GABA(A) receptors containing the α2 subunit are critical for direction-selective inhibition in the retina

Far from being a simple sensor, the retina actively participates in processing visual signals. One of the best understood aspects of this processing is the detection of motion direction. Direction-selective (DS) retinal circuits include several subtypes of ganglion cells (GCs) and inhibitory interneurons, such as starburst amacrine cells (SACs). Recent studies demonstrated a surprising complexity in the arrangement of synapses in the DS circuit, i.e. between SACs and DS ganglion cells. Thus, to fully understand retinal DS mechanisms, detailed knowledge of all synaptic elements involved, particularly the nature and localization of neurotransmitter receptors, is needed. Since inhibition from SACs onto DSGCs is crucial for generating retinal direction selectivity, we investigate here the nature of the GABA receptors mediating this interaction. We found that in the inner plexiform layer (IPL) of mouse and rabbit retina, GABA(A) receptor subunit α2 (GABA(A)R α2) aggregated in synaptic clusters along two bands overlapping the dendritic plexuses of both ON and OFF SACs. On distal dendrites of individually labeled SACs in rabbit, GABA(A)R α2 was aligned with the majority of varicosities, the cell's output structures, and found postsynaptically on DSGC dendrites, both in the ON and OFF portion of the IPL. In GABA(A)R α2 knock-out (KO) mice, light responses of retinal GCs recorded with two-photon calcium imaging revealed a significant impairment of DS responses compared to their wild-type littermates. We observed a dramatic drop in the proportion of cells exhibiting DS phenotype in both the ON and ON-OFF populations, which strongly supports our anatomical findings that α2-containing GABA(A)Rs are critical for mediating retinal DS inhibition. Our study reveals for the first time, to the best of our knowledge, the precise functional localization of a specific receptor subunit in the retinal DS circuit.     

The oscillation-like activity in bullfrog ON–OFF retinal ganglion cell

Oscillatory activity of retinal ganglion cell (RGC) has been observed in various species. It was reported such oscillatory activity is raised within large neural network and involved in retinal information coding. In the present research, we found an oscillation-like activity in ON–OFF RGC of bullfrog retina, and studied the mechanisms underlying the ON and OFF activities respectively. Pharmacological experiments revealed that the oscillation-like activity patterns in both ON and OFF pathways were abolished by GABA receptor antagonists, indicating GABAergic inhibition is essential for generating them. At the meantime, such activities in the ON and OFF pathways showed different responses to several other applied drugs. The oscillation-like pattern in the OFF pathway was abolished by glycine receptor antagonist or gap junction blocker, whereas that in the ON pathway was not affected. Furthermore, the blockade of the ON pathway by metabotropic glutamate receptor agonist led to suppression of the oscillation-like pattern in the OFF pathway. These results suggest that the ON pathway has modulatory effect on the oscillation-like activity in the OFF pathway. Therefore, the mechanisms underlying the oscillation-like activities in the ON and OFF pathways are different: the oscillation-like activity in the ON pathway is likely caused by GABAergic amacrine cell network, while that in the OFF pathway needs the contributions of GABAergic and glycinergic amacrine cell network, as well as gap junction connections.     

Two-photon imaging reveals somatodendritic chloride gradient in retinal ON-type bipolar cells expressing the biosensor Clomeleon

A somatodendritic gradient of Cl(-) concentration ([Cl(-)](i)) has been postulated to generate GABA-evoked responses of different polarity in retinal bipolar cells, hyperpolarizing in OFF cells with low dendritic [Cl(-)](i), and depolarizing in ON cells with high dendritic [Cl(-)](i). As glutamate released by the photoreceptors depolarizes OFF cells and hyperpolarizes ON cells, the bipolars' antagonistic receptive field (RF) could be computed by simply integrating glutamatergic inputs from the RF center and GABAergic inputs from horizontal cells in the RF surround. Using ratiometric two-photon imaging of Clomeleon, a Cl(-) indicator transgenically expressed in ON bipolar cells, we found that dendritic [Cl(-)](i) exceeds somatic [Cl(-)](i) by up to 20 mM and that GABA application can lead to Cl(-) efflux (depolarization) in these dendrites. Blockers of Cl(-) transporters reduced the somatodendritic [Cl(-)](i) gradient. Hence, our results support the idea that ON bipolar cells employ a somatodendritic [Cl(-)](i) gradient to invert GABAergic horizontal cell input.     

