Subcellular localization of neuronal nitric oxide synthase in the superficial gray layer of the rat superior colliculus

The superficial layers of the rat superior colliculus (sSC) receive innervation from retina and include nitric oxide synthase (NOS)-immunoreactive neurons. We used electron microscopic immunocytochemistry to assess the subcellular localization of neuronal NOS (nNOS) in the sSC. nNOS immunoreactivity was detected on the external membrane of mitochondria, endoplasmic reticulum, in pre- and postsynaptic profiles and also diffusely distributed in the cytosol. Postsynaptic labeled regions were often associated with presumptive retinal unlabeled terminals. Microtubules also appeared intensely labeled. These results show that NOS immunoreactive neurons may be innervated by retinal terminals and suggest an association of nNOS with cytoskeletal elements.     

Subcellular localization of GABAA/benzodiazepine receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus

The monoclonal antibody bd-17, which recognizes the β₂ and β₃-subunits of GABA_A/benzodiazepine receptors, was used to determine the cellular and subcellular localization of receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus. In numerous dendrites, very strong immunostaining was present in the cytoplasm and on the postsynaptic dendritic membrane of synaptic junctions. The extrasynaptic portion of the dendritic membrane also very often showed [β₂ + β₃]-like immunoreactivity. However, due to methodological limitations, it could not be stated with certainty whether presynaptic β₂- and β₃-subunits of GABA_A/benzodiazepine receptors actually occur in this mesencephalic visual structure. In conclusion, these results strongly suggest that synaptic and non-synaptic GABA_A/benzodiazepine receptors are present in the superficial gray layer of the rat superior colliculus. These receptors may modulate neuronal cell activity in different ways, depending on their location.     

Electron microscopic immunocytochemical study of somatostatin neurons in the periventricular nucleus of the rat hypothalamus with special reference to their relationships with homologous neuronal processes

The neurons containing somatostatin in the rat periventricular nucleus were studied by using a modified electron microscopic immunocytochemical technique that improves both the penetration of immunoreagents into unembedded immunostained tissues and the preservation of ultrastructural morphology. Inside perikarya and dendrites, immunostaining was not only associated with neurosecretory granules but also with ribosomes and saccules of the cis face of the Golgi apparatus. In the axonal profiles found in this region the labeling was observed both on neurosecretory granule cores and on the limiting membrane of small synaptic-like vesicles. Throughout the periventricular nucleus, both non-synaptic and synaptic relationships were shown between labeled neurons. Non-synaptic relationships mainly consisted of direct apposition of the membranes of neighboring neurons by dendrosomatic, somasomatic or dendrodendritic contacts. These labeled perikarya and dendrites were also synaptically contacted by labeled axonal endings containing numerous aggregated synaptic-like vesicles. The physiological significance of the synaptic and non-synaptic relationships between somatostatinergic neurons is discussed in terms of possible synchronization between homologous neurons of the somatostatin neuroendocrine system and control of these neurons by a central ultra-short loop feedback mechanism.     

Subcellular localization of GABAA/benzodiazepine receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus

The monoclonal antibody bd-17, which recognizes the β₂ and β₃-subunits of GABA_A/benzodiazepine receptors, was used to determine the cellular and subcellular localization of receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus. In numerous dendrites, very strong immunostaining was present in the cytoplasm and on the postsynaptic dendritic membrane of synaptic junctions. The extrasynaptic portion of the dendritic membrane also very often showed [β₂ + β₃]-like immunoreactivity. However, due to methodological limitations, it could not be stated with certainty whether presynaptic β₂- and β₃-subunits of GABA_A/benzodiazepine receptors actually occur in this mesencephalic visual structure. In conclusion, these results strongly suggest that synaptic and non-synaptic GABA_A/benzodiazepine receptors are present in the superficial gray layer of the rat superior colliculus. These receptors may modulate neuronal cell activity in different ways, depending on their location.     

