically, done throughout the operant study or just during FR1, etc.? Did the mice always eat the pellets when performing the operant task? It's interesting that responding in all of the dopamine mice stayed at the same values when they switched from FR1 to FR5, but in the GABA mice, the control animals fell while the netrin-1 knockdowns stayed the same. Full disclosure of these details would help the reader interpret small observations in the data and later assist in reproducing the results, should they wish to do so.

We apologize for the oversight and have corrected the methods to reflect the food restriction used. We have also included the body weights of the animals prior to calorie restriction.

The evidence for the anxiety behavioral phenotype in the dopamine neuron-specific netrin-1 knockdown is pretty thin, as a time-in-center measure in an open field could be affected by other factors, and other supporting data for instance from elevated mazes were not used. Admittedly, anxiety can be difficult to show in mice. However, Reference 20 which was used to support the "proposed role of dopamine in the modulation of anxiety-related behavior" was a dead-end non-citation. If the authors wish to keep this part of their interpretation they should bolster the explanation of the link between dopamine and anxiety with further evidence (experimental or literature). Also, details about the meaning of "time in center" and "time on edge" should be provided in the Methods.

We apologize for the broken citation. The citation should have read ” Zarrindast MR, Khakpai F. The Modulatory Role of Dopamine in Anxiety-like Behavior. Arch Iran Med. 2015;18(9):591-603.” The reference has been corrected.

We have also provided additional information re: time in center and time on edge in the methods to add clarification.

The extinction result in the GABA mice is presented as "a significant delay in the rate of extinction following reinstatement of FR1." This is hard to interpret because responding immediately before extinction is not given and the data are presented only as raw numbers instead of percent of baseline. If the netrin-1 knockdowns were responding more at FR1 when they were returned to that condition, the shape of the curve would actually indicate no difference in extinction. The same could be true in Figure 6 with the double cell knockdowns. Depending on what the data look like, these graphs may be more accurately presented as normalized numbers. This is a minor issue in the scheme of this paper but it should nonetheless be clarified.

We have provided the data on the reinstatement training and have performed additional analysis of the rates of extinction for each of the mice within a given session to more accurately address this concern.

The schematics in Figure 6A show that the effects on the GABA neurons proceed through the dopamine neurons. While this is entirely plausible, the authors never actually show this experimentally, for instance by locally manipulating GABA input in virus-injected mice. It may be at least as likely that the important interactions occur in the nucleus accumbens, or some other area to which both cell types project. As a minimum, the authors should point out this caveat in the Discussion, or point out any other possibility that could also explain the somewhat surprising data in Figure 6.

We agree that the projection of a subset of GABA neurons to other brain regions may play an important role in the observed phenotypes. We have clarified this in the discussion.

There's some confusion about the Ntn1 co-localization in Figure 1. The language in the caption and Figure 1 itself seem clear. However, the text on page 3 seems to contradict the language in the figure caption. The way it is worded, instead of 64, 30, and 6% shouldn't these numbers be 72, 18, and 10%? Please clarify with the correct information, or point out where the confusion lies.

We apologize for the confusion and have clarified this in our revised manuscript.

In the abstract, "simultaneously" is placed confusingly in the sentence about rescuing the GABA phenotype. This would be more clear if the sentence started "Simultaneous loss… in both cell types."

We agree and have made this change.

Figure 1D has two (identical) scale bars.

We have corrected this inadvertent duplication.

Reviewer #3 (Recommendations for the authors):

In this study, they used genetic strategies to decrease Netrin-1 expression in either dopamine or GABA neurons of the VTA. Reduction of Netrin-1 expression in VTA dopamine neurons decreased excitatory, but not inhibitory postsynaptic currents onto TH+ or GABA VTA neurons. While the loss of netrin-1 in dopamine neurons did not significantly influence behaviour, loss of netrin-1 in GABAergic neurons increased locomotor activity, effort for rewards, extinction delay, and decreased prepulse inhibition. Finally, effects on locomotor activity, reward seeking, and prepulse inhibition observed in GABA-netrin mice were not present when there was the loss of netrin-1 in both dopamine and GABA neurons, suggesting that loss of netrin-1 in both could restore the behavioural effects of loss of netrin-1 in GABA neurons.

