Bleaching of mouse rods: microspectrophotometry and suction-electrode recording.

When a substantial fraction of rhodopsin in a rod photoreceptor is exposed to bright light, the rod is desensitized by a process known as bleaching adaptation. Experiments on isolated photoreceptors in amphibians have revealed many of the features of bleaching adaptation, but such experiments have not so far been possible in mammals. We now describe a method for making microspectrophotometric measurements of pigment concentration and suction-electrode recording of electrical responses over a wide range of bleaching exposures from isolated mouse rods or pieces of mouse retina. We show that if pigment is bleached at a low rate in the presence of bovine serum albumin (BSA), and intermediate photoproducts are allowed to decay, mouse rods are stably desensitized; subsequent treatment with exogenous 11-cis retinal results in pigment regeneration and substantial recovery of sensitivity to the dark-adapted value. Stably bleached wild-type (WT) rods show a decrease in circulating current and acceleration of the time course of decay, much as in steady background light; similar effects are seen in guanylyl cyclase-activating protein knockout (GCAPs(-/-)) rods, indicating that regulation of guanylyl cyclase is not necessary for at least a part of the adaptation produced by bleaching. Our experiments demonstrate that in mammalian rods, as in amphibian rods, steady-state desensitization after bleaching is produced by two components: (1) a reduction in the probability of photon absorption produced by a decrease in rhodopsin concentration; and (2) an equivalent background light whose intensity is proportional to the fraction of bleached pigment, and which adapts the rod like real background light. These two mechanisms together fully account for the 'log-linear' relationship in mammalian retina between sensitivity and per cent bleach, which can be measured in the steady state following exposure to bright light. Our methods will now make possible an examination of bleaching adaptation and pigment regeneration in mouse animal lines with mutations or other alterations in the proteins of transduction.

Replacing the rod with the cone transducin subunit decreases sensitivity and accelerates response decay.

Cone vision is less sensitive than rod vision. Much of this difference can be attributed to the photoreceptors themselves, but the reason why the cones are less sensitive is still unknown. Recent recordings indicate that one important factor may be a difference in the rate of activation of cone transduction; that is, the rising phase of the cone response per bleached rhodopsin molecule (Rh*) has a smaller slope than the rising phase of the rod response per Rh*, perhaps because some step between Rh* and activation of the phosphodiesterase 6 (PDE6) effector molecule occurs with less gain. Since rods and cones have different G-protein alpha subunits, and since this subunit (Talpha) plays a key role both in the interaction of G-protein with Rh* and the activation of PDE6, we investigated the mechanism of the amplification difference by expressing cone Talpha in rod Talpha-knockout rods to produce so-called GNAT2C mice. We show that rods in GNAT2C mice have decreased sensitivity and a rate of activation half that of wild-type (WT) mouse rods. Furthermore, GNAT2C responses recover more rapidly than WT responses with kinetic parameters resembling those of native mouse cones. Our results show for the first time that part of the difference in sensitivity and response kinetics between rods and cones may be the result of a difference in the G-protein alpha subunit. They also indicate more generally that the molecular nature of G-protein alpha may play an important role in the kinetics of G-protein cascades for metabotropic receptors throughout the body.

300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses.

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.

Amplitude, kinetics, and reversibility of a light-induced decrease in guanosine 3',5'-cyclic monophosphate in frog photoreceptor membranes.

The concentration of guanosine 3',5'-cyclic monophosphate (cyclic GMP) has been examined in suspensions of freshly isolated frog rod outer segments using conditions which previously have been shown to maintain the ability of outer segments to perform a light-induced permeability change (presence of calf serum, anti-oxidant, and low calcium concentration). Illumination causes a rapid decrease in cyclic GMP levels which has a half-time approximately 125 ms. With light exposures that bleach less than 100 rhodopsin molecules in each rod outer segment, at least 10(4)-10(5) molecules of cyclic GMP are hydrolyzed for each rhodopsin molecule bleached. Half of the total cyclic GMP in each outer segment, approximately 2 X 10(7) molecules, is contained in the light-sensitive pool. If outer segments are exposed to continuous illumination, using intensities which bleach between 5.0 X 10(1) and 5.0 X 10(4) rhodopsin molecules/outer segment per second, cyclic GMP levels fall to a value characteristic for the intensity used. This suggests that a balance between synthesis and degradation of cyclic GMP is established. This constant level appears to be regulated by the rate of bleaching rhodopsin molecules (by the intensity of illumination), not the absolute number of rhodopsin molecules bleached...

Ca2+-dependent changes in cyclic GMP levels are not correlated with opening and closing of the light-dependent permeability of toad photoreceptors.

We have measured the levels of 3',5'-guanosine monophosphate (cyclic GMP) in isolated retinas from toad to investigate their correlation to the opening and closing of the light-dependent permeability of photoreceptors. When Ca2+-induced changes in cyclic GMP concentration are compared with the Ca2+-induced changes in the permeability of photoreceptor light-dependent channel, four quantitative dissimilarities are noted. First, when extracellular Ca2+ ([Ca2+]o) is reduced from normal physiological levels to between 10(-6) and 10(-7) M, the light-dependent permeability is increased, but cyclic GMP levels are not significantly changed. Second, when [Ca2+]o is increased from 1.8 to 20 mM, the light-dependent permeability is suppressed, but cyclic GMP levels are decreased by only 10-15%, about one-quarter the decrease that can be obtained with bright illumination. Third, when [Ca2+]o is increased from 10(-8) M to 20 mM, the light-dependent permeability is closed rapidly, but the cyclic GMP decrease is slow. Fourth, when [Ca2+]o is lowered to 10(-8) M, the sensitivity of the light-dependent permeability to steady illumination is decreased by three to four orders of magnitude, but the sensitivity of the light-dependent decrease in cyclic GMP is not significantly affected. These observations indicate that there is no simple correlation between cyclic GMP levels and the permeability of the light-dependent channels and that Ca2+ can affect the conductance in the absence of changes in cyclic GMP content.