Retinal W-cell input to the upper superficial gray layer of the cat's superior colliculus: a conduction-velocity analysis

1. I have used several methods to estimate the conduction velocities of retinal afferents innervating the upper 50-100 micron of the stratum griseum superficiale (the upper SGS). The measurements were based on a unitary extracellular potential unique to this sublamina, which was first described by McIlwain (28). He termed it the juxtazonal potential (JZP), and showed that it results when a single spike invades the terminal arbor of a single retinal afferent to the upper SGS, triggering synchronous excitatory postsynaptic potentials in postsynaptic collicular cells. 2. Individual unitary JZPs were evoked at fixed latencies by weak shocks to the optic disk, chiasm, or tract. When the same JZP could be evoked in isolation from two stimulus sites, the conduction velocity of the axon triggering the JZP was estimated by dividing the conduction time between the stimulating electrodes (i.e., the "latency difference") into the distance separating these electrodes. This "latency-difference method" lacked general utility, however, since the same JZP could only rarely be evoked in isolation from two stimulus sites. 3. This limitation was circumvented by means of a collision method. When a stimulus that evoked a JZP in isolation was preceded by a sufficiently intense conditioning shock to a second, more central stimulus site, the conditioning stimulus caused the JZP to fail in an all-or-none fashion. It was assumed that when the JZP failed, the conditioning stimulus had exceeded the spike threshold of the axon mediating the JZP and that an antidromic action potential had collided with the orthodromic spike initiated at the peripheral stimulus site. Assessment of the critical interstimulus interval for producing such a collision, together with measurements of the axon's refractory period and the interelectrode conduction distance, permitted an estimate of the conduction velocity of the JZP-triggering axon. Conduction-velocity estimates generated in this way closely matched those based on the latency-difference technique when both methods could be applied. 4. Conduction velocities of 31 JZP-triggering axons analyzed by the collision method ranged from 2.9 to 6.8 m/s [4.6 +/- 1.0 (mean +/- SD)]. Comparable estimates were obtained for such axons by alternative methods based on the absolute latencies of electrically evoked JZPs or of the field potential to which they contribute. The conduction velocities of JZP-triggering axons fell within the range reported for retinal W-cells and entirely outside those of X- and Y-cells, confirming earlier evidence for W-cell input to the upper SGS (7, 15, 18, 28).(ABSTRACT TRUNCATED AT 400 WORDS)     

Most superficial sublamina of rat superior colliculus: neuronal response properties and correlates with perceptual figure-ground segregation

The uppermost layer (stratum griseum superficiale, SGS) of the superior colliculus (SC) provides an important gateway from the retina to the visual extrastriate and visuomotor systems. The majority of attention has been given to the role of this "visual" SC in saccade generation and target selection and it is generally considered to be less important in visual perception. We have found, however, that in the rat SGS1, the most superficial division of the SGS, the neurons perform very sophisticated analysis of visual information. First, in studying their responses with a variety of flashing stimuli we found that the neurons respond not to brightness changes per se, but to the appearance and/or disappearance of visual shapes in their receptive fields (RFs). Contrary to conventional RFs of neurons at the early stages of visual processing, the RFs in SGS1 cannot be described in terms of fixed spatial distribution of excitatory and inhibitory inputs. Second, SGS1 neurons showed robust orientation tuning to drifting gratings and orientation-specific modulation of the center response from surround. These are features previously seen only in visual cortical neurons and are considered to be involved in "contour" perception and figure-ground segregation. Third, responses of SGS1 neurons showed complex dynamics; typically the response tuning became progressively sharpened with repetitive grating periods. We conclude that SGS1 neurons are involved in considerably more complex analysis of retinal input than was previously thought. SGS1 may participate in early stages of figure-ground segregation and have a role in low-resolution nonconscious vision as encountered after visual decortication.     