Major strengths:

This is a nicely written, clearly illustrated study describing the loss of netrin-1 in VTA dopamine neurons and GABA neurons. The authors use an elegant genetic methodology to knock down netrin-1 in select populations of neurons within the VTA. They use a battery of behavioural assays to examine the effects of this knockdown.

Major weaknesses:

While this study provides a potential physiological mechanism underlying the changed behavioural effects, it doesn't connect these changes to the behaviour or identify the mechanism by which adult netrin-1 influences these changes in synaptic transmission. While netrin-1 is involved in synapse formation during development, it is not clear how netrin-1 is influencing synapses in adulthood. For example, is it necessary for the stabilization of synapses?

We agree with the reviewer and have received a similar concern from reviewer 1. A response to this is provided above and we will summarize our experimental approach below.

Author suggestions:

The authors propose that loss of netrin-1 in dopamine neurons leads to enhanced excitation and decreased inhibition, whereas loss in GABA neurons would lead to enhanced inhibition and decreased excitation, and the E:I ratio would be balanced when netrin is lost from both cell population. However, they do not test this assertion by measuring excitatory:inhibitory ratio in each model. This would support their hypotheses in figure 6.

We thank the reviewer for their comment. We conclude that loss of Ntn1 function in either dopamine neurons or GABA neurons results in a loss of excitatory synaptic connectivity with no loss in inhibitory synaptic connectivity. Loss of excitatory input with no change in inhibitory input will bias towards inhibition in the E:I balance. When this occurs in GABA neurons, because local GABA potently inhibits dopamine neurons, the loss of excitation onto GABA neurons biases towards greater inhibition of these cells which disinhibits the dopamine neurons causing a hyperdopaminergic phenotype. We have provided a detailed discussion of the potential mechanism by which loss of Ntn1 in both cell types would restore the E:I balance.

Some of the discussion focuses on the role of netrin-1 in development. However, the manipulations done in this paper were to remove netrin-1 in adulthood after axon migration and synapse formation occur. They do not discuss what the 'adult function' of netrin-1 is. While it seems to play a role in excitatory synaptic transmission, given its effects on mEPSCs, the authors did not provide sufficient information to conclude if this was a reduction in the number of synapses, a silencing of synapses, or a decrease in release probability with compensatory postsynaptic changes. Experiments addressing how adult netrin-1 signaling in the VTA specifically influences synaptic transmission onto dopamine or GABA neurons of the VTA may highlight how netrin-1 might be contributing to associated behavioral changes.

As mentioned above, we have included recordings of AMPA and NMDA receptor mediated currents (via bath application of the specific agonists) in these cells to quantify the levels of these receptors. Our paired-pulse ratio experiments indicate that presynaptic release probability is not altered with the loss of Ntn1 pointing to a postsynaptic effect.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Essential revisions:

We have a few remaining concerns that can be addressed with an additional analysis of existing data (see R3 point #1) and edits to the text to add clarity, change language, temper conclusions, discuss alternatives, etc.

Please provide a point x point response to each reviewer point along with your revision.

Reviewer #1 (Recommendations for the authors):

This resubmitted manuscript is substantially improved from the previous version. Additions and clarification to methods and interpretations have largely addressed my previous concerns, which were minor. Additional discussion has bolstered the notion that netrin-1 is impotant for maintaining the balance of excitatory and inhibitory inputs to dopamine neurons. The addition of maze data would have bolstered conclusions about the anxiety phenotype, but "time on edge" and locomotor data was added and this was always a minor point that did not detract substantially from the rest of the manuscript.

One item does need to be clarified. The experiment represented in Figure 3 Supplement 3 was performed to address a concern of Reviewer 1, however the text describing this is very confusing. The Results describe these data as evoked EPSCs following bath perfusion of AMPA or NMDA. The data themselves seem to be showing changes in holding current in a voltage clamp experiment, although the axis labels and the lack of sample traces leave this in doubt. If it is holding current, more details should to be provided in the figure (including better axis labels and a description of how long the drug went on and the holding voltage, both of which are currently only in the Methods). If instead it is evoked EPSCs in the presence of bath perfused agonists, then other details also need to be provided to make this result make sense (such as sample traces and an explanation of what they were after).

