Nitric oxide and pathway refinement in the lower vertebrates [35,36].

16 R also recently been shown to occur during the later stages of postnatal development the projections from the retina to pathway refinement in visual system development. This the SC are exuberant in that both the ipsilateral and article focuses upon several retrograde signals, notably contralateral retinal pathways overlap extensively with nitric oxide and the neurotrophins, that contribute to many axons misdirected to inappropriate targets pathway refinement in the visual pathways. We also review [38,42,68,69,95,117,141,143]. Both projections sub- evidence that long term potentiation LTP and long term sequently undergo refinement in which incorrectly targeted depression LTD are involved in the synaptic refinement axons disappear either due to retraction of axon branches process. or to elimination of the parent axon. This process of refinement is thought to be mediated by the NMDA receptor in both rodent SC [142] and in the optic tectum of 2. Nitric oxide and pathway refinement in the lower vertebrates [35,36]. developing visual system This refinement is also partially mediated by NO. Thus, NOS is expressed maximally in neurons within the re- Nitric oxide NO, a free radical gas, is well-established tinorecipient layer of rodent SC superficial gray layer as a neuromodulator in brain [22–24,50,65,66,156]. As a SGL between the ages of P4–P21, the time during gas, NO can move rapidly across the plasma membrane in which the retinocollicular pathway is undergoing massive both anterograde and retrograde directions [94,156] and refinement. Neurons containing NOs synthetic enzyme, can therefore signal back to presynaptic terminals that NOS, increase in number 2–3 fold between P1 and P21, activity has occurred in a postsynaptic neuron [63,114]. and then decrease in number in the adult Fig. 1A,B, as This process is thought to involve a sequence of steps. In demonstrated using both an antibody directed against the post-synaptic neuron the events that result in NO nNOS and by use of the histochemical marker NADPH 21 release include Ca influx through the NMDA receptor diaphorase. Retinorecipient neurons expressing NOS in the which triggers a calcium-calmodulin dependent increase in SGL have a variety of morphologies, including both nitric oxide synthase NOS which in return results in excitatory and inhibitory cell types Fig. 1C,D [40,112]. production and release of nitric oxide NO. Presynaptical- Consistent with the time-course of NOS expression, ly, NO can activate a guanylate cyclase-cGMP second refinement of the ipsilateral retinocollicular pathway is messenger system in the presynaptic terminal which can significantly delayed in gene knockout mice in which the lead to an increase in the release of neurotransmitter endothelial and neuronal isoforms of NOS have been [21,65,107,108]. There are, however, a variety of other disrupted e,nNOS mutants, [79]. In SC, this disruption signal transduction pathways implicated in this process, results in an expanded ipsilateral retinocollicular projection and the release and action of NO is complex and incom- which remains more widely distributed across the superfi- pletely understood [51]. cial layers of SC of e,nNOS knockout mice compared to NO was shown to be involved in some forms of synaptic normal mice from ages P9 until at least P42 [113,159,160]. plasticity, such as long term potentiation and long term Fig. 2 illustrates this effect where it can be seen that depression, beginning nearly ten years ago [3,16,75,116]. multiple patches of labeled fibers are distributed more Its role in pathway refinement in the developing brain, widely in both the rostrocaudal and mediolateral axes of however, has only recently been established. The phenom- SC than in normal C57 BL-6 controls Fig. 2A,B. enon has been best studied in the visual system. In 1994 Multiple patches of labeled fibers are seen in the rostral Wu et al. [157] first reported that NO could alter refine- lateral SC and also in the caudal medial SC in the double ment in the chick ipsilateral retinotectal pathway by knockout Fig. 2B, arrows. Significant differences in the showing that the pathway, which is normally transient and cross-sectional area occupied by these labeled fibers have eliminated during embryogenesis, is partially spared after been found when measured in knockout and C57 BL6 inhibition of NOS, NO’s synthetic enzyme [157,158]. This mice at two ages: P15 [160] and P28 Fig. 2C,D and 159. process is NMDA dependent [58] and may also depend Although there is a significant delay in refinement, the upon interaction with the growth factor BDNF see below, ipsilateral retinocollicular pathway does eventually retract. [59]. The pathway begins to refine in the e,nNOS knockout as Development of the retinogeniculate pathway is also early as P9 and is like that of normals by adulthood altered by inhibition of NOS. Thus, intraperitoneal in- [159,160]. jections of n-v-nitro-l-arginine, a NOS inhibitor, disrupts Recently, Vercelli et al. [152] have confirmed this effect the segregation of the ‘on’ and ‘off’ sublaminae of the by showing that the density and distribution of retinocol- ferret lateral geniculate nucleus [44–48]. This effect is also licular axons is expanded in rats treated for 4–6 weeks dependent upon the NMDA receptor [71] and upon with a NOS inhibitor. Branch length and numbers of impulse activity in the retinal afferents [45]. More recently, synaptic boutons are increased in the inhibited rats [152], we have shown that development of the ipsilateral re- suggesting that the effect involves not only the distribution tinocollicular pathway in the mammalian superior col- of fibers but also the density of branches and synapses. A liculus SC also depends in part upon NO. Early in similar effect is seen in the rodent LGN where there is an R .R. Mize, F.