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The Input-Output Relationship of the Basal

Graphical Abstract Authors Matthew R. Gielow, Laszlo Zaborszky

Correspondence zaborszky@.rutgers.edu

In Brief Monosynaptic viral tracing in transgenic rats reveals that patterns of input cells contacting cholinergic are biased according to their cortical or amygdalar output. Mapping inputs across the entire brain, Gielow and Zaborszky provide structural evidence for networks enabling differential release.

Highlights d Inputs to cholinergic cells are biased, varying by cortical output target d Proportions of input to a given cortical cholinergic output are reproducible d The most prominent input to many cholinergic cells is the caudate d Medial -projecting cholinergic cells receive little caudate input

Gielow & Zaborszky, 2017, Cell Reports 18, 1817–1830 February 14, 2017 ª 2017 The Author(s). http://dx.doi.org/10.1016/j.celrep.2017.01.060 Cell Reports Resource

The Input-Output Relationship of the Cholinergic

Matthew R. Gielow1 and Laszlo Zaborszky1,2,* 1Center for Molecular and Behavioral Neuroscience, Rutgers, the State University of New Jersey, Newark, NJ 07102, USA 2Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2017.01.060

SUMMARY Lesions of the BF in experimental animals or humans cause enhancement of slow oscillations and severe attention and mem- Basal forebrain cholinergic neurons influence cortical ory deficits (Botly and De Rosa, 2012; Buzsa´ ki et al., 1988; Lut- state, plasticity, learning, and attention. They collec- kenhoff et al., 2015), while BF stimulation increases the sponta- tively innervate the entire , differen- neous and visually driven cortical firing rates, improving neuronal tially controlling acetylcholine efflux across different response reliability (Pinto et al., 2013). Similarly, experiments in cortical areas and timescales. Such control might the somatosensory (Maalouf et al., 1998) or in the auditory cortex be achieved by differential inputs driving separable (Leach et al., 2013; Metherate and Weinberger, 1990)suggest that ACh plays a role in improving sensory perception. Release cholinergic outputs, although no input-output rela- of ACh across different cortical areas covaries between tionship on a brain-wide level has ever been and wake states (Jasper and Tessier, 1971; Phillis, 1968; Sarter demonstrated. Here, we identify input neurons to and Bruno, 2000). BF cells projecting to remote regions of cortex cholinergic cells projecting to specific cortical appear to have overlapping dendritic fields (Woolf, 1991), and regions by infecting cholinergic axon terminals cholinergic cells projecting to different cortical targets often inter- with a monosynaptically restricted viral tracer. This mingle across an extended region of the BF. These data together approach revealed several circuit motifs, such as have been taken as evidence that the cholinergic BF is a diffuse central neurons synapsing onto basolat- projection system. However, discrete cholinergic efflux occurs eral amygdala-projecting cholinergic neurons or per cortical region at different time points (Fournier et al., 2004; strong somatosensory cortical input to motor cor- Parikh et al., 2007; Rasmusson, 1993), and recent tracing and op- tex-projecting cholinergic neurons. The presence of togenetic activation of cholinergic neurons in the mouse BF sug- gest that cholinergic neurons can modulate sensory cortical areas input cells in the parasympathetic midbrain nuclei in a modality-specific manner (Kim et al., 2016). Part of this contro- contacting frontally projecting cholinergic neurons versy is based upon the complex organization of BF projection suggest that the network regulating the inner eye neurons: cholinergic and non-cholinergic projections to the muscles are additionally regulating cortical state via are not diffuse but are organized into segregated and acetylcholine efflux. This dataset enables future cir- overlapping pools of neurons that may transmit information from cuit-level experiments to identify drivers of known specific locations in the BF to subsets of cortical areas, themselves cortical cholinergic functions. interconnected (Zaborszky et al., 2015a). Additionally, there are subregional differences in input across the BF (Zaborszky et al., 2015b), although how this affects cortical outputs is unknown. INTRODUCTION The relationship of synaptic inputs to target-identified cell pop- ulations cannot be readily assessed on a whole-brain level with The connectome of the basal forebrain (BF) is not well under- classic anatomical techniques that rely on the combination of con- stood due to the anatomical complexity of the region. Patients ventional anterograde and retrograde tracing (Zaborszky et al., with Alzheimer’s disease and related have a signifi- 2006). Here, we employed output-specific monosynaptic viral cant decrease of acetylcholine (ACh) in the cortex and show tracing in cells expressing choline acetyltransferase (ChAT) to pathological changes in cholinergic neurons in the BF (Za- sample the input-output relationship of the cholinergic BF, and borszky et al., 2008). Thus, a complete understanding of its func- thereby identify on a whole-brain scale the neuronal populations tional organization is warranted. ACh is released in the cortex by likely to drive differential ACh efflux in particular output structures. neurons whose cell bodies are located in the BF. Each of these Genetic access to BF ChAT cells was achieved by injecting Cre- cholinergic neurons supplies a small portion of cortex (Price dependent helper viruses (AAV-EF1a-FLEX-TVAmCherry and and Stern, 1983), and ACh release is controlled precisely across AAV-CA-FLEX-RG) (Watabe-Uchida et al., 2012) at five points different cortical regions and timescales, thought to be respon- across the BF in ChAT::Cre rats (Witten et al., 2011)(Figures 1A sible for the variety of its cognitive effects (for review, see Gritton and 1B). When these same helper viruses were used in the BF et al., 2016; Mun˜ oz and Rudy, 2014). However, the mechanism of the same line of rats, expression of helper viral product only oc- behind this control is unknown. cured in cells immunopositive for ChAT (Chavez and Zaborszky,

