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.

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.

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

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Author details

1. Marcella M Cline

   1. Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, United States
   2. Molecular and Cellular Biology Program, University of Washington, Seattle, United States

   Contribution

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

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9590-3725

2. Barbara Juarez

   1. Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, United States
   2. Department of Pharmacology, University of Washington, Seattle, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

3. Avery Hunker

   Department of Pharmacology, University of Washington, Seattle, United States

   Present address

   Allen Brain Institute, Seattle, United States

   Contribution

   Formal analysis, Methodology

   Competing interests

   No competing interests declared

4. Ernesto G Regiarto

   Department of Pharmacology, University of Washington, Seattle, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Bryan Hariadi

   Department of Pharmacology, University of Washington, Seattle, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Marta E Soden

   Department of Pharmacology, University of Washington, Seattle, United States

   Contribution

   Data curation, Formal analysis, Methodology

   Competing interests

   No competing interests declared

7. Larry S Zweifel

   1. Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, United States
   2. Department of Pharmacology, University of Washington, Seattle, United States

   Contribution

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

   For correspondence

   larryz@u.washington.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3465-5331

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