Descending projections from the substantia nigra pars reticulata differentially control seizures Evan Wickera, Veronica C. Becka, Colin Kulick-Sopera, Catherine V. Kulick-Sopera, Safwan K. Hydera, Carolina Campos-Rodrigueza, Tahiyana Khana,b, Prosper N’Gouemoa,b,c, and Patrick A. Forcellia,b,d,1 aDepartment of Pharmacology & Physiology, Georgetown University, Washington, DC 20007; bInterdisciplinary Program in Neuroscience, Georgetown University, Washington, DC 20007; cDepartment of Pediatrics, Georgetown University, Washington, DC 20007; and dDepartment of Neuroscience, Georgetown University, Washington, DC 20007 Edited by Peter L. Strick, University of Pittsburgh, Pittsburgh, PA, and approved November 20, 2019 (received for review May 13, 2019) Three decades of studies have shown that inhibition of the sub- Here, we sought to address 2 unresolved questions. First, to what stantia nigra pars reticulata (SNpr) attenuates seizures, yet the extent does selective silencing of the nigrotectal as compared to the circuits mediating this effect remain obscure. SNpr projects to nigrotegmental pathway recapitulate the effect of silencing SNpr? the deep and intermediate layers of the superior colliculus (DLSC) Second, are the seizure-suppressive effects of SNpr silencing me- and the pedunculopontine nucleus (PPN), but the contributions of diated by divergent pathways depending on the seizure type? these projections are unknown. To address this gap, we optoge- We optogenetically silenced either SNpr, nigrotectal, or nigro- netically silenced cell bodies within SNpr, nigrotectal terminals tegmental projections in 4 models of experimental epilepsy in rats: within DLSC, and nigrotegmental terminals within PPN. Inhibition the gamma-butyrolactone model of absence (thalamocortical) of cell bodies in SNpr suppressed generalized seizures evoked by seizures, pentylenetetrazole (PTZ)-evoked forebrain tonic-clonic pentylenetetrazole (PTZ), partial seizures evoked from the fore- seizures, piriform cortex-evoked limbic seizures, and brainstem brain, absence seizures evoked by gamma-butyrolactone (GBL), tonic-clonic seizures in the GEPRs. The human epilepsies are and audiogenic seizures in genetically epilepsy-prone rats. Strik- diverse and present with a variety of seizure types; these models ingly, these effects were fully recapitulated by silencing nigrotec- were selected to each reflect a different type of seizure seen in the tal projections. By contrast, silencing nigrotegmental terminals epilepsies, providing a measure of whether our manipulations are reduced only absence seizures and exacerbated seizures evoked seizure type-specific or more generally effective against seizures by PTZ. These data underscore the broad-spectrum anticonvulsant writ large. Optogenetic inhibition of SNpr suppressed seizures in efficacy of this circuit, and demonstrate that specific efferent pro- all models, and, strikingly, these effects were completely re- jection pathways differentially control different seizure types. capitulated by selective silencing of the nigrotectal projection. By contrast, inhibition of nigrotegmental projections was effective seizure | deep brain stimulation | basal ganglia | optogenetics | substantia nigra only against absence seizures and, conversely, exacerbated seizures evoked by PTZ. rain circuits that exert endogenous seizure-suppressive ef- Results Bfects have been a topic of investigation for over 30 y. One We microinjected rAAV8-CAG-ArchT-GFP coding for the in- group of circuits has received particular attention in this regard: hibitory opsin, ArchT, into SNpr of wild-type Sprague–Dawley the basal ganglia. While most commonly considered in the (SD) rats. To verify optogenetic silencing, we performed multiunit context of posture, movement control, and movement disorders, recordings in anesthetized rats. Neurons in SNpr displayed tonic discrete nodes of the basal ganglia can also be harnessed to control seizures in a broad-spectrum, multipotent manner. For Significance example, inhibition of the substantia nigra pars reticulata (SNpr) exerts profound antiseizure effects in models of generalized (both convulsive and absence) and partial seizures (1–10), and a Identifying new targets for deep brain stimulation in epilepsy failure of GABA release in SNpr is associated with seizure sus- requires a deeper understanding of the brain networks engaged ceptibility in genetically epilepsy-prone rats (GEPRs) (11). by seizure activity. For over 30 y, the substantia nigra pars Despite decades of study, the circuit mechanisms by which reticulata has been recognized as a potential target, but the SNpr suppresses seizure activity have remained unclear. The efferent pathways mediating the suppression of seizures have remained obscure. Here, we