Cell Reports 18, 1817–1830, February 14, 2017 ª 2017 The Author(s). 1817 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Figure 1. Monosynaptic Viral Tracing (A) Cre-dependent helper viruses limit starter cells to the cholinergic cell type. Virus placed at cortical terminals retrogradely labels a subset of cholinergic cells that project to the cortical injection site (figure based on Wall et al., 2010). (B and C) 12 subjects (B) received helper virus across five basal forebrain sites and (C) received retrograde virus in a single BF projection target, whether in motor cortex (red), medial prefrontal cortex (green), (blue), or amygdala (yellow) (figure based on Paxinos and Watson, 2007). (D) Cells are visualized by the presence of mCherry (helper virus in cholinergic cells), GFP (afferent cells, monosynaptic inputs to starter cells), or both fluorophores (cholinergic starter cells).

2016). Tracing restricted to ChAT terminals was achieved by sub- sively, resulting in many examples of ChAT populations that proj- sequently injecting a pseudotyped, helper-dependent, replica- ect to different targets yet partially overlap in the BFc space (for tion-defective rabies vector (EnvA-DG-rabies-eGFP) (Wall et al., ref see Zaborszky et al., 2015a). The BF topography of starter 2010; Wickersham et al., 2007) into a single target area (prefrontal, cells in the current study confirms this rule of partial overlap (Fig- orbitofrontal, motor cortex, or amygdala) in each subject (n = 12; ure 2; Table S1; Movie S1). When the monosynaptic inputs to the three subjects per group; Figures 1A and 1C). Complementation starter cells are considered across the three cortical target of the deficient rabies with the rabies glycoprotein occurred in groups and the (a cortex-like structure), BF ChAT cells (starter cells) projecting to the cortical area of rabies we find specific sets of inputs differ significantly between the injection. This allowed for retrograde monosynaptic spread of four groups depending on the viral-injected cholinergic projec- GFP-tagged rabies from these starter cells back to the cell bodies tion target (Wilks’ Lambda one-way multivariate analysis of vari- of their synaptic inputs (Figures 1A and 1D), whereas in the control ance [MANOVA]; F = 21.21, p = 0.008) (Figure S1). To make condition, ChAT::Cre with rabies injection but no helper viruses, sense of this result, we took a detailed look at differential distri- no GFP was detected. Therefore, we could map the inputs to spe- butions of afferents in each input region. cific output populations, regardless of how output somata are distributed across the BF itself. Dorsal Striatal and Accumbens Input Disparity Dorsal is on average the largest input source for RESULTS cholinergic BF cells. The proportion of these inputs is far less in the case of medial prefrontal cortex (mPFC) injection The topography of basal forebrain cholinergic (BFc) cell bodies (Figure 3), with an average of 5% of inputs for mPFC cases projecting to particular cortical targets has been studied exten- versus 47% for the motor cortex injection group (p = 0.002;

1818 Cell Reports 18, 1817–1830, February 14, 2017 lateral shell. Taken together, this demonstrates that different basalocortical cholinergic targets receive distinct inputs from dorsal and ventral striatum.