Local excitatory circuits in the intermediate gray layer of the superior colliculus

We have used photostimulation and whole cell patch-clamp recording techniques to examine local synaptic interactions in slices from the superior colliculus of the tree shrew. Uncaging glutamate 10-75 microm from the somata of neurons in the intermediate gray layer elicited a long-lasting inward current, due to direct activation of glutamate receptors on these neurons, and brief inward currents caused by activation of presynaptic neurons. The synaptic responses occurred as individual currents or as clusters that lasted up to several hundred milliseconds. Excitatory synaptic responses, which reversed at membrane potentials near 0 mV, could be evoked by uncaging glutamate anywhere within 75 microm of an intermediate layer neuron. Our results indicate the presence of extensive local excitatory circuits in the intermediate layer of the superior colliculus and support the hypothesis that such intrinsic circuitry contributes to the development of presaccadic command bursts.     

Contribution of superficial layer neurons to premotor bursts in the superior colliculus

In vitro whole-cell patch-clamp methods were used to examine the contribution of one component of intracollicular circuitry, the superficial gray layer, to the generation of bursts of action potentials that occur in the intermediate layer and that command head and eye movements in vivo. Applying a single brief (0.5 ms) pulse of current to the superficial layer of rat collicular slices evoked prolonged bursts of excitatory postsynaptic currents (EPSCs) in the cells of the intermediate layer. The EPSCs were sufficient to elicit bursts of action potentials that lasted as long as 300 ms and resembled presaccadic command bursts. To examine the contribution of neurons within the superficial layer to the production of these bursts, we determined how superficial neurons respond to the same current pulses that evoke bursts in the intermediate layer. Recordings from 61 superficial layer cells revealed 19 neurons that produced multiple action potentials following stimulation. Nine of these 19 neurons were wide- and narrow-field vertical cells, which are known to project to the intermediate layer and could contribute to producing the EPSC bursts. The remaining cells (n = 42) did not generate trains of action potentials and 21 of these showed only subthreshold potential changes in response to the stimulus. Our results indicate that most superficial cells do not directly contribute to production of the EPSC bursts, but a small number do have the properties necessary to provide a prolonged excitatory drive to the premotor neurons.     

Neurons of the superficial tectal gray an intracellular HRP-study on the kitten superior colliculus in vitro

Summary An en bloc preparation of the mammalian superior colliculus in vitro has been used to study neurons of the superficial gray layer (SGS) with intracellular recording and HRP-technics. Electrophysiological data from kittens at 4–19 days of age suggest that at this stage SGS-neurons possess multiple spike trigger zones which can be activated by synaptic depolarization and are probably located on dendrites. In response to intratectal stimulation SGS-neurons generate EPSP-IPSP sequences or IPSPs. IPSPs are found in all penetrated cells as early as the 4th postnatal day. Ascending projection cells (APCs) and inter-layer cells (ILCs) have been identified based on antidromic activation and/or intracellular labeling with HRP. The extended dendritic arbor of APCs and ILNs (dorsal spread up to 10–20 m below surface, horizontal spread up to 1100–1500 m) enables these cells to sample visual information from a wide area of the visual field. Recurrent collaterals, in conjunction with potent inhibitory mechanisms, could contribute to the formation of receptive field properties of superficial tectal neurons. ILCs establish collateral connections with the intermediate gray layer.     

Uncrossed retinal fibers terminating in the superior colliculus of the albino rat

The site of termination of uncrossed retinal fibers was examined in the superior colliculus of the albino rat. Sixty-eight male and female rats of Gifu, Donryu, Wistar and Sprague-Dawley strains from 9 days to 24 months of age were examined by means of a silver impregnation method for degenerating nerve fibers and the horseradish peroxidase method. In this study, terminals of uncrossed retinal fibers were observed in a small region from the posteromedial to the posterior part of the superficial gray stratum of the ipsilateral superior colliculus.     