Disynaptic inhibition between neocortical pyramidal cells mediated by Martinotti cells

Reliable activation of inhibitory pathways is essential for maintaining the balance between excitation and inhibition during cortical activity. Little is known, however, about the activation of these pathways at the level of the local neocortical microcircuit. We report a disynaptic inhibitory pathway among neocortical pyramidal cells (PCs). Inhibitory responses were evoked in layer 5 PCs following stimulation of individual neighboring PCs with trains of action potentials. The probability for inhibition between PCs was more than twice that of direct excitation, and inhibitory responses increased as a function of rate and duration of presynaptic discharge. Simultaneous somatic and dendritic recordings indicated that inhibition originated from PC apical and tuft dendrites. Multineuron whole-cell recordings from PCs and interneurons combined with morphological reconstructions revealed the mediating interneurons as Martinotti cells. Martinotti cells received facilitating synapses from PCs and formed reliable inhibitory synapses onto dendrites of neighboring PCs. We describe this feedback pathway and propose it as a central mechanism for regulation of cortical activity.     

The influences of somatic and dendritic inhibition on bursting patterns in a neuronal circuit model

The balance between inhibition and excitation plays a crucial role in the generation of synchronous bursting activity in neuronal circuits. In human and animal models of epilepsy, changes in both excitatory and inhibitory synaptic inputs are known to occur. Locations and distribution of these excitatory and inhibitory synaptic inputs on pyramidal cells play a role in the integrative properties of neuronal activity, e.g., epileptiform activity. Thus the location and distribution of the inputs onto pyramidal cells are important parameters that influence neuronal activity in epilepsy. However, the location and distribution of inhibitory synapses converging onto pyramidal cells have not been fully studied. The objectives of this study are to investigate the roles of the relative location of inhibitory synapses on the dendritic tree and soma in the generation of bursting activity. We investigate influences of somatic and dendritic inhibition on bursting activity patterns in several paradigms of potential connections using a simplified multicompartmental model. We also investigate the effects of distribution of fast and slow components of GABAergic inhibition in pyramidal cells. Interspike interval (ISI) analysis is used for examination of bursting patterns. Simulations show that the inhibitory interneuron regulates neuronal bursting activity. Bursting behavior patterns depend on the synaptic weight and delay of the inhibitory connection as well as the location of the synapse. When the inhibitory interneuron synapses on the pyramidal neuron, inhibitory action is stronger if the inhibitory synapse is close to the soma. Alterations of synaptic weight of the interneuron can be compensatory for changes in the location of synaptic input. The relative changes in these parameters exert a considerable influence on whether synchronous bursting activity is facilitated or reduced. Additional simulations show that the slow GABAergic inhibitory component is more effective than the fast component in distal dendrites. Taken together, these findings illustrate the potential for GABAergic inhibition in the soma and dendritic tree to play an important modulatory role in bursting activity patterns.     

Crossover inhibition in the retina: circuitry that compensates for nonlinear rectifying synaptic transmission

In the mammalian retina, complementary ON and OFF visual streams are formed at the bipolar cell dendrites, then carried to amacrine and ganglion cells via nonlinear excitatory synapses from bipolar cells. Bipolar, amacrine and ganglion cells also receive a nonlinear inhibitory input from amacrine cells. The most common form of such inhibition crosses over from the opposite visual stream: Amacrine cells carry ON inhibition to the OFF cells and carry OFF inhibition to the ON cells (”crossover inhibition”). Although these synapses are predominantly nonlinear, linear signal processing is required for computing many properties of the visual world such as average intensity across a receptive field. Linear signaling is also necessary for maintaining the distinction between brightness and contrast. It has long been known that a subset of retinal outputs provide exactly this sort of linear representation of the world; we show here that rectifying (nonlinear) synaptic currents, when combined thorough crossover inhibition can generate this linear signaling. Using simple mathematical models we show that for a large set of cases, repeated rounds of synaptic rectification without crossover inhibition can destroy information carried by those synapses. A similar circuit motif is employed in the electronics industry to compensate for transistor nonlinearities in analog circuits.     

Cell-type specific dendritic contacts between retinal ganglion cells during development.