Cortical Feedback to Cholinergic Neurons Direct cortical input to BF ChAT cells was found in a variety of cortical locations in all subjects, arising mostly from deep cortical layers, with occasional labeling in layers II–III. An average across all subjects shows 9% of inputs to cholinergic cells came from cortex. This number drops to just 1.5% from the mPFC in particular, with most cells found in layers 5 and 6. Although mPFC control of BF cholinergic cells has been suggested else- where (Golmayo et al., 2003; Rasmusson et al., 2007), the pre- sent results suggest a role for other cortical areas as well. For instance, the somatosensory cortex regulates cholinergic motor efflux, since in the motor cortex injected cases, somatosensory cortices S1 and S2 supply an average of 2.3% of inputs, versus 0.5% for subjects with rabies injection elsewhere. The inputs from cortex are spatially sparse, a pattern also seen when cholinergic afferents are mapped without regard for output specificity (Do et al., 2016; Hu et al., 2016). Thus, for simplicity, we categorized all cortical areas into either isocortex, mesocor- tex, or allocortex according to McGeorge and Faull (1989) (see classification scheme in Table S3). Although low cortical cell numbers increase within-group variance and prohibit highly spe- cific conclusions, it seems each group of target-identified cholin- ergic cells receives a specific combination of cortical input: as alluded above, the S1-S2 contribution of the total cortical input in case of motor cortex group is 24%–36% (1.6%–2.7% of all in- puts), practically zero in the mPFC group and similarly strong in VO/LO cases (Figures S2 and S7). Also note the allocortical contribution is about 10% of the total input in the mPFC and Figure 2. Topography of Starter Cells less than 1% in the primary/secondary motor cortex (M1/M2) Cholinergic cells capable of spreading virus monosynaptically (mCherry+/ cases. From the allocortical areas, the most numerous cells GFP+) in 12 subjects following retrograde viral injection in M1/M2 (red), mPFC were found in the piriform cortex-endopiriform . In the (green), VO/LO (blue), or amygdala (yellow). Starter cells from individual cases hippocampal complex, a few input cells were found in the stra- (labeled with differently shaped symbols) were warped into a common tem- tum oriens of CA1 and CA3 and the in cases with plate brain. ac, ; BLA, anterior basolateral amygdala; f, ; GP, ; HDB, horizontal diagonal band; ic, internal mPFC or basomedial (BMP) amygdala injections. Occasional la- capsule; lo, lateral ; LV, lateral ventricle; mt, mammillothalamic beling was found in the entorhinal cortex as well. Images of tract; MS/VDB, medial septum/ vertical diagonal band; opt, optic tract; sm, cortical cells appear in Figure 5. stria medullaris; SI/EA, / extended amygdala. Amygdala Feedback to Cholinergic Neurons We found the amygdala holds 6% of all inputs to cholinergic cells independent-samples t test). There is a topography of inputs across all subjects (imaged cells in Figure 6). Notably, this figure from the caudate-putamen (CPu) depending on cholinergic increases to 12% when only amygdala-injected subjects are target site; ventral orbital and lateral orbital cortex (VO/LO)-pro- considered (including two subjects with a majority of rabies in jecting cholinergic cells receive CPu inputs from a medial area the anterior basolateral amygdala [BLA] and a third in the poste- rostrally and more diffusely caudally, while motor cortex-projec- rior basomedial amygdala [BMP]). Although the medial nucleus ting cholinergic cells receive input mainly from ventral CPu, and and the anterior amygdaloid area contained a few input cells, amygdalopetal cholinergic cells receive a majority of input from the largest input source was seen in all compartments of the cen- caudal CPu (Figure 4). tral nucleus, a structure known to have GABAergic outputs (Fig- Inputs to ChAT cells also arise from the ventral striatum (Za´ - ure 3). These data demonstrate that the amygdala influences its borszky and Cullinan, 1992) and occupy a differing topography own cholinergic innervation monosynaptically via a central nu- depending on the target of cholinergic starter cells (Figure S3). cleus feedback. Afferents from the medial division of the central Amygdalopetal ChAT cells receive more inputs from medial nu- nucleus, excited during expression, are likely suppressing cleus accumbens core/shell, while motor-projecting ChAT cells ACh efflux in BLA under conditions of learned fear expression, receive more from lateral core/shell, and VO/LO and mPFC-tar- whereas interpreting the influence of lateral central amygdala geting cholinergic cells receive only scattered cells from the (CeL) input cells will require further study, as CeL contains

Cell Reports 18, 1817–1830, February 14, 2017 1819 Figure 3. Distribution of Inputs across All Brain Regions Percentage of labeled inputs to cholinergic starter cells across all brain regions is shown per subject (individual bars clustered in groups of three). Regions of sparse input (where no injection group averaged >0.5%) are not shown. See also Figures S3 and S4 and Table S2. M1/M2, motor cortex; mPFC, medial prefrontal cortex; VO/LO, ventral/lateral orbitofrontal cortices. both fear-excited and fear-inhibited cells (Ciocchi et al., 2010). 2002; Unal et al., 2015), cholinergic input to the BLA may facili- As the action of ACh in BLA is dependent on both motivational tate memory formation by biasing the synaptic competition in state and state of the BLA principal cells (Power and McGaugh, favor of the strongly activated neurons (Jiang et al., 2016).

1820 Cell Reports 18, 1817–1830, February 14, 2017 Figure 4. Distribution of Striatal Input Cells There is a topography of afferents originating across different levels (rows) in the CPu when comparing three subjects with different rabies injection sites: the amygdala (left column), motor cortex (middle), or ventral orbitofrontal cortex (right column). Various contours delineate cytoarchitectonic areas where cells appear. BFc, basal forebrain cholinergic cells; BMP, posterior basomedial amygdala; M1/M2, primary/secondary motor cortex; VO, ventral orbitofrontal cortex.

Hypothalamus, , and Brainstem (LPO-PLH), but numerous input cells were also found in Several regions within thalamus, , and brainstem the supramammillary nuclei, ventrolateral preoptic nucleus can be viewed as a common source of inputs to cholinergic (VLPO), supraoptic nucleus, and occasionally in the paraven- cells, with apparent disregard to the cholinergic output target. tricular, ventromedial, ventrolateral, and suprachiasmatic nuclei Some notable exceptions to this, where apparent topography (Figure S2). The LPO-PLH area, especially around the fornix, exists between groups, are also described here. Hypothalamic contains orexin and MCH cells that oppositely regulate the inputs make up 2%–11% of all inputs to cholinergic cells (Table sleep-wake cycle (Luppi et al., 2013). Although orexin S2; Table S3). A substantial proportion of hypothalamic cells in the septum synapse with cholinergic neurons and depolarize were found in the lateral preoptic-hypothalamic continuum them (Wu et al., 2004), GFP+ input cells in the perifornical