Postnatal development of the superficial layers in the rat superior colliculus: a study with Golgi-Cox and Klüver-Barrera techniques

Summary The postnatal development of the superficial (optic) layers of the rat superior colliculus has been studied using Klüver-Barrera staining and Golgi impregnation in rats aged 3–45 days. The Klüver-Barrera staining reveals that the SC of 3 day old rats is morphologically immature with no obvious lamination. It contains densely packed cells of uniform size. The packing density of the cells gradually decreases between 9 and 15 days as the thickness of the layers increases. The first myelinated fibres in the SC appear at 15 days but the stratum opticum is still not recognizable. By 30 days, the SC has a distinctly laminated appearance, but the thickness of the superficial layers continues to increase until day 45 postnatal. Golgi-Cox impregnation displays the range of neuronal types in the superficial layers of the SC previously described by Langer and Lund (1974). Using the morphological criteria of these authors for classification of the neurons, the developmental changes of the marginal cells, horizontal cells, ganglion cells types I, II, III and stellate cells have been followed. The SC of 3 day old rats contains immature neurons; only a few larger cells have branched dendrites. In 9 days old SC the neuronal types present in the adult are recognizable, although their appearances are still immature. By 15 days neurons have adult-looking dendritic trees but dendritic growth continues beyond 30 days. The visual part of the SC has a protracted period of postnatal development, the sequence of developmental changes being similar for the different types of collicular neurons. Features common to development are the increasing size of neuronal somata, the increasing length of dendrites and the acquisition of a complex pattern of dendritic arborization. Larger cells appear to commence development earlier than small cells, although the rate of developmental changes is different for each of the various types of collicular neurons.     

Light‐dependent retinal innervation of the rat superior colliculus

Mammalian retinal projections are divided into two anatomically and functionally distinct systems: the primary visual system, which mediates conscious visual processing, and the subcortical visual system, which mediates nonconscious responses to light. Light deprivation during a critical period in development alters the anatomy, physiology, and function of the primary visual system in many mammalian species. However, little is known about the influence of dark‐rearing on the development of the subcortical visual system. To evaluate whether the early lighting environment alters the anatomy of the subcortical visual system, we examined the retinas and retinofugal projections of rats reared in a 12:12 light/dark cycle or in constant dark from birth to 4 months of age. We found that dark‐rearing was associated with a reduction in the distribution of retinal fibers in the stratum opticum of the contralateral superior colliculus. In contrast to the plasticity of the retinocollicular projection, retinal input to sleep, circadian, and pupillary control centers in the hypothalamus, pretectum, and lateral geniculate complex was unaffected by dark‐rearing. A decrease in retinal innervation of the stratum opticum and intermediate layers of the superior colliculus may account for some of the deficits in multisensory integration that have been observed in dark‐reared animals of several species. Anat Rec 2007. © 2007 Wiley‐Liss, Inc.     

Synaptic connections of cortical and retinal terminals in the superior colliculus of the rabbit: an electron microscopic double labelling study

Summary Retinal and cortical afferents to the superior colliculus of the rabbit were labelled simultaneously by injecting ³H-leucine into the right eye and HRP into the left visual cortex. It could be shown that there is some convergence of retinal and cortical input onto common postsynaptic elements in the superficial grey, but these cases were found to be rather rare indicating that most afferents from the retina and the visual cortex terminate either on different postsynaptic cells or on different parts of common postsynaptic cells.     

GABA-immunoreactive neurons and terminals in the cat periaqueductal gray matter: a light and electron microscopic study