The extent of a neuron's dendritic field defines the region within which information is processed. The dendritic fields of functionally distinct ON and OFF center retinal ganglion cells (RGCs) form separate mosaics across the retina. Within each mosaic, neighboring dendritic fields overlap by a constant amount, sampling the visual field with the appropriate coverage. Contact-mediated lateral inhibition between neighboring RGCs has long been thought to regulate both the extent and overlap of dendritic fields during development. Here we show that dendro-dendritic contact exists between developing RGCs and occurs in a manner that would regulate the formation of ON and OFF mosaics separately. Dye-filled neighboring ON and OFF ferret alpha RGCs were reconstructed using multiphoton microscopy. At all neonatal ages examined, we observed dendro-dendritic contacts between RGCs of the same sign (ON/ON; OFF/OFF), but never between cells of opposite signs (ON/OFF). Terminal dendrites of one cell often touched a dendrite of its neighbor as they intersected. In some instances, the distal dendrite of one cell formed a fascicle with the proximal process of its neighbor. Alpha cells did not form contacts with neighboring beta cells of the same sign. Together, these observations suggest that dendro-dendritic contact between RGCs is cell-type specific. Dendritic contacts were observed even before the alpha cell arbors were completely stratified, suggesting that cell-cell recognition may take place early in their development. For each cell type, the relative overlap of dendritic fields was constant with age, despite a two-fold increase in field area. We suggest that dendro-dendritic contacts may be sites of intercellular signaling that could regulate local extension of dendrites to maintain the relative overlap of RGCs within a mosaic during development.     

Parallel mechanisms encode direction in the retina

In the retina, presynaptic inhibitory mechanisms that shape directionally selective (DS) responses in output ganglion cells are well established. However, the nature of inhibition-independent forms of directional selectivity remains poorly defined. Here, we describe a genetically specified set of ON-OFF DS ganglion cells (DSGCs) that code anterior motion. This entire population of DSGCs exhibits asymmetric dendritic arborizations that orientate toward the preferred direction. We demonstrate that morphological asymmetries along with nonlinear dendritic conductances generate a centrifugal (soma-to-dendrite) preference that does not critically depend upon, but works in parallel with the GABAergic circuitry. We also show that in symmetrical DSGCs, such dendritic DS mechanisms are aligned with, or are in opposition to, the inhibitory DS circuitry in distinct dendritic subfields where they differentially interact to promote or weaken directional preferences. Thus, pre- and postsynaptic DS mechanisms interact uniquely in distinct ganglion cell populations, enabling efficient DS coding under diverse conditions.     

Axonal Synapses Utilize Multiple Synaptic Ribbons in the Mammalian Retina

In the mammalian retina, bipolar cells and ganglion cells which stratify in sublamina a of the inner plexiform layer (IPL) show OFF responses to light stimuli while those that stratify in sublamina b show ON responses. This functional relationship between anatomy and physiology is a key principle of retinal organization. However, there are at least three types of retinal neurons, including intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells, which violate this principle. These cell types have light-driven ON responses, but their dendrites mainly stratify in sublamina a of the IPL, the OFF sublayer. Recent anatomical studies suggested that certain ON cone bipolar cells make axonal or ectopic synapses as they descend through sublamina a, thus providing ON input to cells which stratify in the OFF sublayer. Using immunoelectron microscopy with 3-dimensional reconstruction, we have identified axonal synapses of ON cone bipolar cells in the rabbit retina. Ten calbindin ON cone bipolar axons made en passant ribbon synapses onto amacrine or ganglion dendrites in sublamina a of the IPL. Compared to the ribbon synapses made by bipolar terminals, these axonal ribbon synapses were characterized by a broad postsynaptic element that appeared as a monad and by the presence of multiple short synaptic ribbons. These findings confirm that certain ON cone bipolar cells can provide ON input to amacrine and ganglion cells whose dendrites stratify in the OFF sublayer via axonal synapses. The monadic synapse with multiple ribbons may be a diagnostic feature of the ON cone bipolar axonal synapse in sublamina a. The presence of multiple ribbons and a broad postsynaptic density suggest these structures may be very efficient synapses. We also identified axonal inputs to ipRGCs with the architecture described above.     