Cell Reports 18, 1817–1830, February 14, 2017 1821 Figure 5. Input Cells from Cortex In each pair of panels, GFP appears on DAPI (blue, left) and thionin Nissl (violet, right). (A) S1 cells synapsing onto M1/M2-targeted ChAT. (B) Labeled cell in the medial entorhinal cortex (mEC) synapsing onto mPFC-targeted ChAT. (C) Endopiriform cell synapsing onto mPFC-targeted ChAT. (D) Retrosplenial cell synapsing onto mPFC-targeted ChAT. (E, F, and I) CA1 cells of differing morphologies synapsing onto mPFC-targeted ChAT. (G) VO cell synapsing onto VO-targeted ChAT. (H) CA3 cell synapsing onto mPFC-targeted ChAT. Scale bars, 100 mM. cc, corpus callosum; cg, cingulum; CPu, caudate putamen; DG, ; En, endopiriform nucleus; fi, fimbria; or, oriens; PaS, par- asubiculum; Pir, piriform cortex; rad, radiatum; S1, primary somatosensory cortex; VO, ventral orbitofrontal cortex. regions were not colocalized with orexin immunoreactivity labeled cells and axons in the supraoptic nucleus (Figures following mPFC rabies injection, as assessed in the subject S2C and S2C0) in several cases, including prelimbic, BLA and with the largest number of hypothalamic input cells. We also VO/Cl rabies injections. While a supraopic-cholinergic projec- checked for the TH immunoreactivity, abundant in many hypo- tion has not been described before, axons emanating thalamic areas (see Chan-Palay et al., 1984) but found no from the supraoptic nucleus and terminating in CeL has colocalized GFP cells. Interestingly, we found large number of been suggested to be involved in attenuating fear response

1822 Cell Reports 18, 1817–1830, February 14, 2017 Figure 6. Input Cells from Amygdala In each pair of panels, GFP appears on DAPI (blue, left) and thionin Nissl (violet, right). (A) AA cells synapsing onto mPFC-targeted ChAT. (B) BMP cells synapsing onto mPFC-targeted ChAT. (C) CeC cells synapsing onto BMP-targeted ChAT. (D) ACo cells synapsing onto mPFC-targeted ChAT. (E) CeL cells synapsing onto M1/M2-targeted ChAT. (F) AHiPM cell synapsing onto M1/M2-targeted ChAT. Scale bars, 100 mM. AA, anterior amygdaloid area; ACo, anterior cortical amygdaloid nucleus; AHiPM, posteromedial amygdalohippocampal area; BLA, anterior basolateral amygdala; CA3, hippocampal field CA3; Ce, central nucleus, amygdala; CeC, central nucleus, amygdala, capsular part; CeL, central nucleus, amygdala, lateral part; cp, cerebral peduncle; CPu, caudate putamen; cst, commissural ; En, endopiriform nucleus; I, intercalated nucleus, amygdala; La, lateral amygdala; LOT, nucleus of the lateral olfactory tract; LV, lateral ventricle; opt, optic tract.

(Knobloch et al., 2012). Knowing the massive input from the The thalamic input to target-identified cholinergic cells is a central amygdala to cholinergic projection neurons (above), relatively minor component (1.3%–8.6%) of all afferents. Notable the supraoptic-cholinergic link may be involved in the same nuclei that contain input cells include the parafascicular (PF), behavioral response. mediodorsal, ventromedial, and paraventricular. Interestingly,

Cell Reports 18, 1817–1830, February 14, 2017 1823 Figure 7. Input Cells from Brainstem (A) GFP on DAPI background shows inputs in RIP, synapsing onto mPFC-targeted ChAT cells. (A0) The same cells from (A) on thionin Nissl (violet). (B) GFP on DAPI background shows inputs in dorsal raphe, synapsing onto M1/M2-targeted ChAT cells. (B0) The same cells from (B) on thionin Nissl (violet). (C and D) GFP inputs in VTA on Nissl (violet) background synapsing onto mPFC-targeted (C) or M1/M2-targeted ChAT cells (D), respectively. (E) GFP inputs in nucleus Darkschewitsch (Dk) projecting to mPFC-targeted ChAT cells. (E0) The same cells from (E) on thionin Nissl (violet). (F) Macro view of GFP inputs in PAG and PPT synapsing onto M1/M2-targeted ChAT cells. Scale bars, 200 mM. 3N, oculomotor nucleus; 7, facial nucleus; g7, facial nerve; Gi, gigantocellular reticular nucleus; IP, interpeduncular nucleus; isRt, isthmic ; MB, mammil- lary body; MG, medial geniculate nucleus; ml, medial lemniscus; mRt, mesencephalic reticular formation; PAG, periaqueductal gray; Po, poste- rior thalamic nuclei; PPT, pedunculopontine ; PT, pretectal nucleus; RIP, raphe in- terpositus; SNC, ; SNR, substantia nigra pars reticulata; VTA, .