We apologize for the confusion and lack of clarity regarding these experiments. These data were from a voltage clamp experiment in which agonist was bath applied for 30 seconds, and holding current was recorded. The figure legends and axis have been amended to reduce confusion, and the experiment has been better clarified in the Results section.

The addition of body weight data and food restriction information is appreciated. However in the Response to Reviewers the level of restriction was listed as 85% of initial body weight, whereas in the manuscript this is given as 80%. Please make sure the correct number appears in the manuscript.

The addition of body weight data and food restriction information is appreciated.

However in the Response to Reviewers the level of restriction was listed as 85% of initial body weight, whereas in the manuscript this is given as 80%. Please make sure the correct number appears in the manuscript.

Reviewer #2 (Recommendations for the authors):

The revised version improved the interpretation of the results, and I am pleased that the authors replied to most of my comments on the previous version. They added needed citations, clarification on their manipulation, and additional experiments to understand the changes in synaptic connectivity better and clarify the electrophysiological changes that occur with Ntn1 mutagenesis, as well as their proposed model.

The following issues remain unresolved:

– Since the study does not assess alterations in neuronal structure and connectivity, we suggest the word "connectivity" to be dropped or modified.

We thank the reviewer for their comment. Although we disagree that the measurement of miniature inhibitory and excitatory postsynaptic currents is not a direct measure of synaptic connectivity, we have changed the title to “Netrin-1 regulates the balance of synaptic glutamate signaling in the adult ventral tegmental area”.

– It appears that stereology was not performed to calculate the number of neurons expressing netrin-1 in TH+ or GABA+ cells in the mice with conditional netrin-1 KO. The statement "Total Th-positive cells were recorded for all images and averaged across all slices for each mouse to give a total number of Th-positive cells" needs to be revised, because total number of cells can only be assessed using stereological analysis. It remains unknown whether NTn1 deletion leads to dopamine or GABA neuronal loss. This is an important issue that needs to be acknowledged in the manuscript. Please see

https://www.embopress.org/doi/full/10.15252/embj.2020105537

We thank the reviewer for this comment. We have addressed this with the following statement in the Results section: “Following Ntn1 deletion the average number of TH+ cells per slice was not statistically different in DAT-Cre Ntn1 cKO mice compared to controls (control: 178.2±12.68 and Ntn1 cKO 173.3±10.75). Although this result is consistent with Ntn1 inactivation not compromising cell viability, without a complete stereological analysis of every neuron within the VTA, we cannot definitively conclude that some cell loss did not occur.”

– Regarding the increase in locomotor activity observed after the downregulation of Ntn1 in GABA neurons, the authors argue that the level of responding in the FR1 task is not altered in GABA-Ntn1 mutant mice, suggesting that elevated responding in the FR5 and PR assays is not likely a reflection of hyperactivity. Yet, increasing the response rate in the FR1 protocol is no possible. Once the animals respond to the reward in the FR1, the lever retracts, and no more lever-pressing is possible until a reward is collected. The statement added by the authors "however, we did not observe differences in operant responding during FR1 training or reinstatement indicating that the observed effects are likely a reflection of altered motivational states rather than general hyperactivity" needs to be modified because hyperactivity may be influencing the findings reported

We appreciate this suggestion and have modified the phrasing in the manuscript in response. The phrasing has been changed to: While these data likely reflect altered motivational state with loss of Ntn1, it is also possible that the hyperactivity observed in Vgat-Cre Ntn1 cKO mice contributes to the elevated lever press rates during FR5, PR, and extinction.

– Regarding the lever pressing extinction data, (1) the authors acknowledge that mice with Ntn1 cKO in GABA VTA neurons have similar extinction rates than WT. They now need to mention that this finding suggests that the increase in the number of lever-pressing during the extinction phase in Ntn1 cKO in GABA mice is most likely associated with an increase in baseline responding rather than with extinction deficits. This same point needs to be highlighted for the Ntn1 cKO in DATIRES::Vgat-Cre mice too. (2) the rate of extinction is not analyzed nor discussed. (3) it is unclear how plotting the extinction bin data for day1 and day 2 and for day 4 and day 5, for suppl Figures5 and 6 respectively, addresses this issue. The authors need to consider removing these data because they do not seem to provide relevant information.