-S. Lo Brain Research 886 2000 15 –32 17 Fig. 1. Distribution of nitric oxide in the developing rodent superior colliculus. A Computer plots illustrating the distribution of NO containing neurons within the mouse SC at varying ages after birth. Neurons were labeled by an antibody to nNOS. Onset of expression in the retinorecipient layers occurs by P5, with the numbers of neurons increasing thereafter, reaching a peak at P21. There is a decrease in the adult. Modified from Cork et al. [40]. B Histogram showing the ventral to dorsal progression in development of nNOS immunoreactivity in the rodent SC. Note that the peak distribution within the superficial gray layer SGL is at P21. There is a substantial decrease in the numbers of labeled neurons in all layers in the adult. Modified from Cork et al. [40]. C,D Micrographs showing the dense distribution of NO containing neurons within the rodent SC at P14 and P21. Neurons are labeled NADPHd. There is a very dense distribution of labeled neurons within the superficial gray layer SGL at both ages. There are few labeled neurons within the optic layer OL, but some labeled neurons are also scattered within the deep layers asterisks. Modified from Scheiner and Mize [131] and Mize et al. [112]. 18 R Fig. 2. Development of the retinocollicular pathway in normal and e,nNOS double knockout mice. A,B Stacked sections of SC showing the rostral bottom to caudal top distribution of the ipsilateral left and contralateral right retinocollicular pathways in normal A and B e,nNOS double knockout mice at age P21. Note the more extensive distribution of fibers with multiple patches of label seen in more lateral and caudal regions of SC arrows in the e,nNos knockouts. Modified from Wu et al. [160]. C,D Histograms showing the area of labeling of the ipsilateral retinocollicular pathway in e,nNOS double knockout mice C and normal C57 BL-6 mice D at age P28. The area occupied by the ipsilateral patches is expressed as a percent of the contralateral label found in the same section. Significant differences were observed at the 500–600 mm intervals P,0.005 and at the 800–900 and 1100 mm intervals P,0.05. Modified from Wu et al. [159]. increase in distribution and density of ipsilateral retinal below and [56,73,90,91] do not depend upon NO [73,91]. fibers which expand into the territory of the contralateral Thus, there is consistent anatomical and physiological representation of that nucleus [152]. There is thus evidence evidence that NO is not involved in neonatal cortical from several laboratories showing that nitric oxide medi- plasticity. ates pathway refinement in both the retinogeniculate and NO inhibition also fails to modify refinement in several retinocollicular pathways of some mammals. other visual system pathways. Thus, chronic application of NO appears not to mediate pathway refinement in some an NO inhibitor does not disrupt the formation of eye other regions of the brain. For example, inhibition of NOS specific stripes that occurs in the optic tectum of three- does not block the formation of ocular dominance columns eyed frogs [124] even though NOS is expressed in tectal in ferret visual cortex even though NO is expressed in neurons during the formation of these stripes and applica- visual cortical subplate neurons during the time that these tion of NO donors results in growth cone collapse and columns are established [61]. NOS inhibition also fails to retraction of retinal ganglion cell axons in this species block the shift in ocular dominance that occurs in primary [123]. Finally, inhibition of NOS does not block the visual cortex neurons after monocular deprivation, pro- segregation of retinal fibers into ipsilateral and contralater- viding further evidence that NO does not mediate synaptic al laminae of the ferret LGN [48]. Thus, NO has an effect plasticity of ocular dominance columns [122,129]. In on only some visual system pathways and only in certain addition, cellular correlates of synaptic plasticity LTP, species, and the factors which determine whether NO LTD which are present in developing visual cortex see mediates refinement are as yet incompletely understood. R .R. Mize, F.-S. Lo Brain Research 886 2000 15 –32 19 3. The role of impulse activity in visual pathway activity in the nerve terminal. Inhibitors of NOS block this refinement decrease in amplitude, showing that the effect is NO specific. The source of NO is thought to be from the Impulse activity in presynaptic afferents and post-synap- post-synaptic muscle because intracellular injection of the tic neurons has long been known to be a factor contribut- NOS inhibitor into the myocyte also blocks the effect. This ing to the formation of precise retinotopic connections in evidence shows directly that NO release can produce brain [70,86,139]. The role of patterned vision in forming depression of presynaptic activity in developing synapses. connections within the visual cortex was established over The effect of NO appears to be specific to synapses with 40 years ago by showing that deprivation of vision in one low spontaneous activity because active nerve terminals eye diminishes the influence of that eye so that only the are less affected by application of NO donors [153]. non-deprived eye can drive cortical neurons. Ocular domi- Other evidence suggests that the action of NO in nance columns