located that receive STN input and project to the motor or VO/LO cortices, it is likely that these cholinergic cells are distributed in SI/EA/GP/ic regions, that contain the majority of starter cholinergic projection neurons in these cases (Table S1). Thus, our results are compatible with recent electrophysiological data (Sa- unders et al., 2015), indicating that cholin- ergic cells in and around the globus pallidus are functionally integrated into almost all cases display input cells in a narrow triangular area, circuitry. It remains for future studies to inquire medial from medial geniculate body, occupying the SG (supra- whether this subthalamic input is specific for motor-projecting geniculate), MGM (medial geniculate body, medial), the posterior ChAT cells around the GP or within the horizontal diagonal (Po), posterior triangular (PoT), posterior intralaminar (PIL), and band (HDB) as well. subparafascicular parvicellular (SPFPC) nuclei (Figures 7C and The average proportion of brainstem input to cholinergic 7D). Cells in this region are important for auditory plasticity (Dis- starter cells varies between 2% (amygdaloid projecting) and terhoft and Stuart, 1976; Edeline, 1990; Gabriel et al., 1975; Len- 11% (VO/LO innervating) of all labeled input. Substantial compo- nartz and Weinberger, 1992; McEchron et al., 1995; O’Connor nents of these arise from the ventral tegmental area (VTA), sub- et al., 1997; Ryugo and Weinberger, 1978) and most likely, in stantia nigra, , reticular formation, periaqueductal part, corresponds to the area of the peripeduncular nucleus in gray (PAG), and parabrachial nucleus (Figures 7 and S5). Inter- primates from which a heavy projection was described to the estingly, following motor and orbitofrontal rabies injections, a ‘‘basal nucleus of Meynert-substantia innominata’’ (NBM) com- consistent labeling of input cells were observed in the Edinger- plex (Jones et al., 1976). A topography exists both in the PF Westphal (EW) nucleus, the site of origin of parasympathetic and the posterior intralaminar complex such that its medial fibers to the inner ocular muscles, as well as in the interstitial portion is labeled in amygdaloid- and mPFC-injection cases, nucleus of Cajal (InC) and Darkschewitsch (Dk) nuclei, that while its lateral portion contains input cells in motor cases. are associational oculomotor centers. Labeled brainstem cells In most cases, the subthalamic (STN) nucleus, part of the were often distributed diffusely, lacking a clear overall topog- basal ganglia circuitry, contained labeled cells with heaviest la- raphy between groups. However, differences can be seen beling found for motor and VO/LO injection cases. Although when isolating particular nuclei. In the reticular formation from our material it is unclear where the cholinergic cells are mPFC- and VO/LO-projecting cholinergic neurons receive

1824 Cell Reports 18, 1817–1830, February 14, 2017 brainstem input from more medially located areas than do M1/ likely receive local inputs primarily from neighboring rather than M2-projecting cholinergic neurons. Another example is the dis- remote regions of the BF itself. Local neurons contacting cholin- tribution of input cells in the ventral midbrain: mPFC-projecting ergic starter neurons may contain Y (NPY), as such cholinergic cells receive input more from the VTA, while motor neurons have been shown to synapse with cholinergic neurons cortex-projecting cholinergic neurons receive from the substan- using electron microscopic (EM) (Zaborszky and Duque, 2000) tia nigra (Figures 7C and 7D). Additionally, amygdala-projecting and optogenetic strategies (Nelson and Mooney, 2016). Local cholinergic neurons receive input from more ventral brainstem BF afferents may also contain GABA and/or glutamate. Local in- areas than do prefrontal- or motor-projecting ChAT neurons. puts to cholinergic outputs both drive and inhibit ChAT cells, de- The mix of discrete and common sources of input from thalamus, pending on afferent cell identity (Xu et al., 2015). Recent awake hypothalamus and brainstem signals that in future studies, nuclei behaving physiological data indicate heterogeneous response from these regions should be considered as independent actors patterns among BF units becoming activated and inhibited to on the cholinergic system rather than one homogeneous block different phases of a directed attention task (Tingley et al., per region. 