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Nitric oxide (NO) has been implicated as a retrograde signal in the process of refining axonal pathways during brain development. To determine some of the factors involved in this process, we have used two model pathway systems in the rat and mouse superior colliculus (SC). The first, the patch-cluster system, consists of clusters of neurons in the intermediate gray layer (igl) which transiently express NO during development and which receive input from a cholinergic pathway from the parabrachial brainstem as well as from other pathways containing different transmitters. The second system, the retinocollicular pathway, consists of glutamatergic fibers that project to the superficial gray layer. We have used both nitric oxide synthase inhibition (nw-nitro-L-arginine, NoArg) and single (nNOS) and double (nNOS and eNOS) gene knockout mice to examine the effect that reduction in NOS has upon the development of these two systems. The onset of NOS expression in rat, as revealed by nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) labeling, occurred in igl cells as early as postnatal day P5, with clusters being well-established by P14. Cholinergic fibers were first visible at P10 and formed obvious patches and tiers by P14. Intraperitoneal injections of NoArg from P1-P22 had no effect upon the development of these cholinergic patches. The pathway also developed normally in both single and double-knockout mice. In contrast, the ipsilateral retinocollicular pathway was altered in the double, but not in the single knockout mouse. This pathway is exuberant during the first week of life, being distributed across much of the mediolateral axis of the rostral SC. By P8-P15, this pathway has retracted to the most mediorostral SC. This refinement was delayed substantially in the double NOS gene knockout mouse. Ipsilateral fibers were found within 3-5 separate medio-lateral patches within the rostral 600 microns of SC at P15, and patches of abnormal size and extent were also seen at P18. We conclude from these results that NO plays a role in pathway development in the rodent SC, but only in glutamatergic pathways and only when both endothelial and neuronal forms of NOS have been deleted. The mechanism of this effect must involve pathway elimination in situations where there is non-correlated electrical activity. It is likely that NO promotes fiber retraction rather than fiber stabilization in these developing nerve fibers.

We have produced a digital atlas of the distribution of nitric oxide synthase (NOS) in the mouse brain as a reference source for our studies on the roles of nitric oxide in brain development and plasticity. NOS was labeled using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry. In addition, choline acetyltransferase (ChAT) immunocytochemistry was used to identify cholinergic cells because many of the NADPHd positive cells were thought to colocalize acetylcholine. Some sections were also labeled with antibodies to either the neuronal (nNOS) or endothelial (eNOS) isoforms of NOS. Series of sections from 11 C57/BL6 mice were collected and labeled for NADPHd and/or ChAT. We collected two types of data from this material: color digital photographs illustrating the density of cell and fiber labeling, and computer/microscope plots of the locations of all the labeled cells in selected sections. The data can be viewed as either a series of single-section maps produced by combining the plots with the digital images, or as 3-D views derived from the cell plots. The atlas of labeled cell maps, together with selected color photographs and 3-D views, is available for viewing via the World Wide Web (http:@nadph.anatomy.lsumc.edu). Examination of the atlas data has revealed several points about the distribution of NOS throughout the mouse brain. Firstly, different populations of NADPHd-positive neurons can be distinguished by different patterns of staining. In some brain areas neurons are intensely stained by the NADPHd technique where label fills the cell bodies and much of the dendritic trees. In other brain regions labeling is much lighter, is principally confined to the cytoplasm of the cell soma, and extends only a short distance within proximal dendrites. Intense labeling is typical of neurons in the caudate/putamen and mesopontine tegmental nuclei. Most of the labeled neurons in the cortex also stain this way. Lighter, "granular" label is found in many other nuclei, including the medial septum, hippocampus, and cerebellum. In addition to staining pattern, we have also noted that different subpopulations of NOS-neurons can be distinguished on the basis of colocalization with ChAT. Substantial overlap of the distributions of these two substances was observed although very little colocalization was found in most cholinergic cell groups except the mesopontine tegmental nuclei. Other points of interest arising from this project include the apparent lack of NADPHd labeling in the CA1 pyramidal cells of the hippocampus or the Purkinje neurons in the cerebellum. This observation is especially relevant given that synaptic plasticity in these regions is reported to be nitric-oxide dependent.