Immunocytochemical and electron microscopic methods were used to study the GABAergic innervation in adult cat periaqueductal gray matter (PAG). A mouse monoclonal antibody against γ -aminobutyric acid (GABA) was used to visualize the inhibitory neuronal system of PAG. At light microscopy, GABA-immunopositive (GABA_IP) neurons formed two longitudinally oriented columns in the dorsolateral and ventrolateral PAG that accounted for 36% of the neuronal population of both PAG columns; their perikaryal cross-sectional area was smaller than that of unlabeled (UNL) neurons found in the same PAG subdivisions. At electron microscopic level, patches of GABA immunoreactivity were readily detected in neuronal cell bodies, proximal and distal dendrites, axons and axon terminals. Approximately 35–36% of all terminals were GABA_IP; they established symmetric synapses with dendrites (84.72% of the sample in the dorsolateral PAG and 86.09% of the sample in the ventrolateral PAG) or with cell bodies (7–10% of the sample). Moreover, 49.15% of GABA_IP axon terminals in the dorsolateral and 52.16% in the ventrolateral PAG established symmetric synapses with GABA_IP dendrites. Immunopositive axon terminals and unlabeled terminals were also involved in the formation of a complex synaptic arrangment, i.e. clusters of synaptic terminals in close contact between them that were often observed in the PAG neuropil. Moreover, a fair number of axo-axonic synapses between GABA_IP and/or UNL axon terminals were present in both PAG subdivisions. Several dendro-dendritic synapses between labeled and unlabeled dendrites were also observed in both PAG subdivisions. These results suggest that in the cat PAG there exist at least two classes of GABArgic neurons. The first class could exert a tonic control on PAG projecting neurons, the second could act on those GABAergic neurons that in turn keep PAG projecting neurons under tonic inhibition. The functional implications of this type of GABAergic synapse organization are discussed in relation to the dishinibitory processes that take place in the PAG.     

Characterisation of rat superficial superior colliculus neurones: firing properties and sensitivity to GABA

Physiological, pharmacological and morphological properties of superficial superior colliculus neurones (n=93) were characterised using whole-cell patch-clamp recordings in rat brain slices. Six cell types (narrow- and wide-field vertical, horizontal, piriform, marginal and stellate) were identified based on Lucifer Yellow labelling but no cell type-specific spike pattern could be identified. Resting membrane potentials were homogeneous (mean: -67.1 +/- 0.7 mV, n=48), and spike frequencies ranged from 10 to 70 Hz (80 pA current injection). About 66% of the cells displayed regular and sustained spike production, throughout all neuronal categories. Rebound spikes and spontaneous activity were observed frequently in all cell types. Synaptically evoked action potentials appeared as single spikes (mean amplitude: 76.0 +/- 3.2 mV, n=34) followed by a fast after-hyperpolarising potential (mean amplitude: 25.4 +/- 1.4 mV, n=34) and variable late potentials (late after-depolarising and/or -hyperpolarising). Pharmacologically, a characterisation using GABA and its subtype-specific agonists indicated a strong inhibitory influence of this transmitter system on >90% of cells. The GABA(A) receptor agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (100 microM), caused a reversible hyperpolarisation (approximately 9 mV) and spike inhibition of all neurones studied. This was more pronounced for intrinsic than for synaptically evoked spikes. Assessment of the GABA(C) receptor agonist, cis-4-aminocrotonic acid (1 mM), also revealed a hyperpolarisation (approximately 3 mV) and an inhibitory action on firing, but this was not as potent and homogeneous, compared to the GABA(A) receptor agonist. Further, the GABA(B) receptor agonist, baclofen (50-100 microM), had more variable (hyperpolarising, depolarising or no change) effects on the membrane potential. It showed little modulation of current-induced action potentials but fully blocked synaptic spikes. Assessment of GABA receptor antagonist actions revealed the presence of weak tonic and strong phasic GABA(A) receptor-mediated inhibition in the superficial superior colliculus: application of the GABA(A) receptor antagonist, bicuculline (100 microM), led to a generally enhanced excitability and depolarisation (approximately 5 mV). Intrinsic firing was somewhat enhanced, but synaptic spiking was drastically potentiated and prolonged. In contrast, 1,2,5,6-tetrahydro-(pyridin-4-yl) methylphosphinic acid (TPMPA; 100 microM), the GABA(C) receptor antagonist, produced little effect on these physiological parameters. The GABA(B) receptor antagonist, CGP35348 (200 microM), caused a partial inhibition of late after-hyperpolarising potentials (approximately 30%). Uptake of GABA contributes little to endogenous inhibition in the superior colliculus slice preparation, as suggested by the action of GABA uptake inhibitors SKF89976 (50-100 microM) and nipecotic acid (200-500 microM), both had no obvious effect on physiological parameters. However, in the presence of these compounds, sub-maximal inhibitory actions of GABA were potentiated.In conclusion, different cell types in the superficial superior colliculus do not display distinct physiological properties and are subject to strong inhibitory modulation. We therefore suggest that signal processing in this brain region does not require cell type-specific encoding of information. In line with evidence provided by previous in vivo investigations, identification of visual stimuli and orientation responses appears to be realised via the network properties of the receptive fields that form topographic maps.     