2014), patterns that may be carried by local subgroups of BF The VTA (A10 cell group), substantia nigra (A9; SN), and input impinging on different cholinergic output targets. Further retrorubral field (A8) all contain dopaminergic, GABAergic and studies into the role of local afferents in contributing to specific (Vglut2-containing) neurons displaying a distinct behaviors are warranted. subregional distribution pattern. In addition, these transmitter- specific neurons contain in various degrees (DAT) DISCUSSION and vesicular monoamine (VMAT2) transporters and D2 recep- tors, thus individual subpopulations of neurons are likely to be Synaptic inputs to BF cholinergic cells used to be studied one differentially involved in drug and various motivational input source at a time with EM (for reference, see Zaborszky and cognitive functions (Faget et al., 2016; Hur and Zaborszky, et al., 2015b), yet the output targets of the cholinergic neurons 2005; Morales and Root, 2014; Nair-Roberts et al., 2008; Tritsch in these studies were rarely investigated, which is important et al., 2012). Cholinergic neurons in the BF receive synapses since heterogeneous cholinergic neurons from any one subre- from the VTA and substantia nigra (Gaykema and Zaborszky, gion in the BF collectively project to wide cortical areas. Mono- 1997; Zaborszky and Cullinan, 1996). Here we found occasional synaptic viral tracing of neuromodulatory systems has been TH-containing input cells in the VTA and retrorubral field, used in the noradrenergic, dopaminergic and serotonergic sys- although a majority of input cells in this region were TH-negative tems (Beier et al., 2015; Faget et al., 2016; Lerner et al., 2015; (Figure S6). Glutamatergic (Hur and Zaborszky, SFN Abstract, Ogawa et al., 2014; Pollak Dorocic et al., 2014; Schwarz et al., 2007) and GABAergic (Gaykema and Zaborszky, 1997) projec- 2015; Watabe-Uchida et al., 2012; Weissbourd et al., 2014). tions from the VTA to BF areas rich in cholinergic cells have BFc monosynaptic tracing was also reported (Do et al., 2016; been described, although it is not known whether these inputs Hu et al., 2016) though without reference to target specificity. target cholinergic neurons. Due to the divergent molecular Here, we sample monosynaptic inputs from across the brain to composition of SN and VTA neurons and their role in various gain insight into the networks able to control cholinergic innerva- functions and diseases, further investigation into the behavioral tion of different cortical regions and the amygdala. Several motifs role of these inputs to BFc neurons is warranted. are described such as common thalamic, hypothalamic, and input to the BF apparently synapses only on parval- brainstem input to the four cholinergic projection populations bumin-containing neurons (Leranth and Vertes, 1999), while examined, as well as inputs distinct to particular projection pop- cholinergic neurons do not seem to receive direct input from ulations, such as strong somatosensory cortical input to motor 5HT axons (Hajszan and Zaborszky, 2000). cortex-projecting cells, versus strong piriform input to mPFC- Although a few input cells in the mesopontine tegmentum projecting cells. Similarly, VTA input is biased toward mPFC- were detected in mPFC and amygdala (BMP) rabies injection innervating cholinergic cells and SN to motor cortex-innervating cases, we found that these cells were not colabeled following cholinergic neurons. Several follow-up circuit-level experiments ChAT immunostaining, and thus we have no evidence to demon- can be designed based on this database to identify drivers of strate a cholinergic brainstem synapse onto cholinergic BF cells. known cholinergic-dependent cortical functions. Additionally, projections from the parabrachial nucleus may contain glutamate (Hur and Zaborszky, SFN Abstract, 2007) Limitations and could participate in the hypercapnia-induced effect In pilot studies, we attempted to image injection sites by mixing (Kaur et al., 2013). rabies with various labeling agents, from India ink to Dye I. Unfor- tunately, all these attempts resulted in no labeling in the brain Local Inputs to Cholinergic Neurons whatsoever. Therefore, we identified the injection site by finding Local afferents to ChAT starter cells are found across the BF the pipette tract and mapping the region of gliosis and other cholinergic volume, often appearing in the vicinity of starter cells debris created by the rabies injection itself. Following this (Figure 1D; Table S3). Across all cytoarchitectonic BFc subre- mapping, Nissl staining of the same section revealed the cy- gions in all subjects, the number of labeled afferents within a toarchitectonic region of injection. Therefore, although the pre- BFc subdivision correlates significantly with the number of cise location of the center of each injection site is known, the starter cells within the same structure (Spearman’s Rho = 0.77; spread (of approximately 100–200 nL of virus) is unknown. Due p < 0.001), suggesting that cholinergic corticopetal neurons to the large size of the rabies virus particles and small volume

