Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation 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. https://doi.org/10.7554/eLife.83760.sa0 Decision letter eLife's review process 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 Download asset Open asset Netrin-1 is present in the adult ventral tegmental area (VTA) and expressed by both dopamine and GABA neurons. 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. https://cdn.elifesciences.org/articles/83760/elife-83760-fig1-data1-v1.xlsx Download elife-83760-fig1-data1-v1.xlsx 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 Download asset Open asset Virally delivered CRISPR-Cas9 complex targeting the Ntn1 locus results in a significant reduction in Ntn1 antibody staining. (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. https://cdn.elifesciences.org/articles/83760/elife-83760-fig2-data1-v1.xlsx Download elife-83760-fig2-data1-v1.xlsx 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 with 3 supplements see all Download asset Open asset 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. https://cdn.elifesciences.org/articles/83760/elife-83760-fig3-data1-v1.xlsx Download elife-83760-fig3-data1-v1.xlsx Figure 3—source data 2 Additional EPSC and IPSC data from non-targeted cells. https://cdn.elifesciences.org/articles/83760/elife-83760-fig3-data2-v1.xlsx Download elife-83760-fig3-data2-v1.xlsx 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 with 1 supplement see all Download asset Open asset 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. https://cdn.elifesciences.org/articles/83760/elife-83760-fig4-data1-v1.xlsx Download elife-83760-fig4-data1-v1.xlsx 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 with 1 supplement see all Download asset Open asset 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. https://cdn.elifesciences.org/articles/83760/elife-83760-fig5-data1-v1.xlsx Download elife-83760-fig5-data1-v1.xlsx 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 with 1 supplement see all Download asset Open asset 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. https://cdn.elifesciences.org/articles/83760/elife-83760-fig6-data1-v1.xlsx Download elife-83760-fig6-data1-v1.xlsx 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

Author Response: Netrin-1 Regulates the Balance of Synaptic Glutamate Signaling in the Adult Ventral Tegmental Area