Non-associative Potentiation of Perisomatic Inhibition Alters the Temporal Coding of Neocortical Layer 5 Pyramidal Neurons

In the neocortex, the coexistence of temporally locked excitation and inhibition governs complex network activity underlying cognitive functions, and is believed to be altered in several brain diseases. Here we show that this equilibrium can be unlocked by increased activity of layer 5 pyramidal neurons of the mouse neocortex. Somatic depolarization or short bursts of action potentials of layer 5 pyramidal neurons induced a selective long-term potentiation of GABAergic synapses (LTPi) without affecting glutamatergic inputs. Remarkably, LTPi was selective for perisomatic inhibition from parvalbumin basket cells, leaving dendritic inhibition intact. It relied on retrograde signaling of nitric oxide, which persistently altered presynaptic GABA release and diffused to inhibitory synapses impinging on adjacent pyramidal neurons. LTPi reduced the time window of synaptic summation and increased the temporal precision of spike generation. Thus, increases in single cortical pyramidal neuron activity can induce an interneuron-selective GABAergic plasticity effectively altering the computation of temporally coded information.     

Environmental enrichment effects on development of retinal ganglion cell dendritic stratification require retinal BDNF

A well-known developmental event of retinal maturation is the progressive segregation of retinal ganglion cell (RGC) dendrites into a and b sublaminae of the inner plexiform layer (IPL), a morphological rearrangement crucial for the emergence of the ON and OFF pathways. The factors regulating this process are not known, although electrical activity has been demonstrated to play a role. Here we report that Environmental Enrichment (EE) accelerates the developmental segregation of RGC dendrites and prevents the effects exerted on it by dark rearing (DR). Development of RGC stratification was analyzed in a line of transgenic mice expressing plasma-membrane marker green fluorescent protein (GFP) under the control of Thy-1 promoter; we visualized the a and b sublaminae of the IPL by using an antibody selectively directed against a specific marker of cholinergic neurons. EE precociously increases Brain Derived Neurotrophic Factor (BDNF) in the retina, in parallel with the precocious segregation of RGC dendrites; in addition, EE counteracts retinal BDNF reduction in DR retinas and promotes a normal segregation of RGC dendrites. Blocking retinal BDNF by means of antisense oligos blocks EE effects on the maturation of RGC dendritic stratification. Thus, EE affects the development of RGC dendritic segregation and retinal BDNF is required for this effect to take place, suggesting that BDNF could play an important role in the emergence of the ON and OFF pathways.     

The neuronal basis of a sensory analyser, the acridid movement detector system. II. response decrement, convergence, and the nature of the excitatory afferents to the fan-like dendrites of the LGMD.

No dendritic spikes occur in the input fan of the lobular giant movement detector (LGMD) neurone. The action potentials are initiated at the point of thickening of the axon, which therefore represents the site of convergence of the retinotopic projection in the MD system. Previous work has shown that the site of decrement in response to repetitive visual stimulation is distal to this point. No change in spiking threshold in the LGMD could be demonstrated, and decrement in the number of LGMD action potentials is completely explained by the observed decrement of EPSPs recorded in the LGMD input dendritic fan. Possible postsynaptic mechanisms which might affect EPSP amplitude are excluded experimentally or shown to be improbable. Latency measurements during electrical stimulation in the second chiasma (which produces a decrementing EPSP in the fan) indicate that the pathway from the chiasma afferents to the LGMD fan is probably monosynaptic. By exclusion, the site of decrement appears to be located at the presynaptic terminal of that synapse. Generalization of habituation of the response to ON and OFF stimuli is demonstrated, showing that the presynaptic neurone at the labile synapse is an ON/OFF unit. The greater part of the previously described sensitivity gradient on the retina, relative to the MD response, appears to be explicable by the geometry of the LGMD fan and of the retinotopic projection. We conclude that the LGMD is fed by a homogeneous population of ON/OFF units running in the second optic chiasma, which form labile synapses on the input fan.     

Dendritic ventriloquism: inhibitory synapses throw their voices

In a theoretical study in this issue of Neuron,Gidon and Segev (2012) identify several new principles governing how inhibition interacts with excitation in active dendrites. They show that inhibitory synapses can interact with excitability at a distance, effectively "throwing their voices" in the dendritic tree, such that distributed inhibitory synapses can act synergistically to provide a global veto of dendritic excitability.     