Cell Reports 18, 1817–1830, February 14, 2017 1825 injected at each position, we feel the size of the spread is not S2). For example, in the rostral forebrain at the level of the likely to stray outside of the cytoarchitectonic areas reported. septum, the amygdala-targeted input cells occupy a middle To this point, we have acted conservatively, since in cases where space between the mPFC-targeted input cells within the septum the injection site encroached upon a bordering area (e.g., claus- and more laterally located motor-targeted input cells occupying trum or central amygdala), we included this bordering area in much of the striatum (see Figure S3). While the distribution of the description of the injection site to err on the side of caution. these three networks is visible rostral to the crossing of the ante- Injection sites in cortex and amygdala are centered on slightly rior commissure, with the appearance of the amygdaloid body different cytoarchitectonic zones when comparing subjects caudally, there is a lateralward shift of the input cells of the amyg- within group, such that different injections targeting the same dala as more of such cells become visible in the central amyg- gross region may receive cholinergic innervation from differing dala and the dorsal striatum. Eventually, the motor-targeted BF neurons. Although each subject therefore can be viewed as input cells in the striatum disappear and between 3.0 and a complete and independent dataset, the grouping of subjects 3.4 mm caudal to bregma, the dorsal striatum is almost exclu- allows the results to be interpreted in a coherent manner, sively occupied by amygdala-targeted input cells (Figure S4). comparing four groups instead of twelve individuals. While Figure S3 clearly demonstrates segregated amygdala- Limitations of this technique are known (Callaway and Luo, and motor-targeted input cells in the , there 2015). Additionally, since only one in every four sections is also a substantial overlap of amygdala-, mPFC- and motor-tar- throughout the brain was mapped and analyzed, each distribu- geted input cells in the dorsal striatum, interstitial nucleus of the tion of input cells should be viewed as a sample of a sample anterior commissure (IPAC), LPO-PLH, and the PF of the thal- for this viral tracing, as previously suggested (Ginger et al., amus, suggesting these latter areas may play more of a role in 2013). In spite of these various methodological limitations, we modulation of global cholinergic tone. still see some within-group similarities and notable significant Septal areas (excluding medial septum) prominently contact between-group difference in inputs collecting three rats per cholinergic cells that project to mPFC, representing on average group for the four regions studied. Since most of the forebrain af- 18% of all inputs, yet are comparatively minor inputs (1%) for ferents, including cortical, amygdaloid and striatal axons appear other output groups (p < 0.0001; independent-samples t test) to have a preferential distribution across cytoarchitectonically (Figure S1). The main component of these septal inputs arises defined BF regions (Zaborszky et al., 2015b), additional speci- from the dorsal lateral septum, a small area that on its own com- ficity might be garnered if helper viruses were introduced prises an average of 7% of all input for mPFC cases. The primate into specific cytoarchitectonic BF subregions, followed by the homolog of this area, the anterodorsal septum is known to code same cortical rabies injections. for reward uncertainty (Monosov and Hikosaka, 2013), a behav- Another potential caveat arises when we consider the possibil- ioral measure disrupted in rats following immunolesion of BFc ity of cholinergic-cholinergic synapses spreading virus locally. neurons (Cordova and Chiba, 2004, SFN Abstract). Furthermore, This does not appear to be the case, given that the BF location reward uncertainty information is carried by basal forebrain units of the majority of starter cells in each case is restricted only to (Ledbetter et al., 2016). This raises the possibility that reward BF subregions known to house cholinergic projection neurons uncertainty is updated in the mPFC with ACh, as driven mono- innervating the cortical region in question. In other words, the synaptically by the likely GABAergic (Hur and Zaborszky, SFN pattern of starter cells across the BF matches the pattern occur- Abstract, 2007) dorsal lateral (anterodorsal) septum. ring following classical retrograde tracer injection in the same cortical area. If, on the other hand, we were to find starter cells Cellular and Circuit Mechanisms of Cortical State in large numbers all across the BF, occurring in areas not known Control to project to the cortical region in question, this would have Classically, three cortical states are differentiated, including caused us concern in our interpretation of the results. However, wake, SWS and REM sleep (for reference, see Brown et al., this was not the case. If we suppose that it is nevertheless 2012; McCormick et al., 2015). However, recent studies suggest possible that spread occurred between connected cholinergic a more complex scenario that is characterized by a continuum cells, and that inputs of these secondarily infected cells were of states rather than sharp transitions and, both in SWS and labeled, the resulting mono- and di-synaptic labeled population waking, finer distinctions can be made in terms of internal still comprises a circuit eventually impinging on the rabies-in- cortical dynamics and responsiveness to external stimuli (Harris jected cortical area. and Thiele, 2011; McGinley et al., 2015; Vyazovskiy et al., 2011). The mechanism of how cortical arousal, movement-related ac- Specific Cholinergic Input-Output Streams tivity (Harrison et al., 2016), and pupil micro-dilations (McGinley Our study demonstrates that although each cortical and amyg- et al., 2015) are linked remains unexplained. daloid area receives the majority of cholinergic input from cell Input cell labeling in the EW, Dk, and InC nuclei in the upper bodies located in partially overlapping areas of the BF (Figure 2), midbrain tegmentum of some of the motor, VO/LO, and PrL/IL each of these target-identified groups of cholinergic output neu- rabies injection cases suggests that BFc neurons receive rons is innervated by a partially unique set of afferents (Figure 3). information about pupil diameter and reflex gaze coordination In addition to numerical differences of input cells in these net- and can broadcast these signals to frontal cortex, potentially works (Table S3), convolved maps of inputs to output-identified to modulate attention. On another level, input cells to BFc neu- cholinergic neurons reveal a grossly segregated topography rons, in the pedunculopontine tegmental (PPT, see Figure 7), supporting the partial separability of these networks (Movie cuneiform, and parabrachial nuclei (see Table S3), largely