in visual cortex which represent the non- pathway refinement may not require or even be related to deprived eye were also shown to expand in size in order to impulse activity. A number of studies have shown that NO favor the eye with normal vision see [81], for review. donors produce arrest and retraction of growth cones and More recently, it was discovered that spontaneous impulse filipoidia in in vitro models in which impulse activity is activity in the retina can also influence the formation of largely absent [59,67,76,77,151,123]. These studies sup- synaptic connections before eye opening. This activity port the conclusion that NO serves as a repellant molecule arises from spontaneous discharges which produce waves that can promote coarse axon arbor retraction that is of correlated electrical activity in neighboring retinal unrelated to patterns of impulse activity see [124]. Thus, ganglion cells [62,109,139]. This correlated activity in the link between impulse activity and NO mediated adjacent regions of retina leads to synaptic strengthening in pathway refinement is based largely upon indirect evidence presynaptic fibers representing a similar region of retina in which activity has been blocked by TTX but not when compared with uncorrelated activity in fibers from manipulated or measured directly at the synapse. A more other retinal sites [15,109]. direct approach to the study of this relationship is an This spontaneous impulse activity has been shown to be important future direction for research on this topic. important in the formation of ocular dominance columns in One promising approach is to artificially induce pre- and visual cortex [32,148] and in the segregation of retinal post-synaptic activity in competing pathways using electri- axons into eye specific layers in the LGN [140]. In both cal stimulation and whole cell recording in a slice prepara- structures, injections of TTX, a sodium channel blocker, tion. This technique permits one to examine potentiation or blocks formation of normal patterned connections, pre- depression of synaptic transmission and how it is modified sumably due to the imbalance in impulse activity in the by pairing stimuli applied to one or both pathways. Recent two eyes. The pattern of activity is also important, since studies by Poo [161] have shown that the temporal order of artificially induced synchronous activity in the ipsilateral activation of synapses in convergent pathways can de- and contralateral optic nerves can also block formation of termine whether a synaptic response is strengthened or ocular dominance columns while asynchronous activity depressed in individual neurons. In the frog retinotectal sharpens these columns [149]. Activity patterns are also system, repetitive stimulation of one region of the retina important in refining the retinotectal pathway in lower just prior to spiking of a postsynaptic neuron potentiates vertebrates [25,121,132–134]. subsequent responses of that neuron to stimulation. Pairing A relationship between impulse activity and the action this stimulus with a weak stimulus to another region of the of NO in development was predicted explicitly by Gally et retina, which also activates the neuron via a convergent al. [63,114]. They showed in modeling experiments that synapse, produces a depression in the response to the NO could alter synaptic efficacy such that synapses whose second stimulus, but only when the pairing falls within a presynaptic activity is correlated with action potentials in critical time frame [161]. Thus, both the temporal sequenc- the postsynaptic neuron would be strengthened by the ing of impulse activity in the convergent inputs and the release of NO while those with uncorrelated activity would level of spiking in the post-synaptic neuron determine be weakened. Impulse activity has subsequently been whether convergent synapses will be potentiated or de- shown to contribute to NO mediated pathway refinement in pressed [161]. several experiments. Thus, TTX injection into one eye We have recently developed an in vitro brainstem blocks the segregation of the on and off sublaminae of the preparation which also allows us to study directly how the ferret LGN [45] in a manner similar to that of NOS timing and amount of impulse activity in the ipsilateral and inhibition [47]. The role of NO in activity dependent contralateral retinocollicular pathways affects the efficacy synaptic depression has also been shown directly in nerve of individual synapses in the rodent SC. This preparation is muscle cultures [153]. In this preparation NO donors an isolated brainstem that includes the midbrain, dien- decrease the amplitude of evoked presynaptic currents cephalon thalamus, and also the optic tracts, optic when the myocyte is depolarized in order to artificially chiasm, and optic nerves that are intact and remain produce activity which is uncorrelated with presynaptic functionally connected to the eye Fig. 3A. The ipsilateral 20 R synaptic field potentials Fig. 3B,C, or extracellular action potentials Fig. 3D,E, or even intracellular post-synaptic responses from individual cells in which the synaptic circuitry remains intact. Preliminary data shows that we can elicit responses from both pathways Fig. 3B,C. Spontaneous activity can also be recorded from single cells in SC Fig. 3F. This activity sometimes displays a rhythmicity suggesting that the retina produces sponta- neous retinal waves in this preparation. We do not yet know how NO will modulate these responses, but NO does affect LTD in a similar preparation see below.

4. NO Mediated pathway refinement and LTP LTD