show that silencing the projection SNpr is a GABAergic projection nucleus with several major from the substantia nigra to the superior colliculus fully reca- output pathways; thus, inhibition of SNpr leads to disinhibition pitulates the antiseizure effects evoked from cell bodies within of multiple target structures. These output targets include the the substantia nigra. By contrast, inhibition of the projection to thalamus, the deep and intermediate layers of the superior col- – the pedunculopontine nucleus exacerbates some seizures, re- liculus (DLSC), and the pedunculopontine nucleus (PPN) (12 duces others, and is without effect on still others. The functional 15). Prior studies using conventional lesion, microinjection, and divergence of these pathways highlights a key role for projec- electrical recording methods have suggested a role for each of tions to the superior colliculus in the control of seizures. these target regions in nigra-evoked seizure control (16–18). However, prenigral hemitransections which sever rostral-going Author contributions: P.N. and P.A.F. designed research; E.W., V.C.B., C.K.-S., C.V.K.-S., projections from SNpr to the forebrain do not impair the anti- S.K.H., C.C.-R., T.K., P.N., and P.A.F. performed research; E.W. and P.A.F. analyzed data; convulsant effect of SNpr inhibition. Therefore, we focused on and E.W., V.C.B., C.K.-S., C.V.K.-S., S.K.H., C.C.-R., T.K., P.N., and P.A.F. wrote the paper. 2 caudal projections: the nigrotectal pathway, terminating in The authors declare no competing interest. DLSC, and the nigrotegmental pathway, terminating in PPN. This article is a PNAS Direct Submission. With conventional microinjection or lesion approaches, it was Published under the PNAS license. impossible to selectively manipulate these highly collateralized 1To whom correspondence may be addressed. Email: [email protected]. projection pathways; pharmacological methods would inhibit or This article contains supporting information online at https://www.pnas.org/lookup/suppl/ disinhibit all of the output pathways of SNpr simultaneously. doi:10.1073/pnas.1908176117/-/DCSupplemental. Optogenetic approaches to silencing have enabled this dissection. First published December 16, 2019. 27084–27094 | PNAS | December 26, 2019 | vol. 116 | no. 52 www.pnas.org/cgi/doi/10.1073/pnas.1908176117 Downloaded by guest on September 25, 2021 baseline firing, which was suppressed by light delivery (Fig. 1A). of our manipulation (Fig. 2 A and B). The suppression in seizure Optogenetic silencing of nigrotectal terminals disinhibited neu- activity observed in active-vector SD rats was evident both be- rons in DLSC, resulting in burst firing during light delivery (Fig. haviorally (Fig. 2A) and electrographically (Fig. 2G vs. Fig. 2H). In 1B). In both cases, moving the optrode 1.5 mm dorsal from the site the absence of light delivery, PTZ administration resulted in high- of virus injection eliminated optogenetic modulation of neuronal amplitude, fast ictal discharges on the cortical EEG (Fig. 2G); by activity. This is in keeping with the modeled decay in power with contrast, in the presence of light delivery, this activity was reduced increasing distance from the tip of the fiber (19, 20), which sug- to short bursts (Fig. 2H). gests that we were inhibiting neurons and terminals within a cone From a clinical neurostimulation standpoint, reduced duty cycle of tissue extending ∼1 mm radially from the tip and ∼1 to 1.5 mm offers advantages such as prolonged battery life. Moreover, re- below the fiber tip. With our fiber placed 0.2 mm dorsal to SNpr at duced total integrated light delivered minimizes concerns re- its approximate midpoint in the rostrocaudal and mediolateral garding heating and tissue damage with optogenetics. To planes, we were thus positioned to deliver light to approximately determine if a strategy that used minimal light delivery would also half of the structure. The light delivery parameters we employed be effective, we injected wild-type SD rats with a step-waveform for DLSC mirror those we had previously used for optogenetic inhibitory channelrhodopsin (rAAVDJ-hSyn-SwiChR-eYFP) into activation experiments (21). While we did not record from DLSC SNpr. This opsin is an anion-conducting variant of channelrho- while inhibiting cell bodies in SNpr, our data inhibiting nigrotectal dopsin with slow off-rate kinetics, allowing brief pulses of blue terminals within DLSC are in keeping with the well-described light to trigger sustained neuronal hyperpolarization (25). We functional architecture of nigrotectal pathways, best described in applied 0.2 Hz light stimulation, and, as with our other experi- the context of saccadic eye movements (22–24), i.e., suppression of ments, detected a significant