Impaired Excitatory Drive to Spinal Gabaergic Neurons of Neuropathic Mice

Adequate pain sensitivity requires a delicate balance between excitation and inhibition in the dorsal horn of the spinal cord. This balance is severely impaired in neuropathy leading to enhanced pain sensations (hyperalgesia). The underlying mechanisms remain elusive. Here we explored the hypothesis that the excitatory drive to spinal GABAergic neurons might be impaired in neuropathic animals. Transgenic adult mice expressing EGFP under the promoter for GAD67 underwent either chronic constriction injury of the sciatic nerve or sham surgery. In transverse slices from lumbar spinal cord we performed whole-cell patch-clamp recordings from identified GABAergic neurons in lamina II. In neuropathic animals rates of mEPSC were reduced indicating diminished global excitatory input. This downregulation of excitatory drive required a rise in postsynaptic Ca²⁺. Neither the density and morphology of dendritic spines on GABAergic neurons nor the number of excitatory synapses contacting GABAergic neurons were affected by neuropathy. In contrast, paired-pulse ratio of Aδ- or C-fiber-evoked monosynaptic EPSCs following dorsal root stimulation was increased in neuropathic animals suggesting reduced neurotransmitter release from primary afferents. Our data indicate that peripheral neuropathy triggers Ca²⁺-dependent signaling pathways in spinal GABAergic neurons. This leads to a global downregulation of the excitatory drive to GABAergic neurons. The downregulation involves a presynaptic mechanism and also applies to the excitation of GABAergic neurons by presumably nociceptive Aδ- and C-fibers. This then leads to an inadequately low recruitment of inhibitory interneurons during nociception. We suggest that this previously unrecognized mechanism of impaired spinal inhibition contributes to hyperalgesia in neuropathy.     

Balanced GABAergic and glutamatergic synapse development in hippocampal neurons

Coordinated development of excitatory and inhibitory synapses is crucial for normal function of neuronal circuits. Using homo- and heterochronic cultures of hippocampal neurons, we compared the formation of glutamatergic and GABAergic synapses at different stages and asked whether the age of dendrites affects their ability to accept new glutamatergic and GABAergic synapses. Neurons were transfected with either CFP-actin as a dendritic marker or GFP-synaptophysin as a presynaptic marker. We found that GFP-synaptophysin clusters formed on CFP-actin-labeled dendrites at similar density regardless of pre- and postsynaptic cell type or the age of dendrites (0-2 weeks) upon co-culturing. Therefore, the age of mature dendrites does not affect their ability to accept new synapses. Because GABAergic transmission switches from depolarizing to hyperpolarizing during 1-2 weeks in these cultures, our observations also suggest that this developmental switch does not alter the formation of GABAergic synapses.     

A descending contralateral directionally selective movement detector in the praying mantis <Emphasis Type="Italic">Tenodera aridifolia</Emphasis>

Extracellular recordings were made from a directionally selective neuron in the ventral nerve cord of mantises. The neuron’s preferred direction of motion was forward and upward over the compound eye contralateral to its axon at the cervical connective. The neuron was sensitive to wide-field motion stimuli, resistant to habituation, and showed transient excitation in response to light ON and OFF stimuli. Its responses to drifting gratings depended on the temporal frequency and contrast of the stimulus. These results suggest that the neuron receives input from correlation-type motion detectors.     

神经毒素ECMA对家鸽视网膜神经节单元反应特性的影响

从家鸽视差表层总共记录了１０１个视网膜神经节单元,并定量分析研究ＥＣＭＡ损伤对其反应特性的影响,在对照组中,神经节单元都没有自发放电,而需要视觉刺激才能引起反应,对闪光刺激的反应,分别为ＯＮ—ＯＦＦ,ＯＮ,ＯＦＦ三种,其反应均是瞬变的,而且也都对在感受野内运动的小条纹起反应。４２个单元中有１４个是方向选择性单元。其它的则为运动敏感单元。方向选择性单元的无效方向不是均等分布的,其中有８个单元的无效是从前向后的,但没有发现其无效方向是从后向前的单元。与对照组相比,经ＥＣＭＡ损伤后的实验组中只记录到ＯＮ－ＯＦＦ反应和ＯＮ反应单元,未能找到单独的ＯＦＦ反应单元。神经节单元的ＯＮ反应部分为持续成分。所有的单元对运动条纹刺激都失掉了方向选择性,这些现象的机理可能是由于ＥＣＭＡ去除了胆碱能无足细胞所致。     