Organization of the intermediate gray layer of the superior colliculus. I. Intrinsic vertical connections

A pathway from the superficial visual layers to the intermediate premotor layers of the superior colliculus has been proposed to mediate visually guided orienting movements. In these experiments, we combined photostimulation using "caged" glutamate with in vitro whole cell patch-clamp recording to demonstrate this pathway in the rat. Photostimulation in the superficial gray and optic layers (SGS and SO, respectively) evoked synaptic responses in intermediate gray layer (SGI) cells. The responses comprised individual excitatory postsynaptic currents (EPSCs) or EPSC clusters. Blockade of these EPSCs by TTX confirmed that they were synaptically mediated. Stimulation within a column (approximately 500 microm diam) extending superficially from the recorded cell evoked the largest and most reliable responses, but off-axis stimuli were effective as well. The EPSCs could be evoked by stimuli 1,000 microm off-axis from the postsynaptic neuron. The dimensions of this wider region (approximately 2 mm diam) corresponded to those of the dendrites of superficial layer wide-field neurons. SGI neurons differed in their input from SGS and SO; neurons in the middle of the intermediate layer (SGIb) were less likely to respond to visual layer photostimulation than were those in sublayers just above and below them. However, focal stimulation within SGIa did evoke responses within SGIb, indicating that SGIb neurons may receive input from the visual layers indirectly. These results demonstrate a columnar pathway that may mediate visually guided orienting movements, but the results also reveal spatial attributes of the pathway which imply that it also plays a more complex role in visuomotor integration.     

Sometimes you see them,sometimes you don't: IPSCs in the rat superficial superior colliculus

Superficial superior colliculus (SSC) neurones were voltage-clamped and the current-voltage relationship of synaptically evoked currents analyzed in vitro. A strong interplay between excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) was identified. Glutamate receptor antagonists not only fully blocked EPSCs but IPSCs were also frequently reduced by the specific d,l,-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist (by 66.9%), indicative of glutamate-driven inhibitory projections. The GABA_A receptor antagonist bicuculline enhanced EPSCs and either abolished or reduced (by 79.3%) IPSCs. The GABA_C receptor antagonist 1,2,5,6-tetrahydro-(pyridin-4-yl)methylphosphinic acid decreased IPSCs in 80% of cells tested (by 24.1%), but had no effect on EPSCs. Varying the recording conditions influenced postsynaptic currents. At a holding potential of –60 mV, IPSCs were generally produced with intracellular chloride concentrations of both 5 and 10 mM (total n=24/30). However, with perforated-patch recordings using gramicidin, IPSCs were less frequently encountered (n=5/21), suggesting a higher intracellular chloride concentration in a large proportion of SSC neurones. Further assessment of experimental conditions revealed that two frequently used sodium channel blockers, QX-314 (bromide salt, intracellular) or tetrodotoxin (extracellular), shifted the IPSC reversal potential towards more positive values. Hence, IPSCs were not encountered at −60 mV in their presence. The level of stimulation intensity (minimal or maximal) did not influence IPSC production in these conditions. Thus, the current study describes the pharmacological properties of PSCs in the SSC and highlights the impact of experimental conditions on synaptic transmission, which requires consideration for past and present data reported in this preparation. Electronic Publication