1826 Cell Reports 18, 1817–1830, February 14, 2017 corresponding to the mesencephalic locomotor region, are good end, we suggest two, not mutually exclusive principles: (1) candidates to convey fast movement-related information that segregated inputs to the BFc convey specific cognitive opera- accompany cortical desynchronization and arousal (Bennett tions, and overlapping, common inputs mediate state-related et al., 2014; Kaur et al., 2013; Lee et al., 2014; Nelson and changes; alternatively, (2) the complex set of inputs described Mooney, 2016). The brainstem motor-related inputs together in our study reflect multiple inbuilt templates for flexible control with inputs from the and dorsal raphe neurons, of cortical states in a modality-specific fashion via the tonic known to show state-related activity (Jones, 2005; Luppi et al., and phasic firing of cholinergic neurons (Hangya et al., 2015; 2013), as well as top-down input from M2 cortex to cholinergic Harrison et al., 2016). neurons may be a common mechanism by which sensory cortical neurons, including those in the auditory cortex, receive EXPERIMENTAL PROCEDURES bottom-up and top-down signals (Chavez and Zaborszky, 2016; Nelson and Mooney, 2016). Subjects Animals were treated in accordance with the National Research Council’s ‘‘Guide for the Care and Use of Laboratory Animals.’’ Experiments were per- Putative GABAergic Circuits formed with the approval of the Institutional Animal Care and Use Committee Further extrapolation of our results can be gained when consid- of Rutgers University. ChAT::Cre rats expressing cre under the ChAT promoter ering afferent groups with known transmitter content. The largest (Witten et al., 2011), a gift from Dr. Karl Deisseroth at Stanford University, were input source we found is the dorsal striatum, known to elicit sus- backcrossed with wild-type Long-Evans (Harlan). 13 naive ChAT::Cre adults tained GABAergic output activated by a variety of stimuli and ac- (3+ months of age) were used in virus tracing experiments; 12 were used for tions, including by outcome-predictive cues (Adler et al., 2013; cortically targeted tracing, and one was used as a no-helper-virus control. All were housed in cages of one to three animals on a 12-hr-light/12-hr-dark cycle. Kita and Kitai, 1988). While the accumbens, also GABAergic in its output, is contacting cholinergic outputs across all four Viruses and Surgeries groups in the present study, the relative absence of CPu input Subjects were anesthetized with 1%–4% isoflurane inhalation in O2. 12 rats for mPFC-projecting ChAT cells, compared with the abundance received intracranial injection of 2.64 mL of each helper virus (both from Univer- of this projection in the other three groups (Figure 3), under- sity of North Carolina [UNC] vector core: rAAV5/EF1a-Flex-TVA-mCherry, titer scores the special role ACh plays in the mPFC. We can now 4.3e12 VP/mL; rAAV5/CA-Flex-RG, titer 2e12 VP/mL) (Watabe-Uchida et al., hypothesize that while suppression of cholinergic outputs to 2012), injected via micropipette across five locations filling the right BF (coor- multiple brain areas is likely highly dependent on the CPu, at dinates in mm relative to bregma, from pia, +0.5 AP, 1.05 mediolateral [ML], 6.7 dorsoventral [DV] for males, 6.0 DV females; 0.0 AP, 1.65 ML, 7.1 DV males, 6.5 the same time the dorsal striatum is likely playing little to no DV females; 0.87 AP, 2.3 ML, 5.3 and 7.1 DV males, 5.2 and 6.5 DV role in the inhibition of cholinergic tone in the mPFC. Given that females; 1.72 AP, 3.6 ML, 5.8 DV males, 1.7 AP, 3.4 ML, 5.6 DV females). discrete cholinergic depletion of the PFC disrupts working mem- 21 days later, EnvA-DG-rabies-eGFP (Salk vector core, titer 4.3e8 transducing ory and attentional processes (Croxson et al., 2011; Dalley et al., units [TU]/mL) (Wickersham et al., 2007) was injected ipsilaterally into motor 2004), this putative striatal dichotomy of control over cholinergic cortex (111nL at +1.1 AP, 3.2 ML, 1.0 and 1.3 DV from pia), mPFC (222 nL output modules is a prime candidate of topic for future experi- at +3.15 AP, 0.5 ML, 3.9 and 4.7 DV from skull), VO/LO (111 nL at +3.6 AP, 2.23 ML, 4.7 DV from skull), or amygdala (222 nL at 2.16 AP, 4.756 ML, ments. Another input source known to be GABAergic (Hur and 7.55 DV from pia). Each litter of animals was spread as evenly as possible Zaborszky, SFN Abstract, 2007) arises from the lateral septum, across all injection groups, and both females and males appear in each group. constituting the largest source of afferents outside the BF in One additional control animal (ChAT::Cre+) received rabies injections in all four the mPFC injection cases and containing few to no cells in other of the above cortical/amygdalar loci, without receiving any helper viruses. groups (Figure 3). Further inputs, most likely GABAergic (Nair- Roberts et al., 2008), arise from the VTA and SN pars compacta Tissue Preparation and Imaging contacting cholinergic outputs in all experimental groups. 7 days following rabies injections, animals were transcardially perfused with Other afferents arising from the ventral anterior and ventrolat- 0.1 M PB followed by 4% paraformaldehyde in 0.1 M PB. Brains were kept in this fixative overnight at 4C and then sunk in 30% sucrose in 0.1 M PB. eral thalamic nuclei, the glutamatergic thalamocortical nuclei 50-mm sections were collected via sliding microtome, and every fourth section receiving basal ganglia output, selectively contact cholinergic was mounted onto gelatin-subbed slides. All experimental animals showed outputs to motor cortex. Therefore, different cholinergic output good fluorescence, whereas the single control animal (rabies only; no helper vi- streams may be individually controlled subcortically by differing rus injected) showed no fluorescence anywhere in the brain. Fluorescent images loops of the basal ganglia, including large numbers of different were acquired via light or confocal microscope, and the location of cell bodies GABAergic cells. This hypothesis is in agreement with data was mapped with Neurolucida software (MBF Bioscience). Mapping was per- formed with the experimenter blind to the rabies injection location. Coverslips demonstrating BF cells reliably responding to particular events were then removed, and the same slides were prepared with a thionin Nissl stain. in a behavioral task, while some cells seem to be active during Nissl-stained sections were illuminated with bright-field and aligned to existing the exact same periods when others are silent (Tingley et al., images or to existing Neurolucida maps previously created under fluorescence, 2014). In this way, differing input-output streams of the cholin- and cytoarchitectonic boundaries were created in Neurolucida, allowing for pre- ergic BF may comprise modules important for differing behav- cise identification of the regions where fluorescent cells were mapped. The raw ioral demands. cell counts per cytoarchitectonic area appear in Table S3. For the purpose of comparing cell distributions across subjects, maps of individual subjects were warped into a common volume using Neurolucida software. Conclusions Future optogenetic studies utilizing viral tracing and genetic Data Availability methods are necessary to uncover the fine functional details Raw cell count data appear in Table S3. Any other relevant data are available that this BF input-output organization subtends. Toward this from the authors.

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