Emerging methodologies for the study of hypothalamic gonadotropin-releasing-hormone (GnRH) neurons

Gonadotropin-releasing-hormone (GnRH) neurons form part of a central neural oscillator that controls sexual reproduction through intermittent release of the GnRH peptide. Activity of GnRH neurons, and by extension release of GnRH, has been proposed to reflect intrinsic properties and synaptic input of GnRH neurons. To study GnRH neurons, we used traditional electrophysiology and computational methods. These emerging methodologies enhance the elucidation of processing in GnRH neurons. We used dynamic current-clamping to understand how living GnRH somata process input from glutamate and GABA, two key neurotransmitters in the neuroendocrine hypothalamus. In order to study the impact of synaptic integration in dendrites and neuronal morphology, we have developed full-morphology models of GnRH neurons. Using dynamic clamping, we have demonstrated that small-amplitude glutamatergic currents can drive repetitive firing in GnRH neurons. Furthermore, application of simulated GABAergic synapses with a depolarized reversal potential have revealed two functional subpopulations of GnRH neurons: one population in which GABA chronically depolarizes membrane potential (without inducing action potentials) and a second population in which GABAergic excitation results in slow spiking. Finally, when AMPA-type and GABA-type simulated inputs are applied together, action potentials occur when the AMPA-type conductance occurs during the descending phase of GABAergic excitation and at the nadir of GABAergic inhibition. Compartmental computer models have shown that excitatory synapses at >300 microns from somtata are unable to drive spiking with purely passive dendrites. In models with active dendrites, distal synapses are more efficient at driving spiking than somatic inputs. We then used our models to extend the results from dynamic current clamping at GnRH somata to distribute synaptic inputs along the dendrite. We show that propagation delays for dendritic synapses alter synaptic integration in GnRH neurons by widening the temporal window of interaction for the generation of action potentials. Finally, we have shown that changes in dendrite morphology can modulate the output of GnRH neurons by altering the efficacy of action potential generation in response to after-depolarization potentials (ADPs). Taken together, the methodologies of dynamic current clamping and multi-compartmental modeling can make major contributions to the study of synaptic integration and structure-function relationships in hypothalamic GnRH neurons. Use of these methodological approaches will continue to provide keen insights leading to conceptual advances in our understanding of reproductive hormone secretion in normal and pathological physiology and open the door to understanding whether the mechanisms of pulsatile GnRH release are conserved across species.     

Fast recruitment of recurrent inhibition in the cat visual cortex

Neurons of the same column in L4 of the cat visual cortex are likely to share the same sensory input from the same region of the visual field. Using visually-guided patch clamp recordings we investigated the biophysical properties of the synapses of neighboring layer 4 neurons. We recorded synaptic connections between all types of excitatory and inhibitory neurons in L4. The E-E, E-I, and I-E connections had moderate CVs and failure rates. However, E-I connections had larger amplitudes, faster rise-times, and shorter latencies. Identification of the sites of putative synaptic contacts together with compartmental simulations on 3D reconstructed cells, suggested that E-I synapses tended to be located on proximal dendritic branches, which would explain their larger EPSP amplitudes and faster kinetics. Excitatory and inhibitory synapses were located at the same distance on distal dendrites of excitatory neurons. We hypothesize that this co-localization and the fast recruitment of local inhibition provides an efficient means of modulating excitation in a precisely timed way.     

Pharmacological evaluation of GABAergic and glutamatergic inputs to the nucleus basalis--cortical and the septal-hippocampal cholinergic projections

The presence of different receptor populations within a given brain area can be effectively evaluated via the local injections of defined receptor agonists and antagonists. Using this approach, it has become evident that the nucleus basalis - cortical cholinergic pathway possesses an inhibitory GABAergic input to the nucleus basalis from the nucleus accumbens as well as a positive glutamatergic feedback from the cortex. The septal-hippocampal cholinergic pathway also possesses an inhibitory GABAergic regulation which consists of a large GABAergic interneuron population in the septum. A glutamatergic feedback from the hippocampus is also present. These regulatory inputs to cholinergic cells are not tonically active but appear to function as phasic modulators of cholinergic transmission in both pathways.