The cat superior colliculus (SC) plays an important role in visual and oculomotor functions, including the initiation of saccadic eye movements. We have studied the organization of neurochemical specific circuits in SC that underly these functions. In this chapter we have reviewed three microcircuits that can be identified by cell type, chemical content, and synaptic input from specific afferents. The first is located within the upper sgl and is related to the W retinal pathway to this region of SC. This circuit includes relay and interneurons that contain the calcium binding protein calbindin (CB), GABA containing presynaptic dendrites, and retinal terminals that have a distribution and size typical of W retinal terminals in the cat SC. This circuit is a typical synaptic triad that mediates feedforward inhibition, possibly to regulate outflow of the W pathway to the lateral geniculate nucleus. CB neurons in SC and other structures may be uniquely related to low threshold calcium currents in these neurons. The second microcircuit consists of neurons that contain parvalbumin (PV), another calcium binding protein. These neurons are located in a dense tier with the deep sgl and upper ol and they receive input from retinal terminals that are likely from 'Y' retinal ganglion cells. Some of these neurons also project to the lateral posterior nucleus and some colocalize glutamate. We speculate that these neurons also receive cortical 'Y' input although we have yet to prove this experimentally. The role of PV in these cells is unknown, but PV has been shown to be contained in fast spiking, non-accomodating neurons in visual cortex which have very rapid spike discharges that are also characteristic of SC neurons innervated by 'Y' input. The third microcircuit consists of a group of clustered neurons within the igl of the cat SC that overlaps the patch-like innervation of afferents to this region that come from the pedunculopontine tegmental and lateral dorsal tegmental nuclie, the substantia nigra, and the cortical frontal eye fields. These clustered neurons project through the tectopontobulbar pathway and terminate within the cuneiform region (CFR) of the midbrain tegmentum. They transiently express NOS during development. Ongoing studies in our laboratory suggest that these cells receive synaptic inputs directly from the PPTN and SN and may represent functional modules involved in the initiation of saccadic eye movements.

Neurons within the superior colliculus (SC) contain a variety of neurochemicals, including the amino acid neurotransmitters GABA and glutamate, the calcium binding proteins calbindin and parvalbumin, and the neuromodulator nitric oxide. We have examined the development of expression of these substances using antibody immunocytochemistry. These results are summarized in Fig. 10. GABA and calbindin are expressed very early in development, at a time when cells are still dividing and migrating from the subventricular zone. The expression of both GABA and CB is maximal at around E40-46, the age at which these cells have just established their adult lamination and extrinsic afferents have begun to grow into the tectum. GABA and CB likely play diverse roles during this stage of development, including the regulation of intracellular calcium during cell migration and neurite outgrowth. Glutamate is expressed somewhat later in development while parvalbumin immunoreactivity does not appear until shortly after birth. These two substances continue to increase in density throughout the period of postnatal growth, at a time when synapse formation and evoked electrical activity are beginning to develop. Both PV and glutamate may be involved in one or both of these activity-dependent processes. Nitric oxide synthase (NOS) is expressed at different times in different cell groups. NOS appears very early in prenatal development in cells within the SVZ and in the deep gray layer of SC. On the other hands, cells within the intermediate gray layer of SC do not express NOS until shortly before birth. The igl cells that express NOS at this age are clustered neurons similar to those that project to the CFR in the adult. NOS expression occurs in these cells at precisely the time when axons begin to form patches that innervate these clusters. Based upon this temporal correlation, we hypothesize that nitric oxide may regulate synapse formation in this cell group.

GABA is an important inhibitory neurotransmitter in the mammalian superior colliculus. As in the lateral geniculate nucleus, GABA immunoreactive neurons in SC are almost all small and are distributed throughout the structure in all mammalian species studied to date. Unlike the LGN, GABA-labeled neurons in SC have a variety of morphologies. These cells have been best characterized in cat, where horizontal and two granule cell morphologies have been identified. Horizontal cells give rise to one class of presynaptic dendrite while granule C cells give rise to another class of spine-like presynaptic dendrite. Granule A cells may be the origin of some GABAergic axon terminals. GABA containing synaptic profiles form serial synapses, providing a possible substrate for disinhibition. The distribution of GABAA and GABAB receptor subtypes appears similar to that of GABA neurons, with the densest distribution found within the superficial gray layer. However, antibody immunocytochemistry of the beta 2 and beta 3 subunits of the GABAA receptor reveals that it is located at both synaptic and non-synaptic sites, and may be associated with membrane adjacent to terminals with either flattened or round vesicles. A few GABA containing neurons in SC colocalize the pentapeptide leucine enkephalin or the calcium binding protein calbindin. However, none appear to co-localize parvalbumin, a situation different from GABA containing interneurons in the LGN and visual cortex. The diversity of GABA neurons in SC rivals that found in visual cortex, although unlike visual cortex, the pattern of co-occurrence does not distinguish GABA cell types in SC. The superior colliculus also differs from both LGN and visual cortex in that GABA and calbindin immunoreactivity is not altered by either long-term occlusion and/or short-term enucleation in adult Rhesus monkeys. No consistent differences have been found in the optical density of GABA labeling in either cells or neuropil. To conclude, GABA neurons in the superior colliculus share some properties like those in LGN and others like those in visual cortex. In other properties, they differ from GABA neurons in both the LGN and visual cortex. The GABA systems in the superior colliculus are similar in all mammalian species studied, suggesting that they are phylogenetically conserved systems which are not amenable to plastic alterations, a situation different to that in the geniculostriate system.