We thank the reviewer for this comment and we believe that this is addressed in the response above. With regard to the inclusion of the additional extinction analysis previously requested by the reviewer, we believe that this data provides additional information for the reader to better understand the extinction responding in control and Ntn1 mutant mice.

Reviewer #3 (Recommendations for the authors):

I appreciate the addition of the bath application of AMPA or NMDA experiments. While these add support to the effect of netrin being postsynaptic, it should be indicated that these experiments do not distinguish between synaptic responses and extrasynaptic responses. Further a change in holding current after bath application of AMPA could be due to reduced synapse number that is associated with a reduced postsynaptic AMPA receptor complement. Further, while they show no change in PPR consistent with no effect on release probability (Pr), their decrease in mEPSC amplitude and frequency could be consistent with a reduction in synapse number (number of release sites N) or a change in number or function of postsynaptic AMPA receptors. To distinguish this, you can measure 1/CV2 which depends on N and Pr but is independent of quantal size. Given that there is no change in Pr, if you see a change in 1/CV2 it might be consistent with a decrease in release sites as opposed to a change in postsynaptic receptor number or function. Alternatively, if there is no change in 1/CV2, you can make a stronger conclusion that the netrin manipulation is altering number or function of postsynaptic AMPA receptors.

This is an excellent suggestion. We calculated 1/CV2 for mEPSCs based on this suggestion and have presented that data in the results and as figure 3 supplemental 3.

Regarding the shift in the E:I balance, while I do not disagree with their model, I was proposing that they could test their model directly by recording mIPSCs at the reversal potential for EPSCs (0 to 10mV) and mEPSCs at the reversal potential for GABA mIPSCs (~-60 to -70mV) and converting the average peak current amplitude into conductance. This way, you can directly measure the amount of inhibitory current and amount of excitatory current the same cell is receiving. Perhaps this experiment is beyond the scope of the current manuscript, but when discussing their model, they could indicate that they could test the validity of this hypothesis in future experiments.

We appreciated the suggestion and do agree that this experiment is beyond the scope of the current manuscript, but we have included this as a suggestion for future experiments in the discussion of this paper.

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    Figure 1—source data 1. Cell counts.
    elife-83760-fig1-data1.xlsx (10.8KB, xlsx)
    Figure 2—source data 1. Cell counts.
    elife-83760-fig2-data1.xlsx (8.8KB, xlsx)
    Figure 3—source data 1. EPSCs and IPSCs from targeted cells.
    elife-83760-fig3-data1.xlsx (53.3KB, xlsx)
    Figure 3—source data 2. Additional EPSC and IPSC data from non-targeted cells.
    elife-83760-fig3-data2.xlsx (19.8KB, xlsx)
    Figure 4—source data 1. Behavioral data for Figure 4.
    elife-83760-fig4-data1.xlsx (101.2KB, xlsx)
    Figure 4—figure supplement 1—source data 1. Behavioral data for Figure 4—figure supplement 1.
    elife-83760-fig4-figsupp1-data1.xlsx (15.5KB, xlsx)
    Figure 5—source data 1. Behavioral data for Figure 5.
    elife-83760-fig5-data1.xlsx (95.9KB, xlsx)
    Figure 5—figure supplement 1—source data 1. Behavioral data for Figure 5—figure supplement 1.
    elife-83760-fig5-figsupp1-data1.xlsx (19KB, xlsx)
    Figure 6—source data 1. Behavioral data for Figure 6.
    elife-83760-fig6-data1.xlsx (109.2KB, xlsx)
    Figure 6—figure supplement 1—source data 1. Behavioral data for Figure 6—figure supplement 1.
    elife-83760-fig6-figsupp1-data1.xlsx (20.2KB, xlsx)
    MDAR checklist
    elife-83760-mdarchecklist1.docx (99.6KB, docx)

    Data Availability Statement

    All data generated or analysed during this study are included in the manuscript and supporting file.

    Articles from eLife are provided here courtesy of eLife Sciences Publications, Ltd

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