Light-dependent ion influx into toad photoreceptors.

To measure the influx of Na+ and other ions through the light-dependent permeability of photoreceptors, we superfused the isolated retina of the toad, Bufo marinus, with a low-Ca2+ (10(-8) M), low-Cl- Ringer's solution containing 0.5 mM ouabain. Under these conditions, the membrane potential of the rod is near zero and there is no light-induced potential change either in the rod or in more proximal neurons. The photoreceptors, however, continue to show a light-dependent increase in membrane resistance, which indicates that the light-sensitive channels still close with illumination. Dark-adapted retinas show a larger 22Na+ accumulation than do light-adapted retinas. The extra accumulation of 22Na+ into dark-adapted retinas can be removed if the retinas are washed in darkness with low-Ca2+ Ringer's solutions, or if the ionophore gramicidin D is present in the perfusate. The additional accumulation in dark retinas corresponds to a flux of at least 10(9) Na+ per receptor per second, which is close to the value of the photoreceptor dark current. The light-dependent uptake of 22Na+ can be prevented by exposing the retinas to Ca2+ during the incubation period, but is restored if the phosphodiesterase inhibitor IBMX is added to the perfusate. A significant light-dependent ion accumulation can be observed for the cations K+, Rb+, Cs+, and Tl+, in addition to Na+, but not for methylamine, choline, or tetraethylammonium.

Guanosine 3',5'-cyclic monophosphate and the in vitro physiology of frog photoreceptor membranes.

Frog rod outer segments freshly detached from dark-adapted retinas contain approximately 1-2 molecules of guanosine 3',5'-cyclic monophosphate (cyclic GMP) for every 100 molecules of visual pigment present. This cyclic GMP decays to 5'-GMP, and the conversion is accelerated upon illumination of the outer segments. Bleaching one rhodopsin molecule can lead to the hydrolysis of 1,000-2,000 molecules of cyclic GMP within 100-300 ms. The decline in cyclic GMP concentration becomes larger as illumination increases, and varies with the logarithm of light intensity at levels which bleach between 5 X 10(2) and 5 X 10(5) rhodopsin molecules per outer segment-second. Light suppression of plasma membrane permeability, assayed in vitro as light suppression of outer segment swelling in a modified Ringer's solution, occurs over this same range of light intensity. The correlation between cyclic GMP and permeability or swelling is maintained in the presence of two pharmacological perturbations: papaverine, a phosphodiesterase inhibitor, increases both cyclic GMP levels and the dark permeability of the plasma membrane; and beta,gamma-methylene ATP increases the effectiveness of light in suppressing both permeability and cyclic GMP levels.

Lesions of the dorsal spinal cord decrease the duration of contact defensive immobility (animal hypnosis) in the rabbit.

Rabbits received either bilateral dorsal or unilateral dorsolateral spinal cord lesions. The duration and incidence of contact defensive immobility (CDI; animal hypnosis) were tested in these rabbits and in intact controls. Neither of the spinal cord lesions affected the number of CDI inductions, but rabbits with lesions of the dorsal spinal cord exhibited significantly shorter durations of CDI than either of the other groups which did not differ from each other. These results are interpreted to indicate that the somesthetic systems that ascend in the dorsal spinal cord are important for the maintenance, but not the initiation, of CDI.

Fornix lesions, plasma ACTH levels, and shuttle box avoidance in rats.

Male hooded rats served as subjects for the two experiments conducted in this study. In Experiment 1, the consequences of fornix lesions for partial acquisition, and performance at three postacquisition time intervals, of shuttle box avoidance were tested. Fornix-damaged rats achieved criterion performance more rapidly than controls. The performance of control rats was depressed when they were tested 1 hr after acquisition ("Kamin effect"), but the performance of fornix-damaged rats was not impaired. The possibility that fornix lesions altered plasma ACTH concentrations at the intervals after avoidance training used in Experiment 1 was examined in Experiment 2. Plasma ACTH concentrations were elevated in rats with fornix lesions, compared with those in unoperated and sham-operated controls, both immediately after and 1 hr after avoidance training. Plasma ACTH levels were not elevated in fornix-damaged rats following either stress alone or 24 hr after avoidance training. These results are interpreted to indicate that fornix lesions cause elevated ACTH levels in rats after avoidance training and that the elevated plasma ACTH concentrations in these rats are responsible for elimination of the Kamin effect. In addition, these data support the contention that enhanced acquisition of two-way active avoidance by rats with fornix or hippocampal damage might be attributable to increased plasma ACTH levels.

Effects of transplantation of fetal hippocampal or hindbrain tissue into the brains of adult rats with hippocampal lesions on water maze acquisition.

Rats were given bilateral aspiration lesions of the hippocampus. Some of these rats then received bilateral transplants of fetal hippocampal or dorsal ventricular ridge tissue that was dissected from embryonic rat brains at 16 or 17 days of gestation. The remaining rats with hippocampal lesions did not receive fetal brain transplants. Rats with neocortical aspiration lesions, but without transplants, and rats without brain damage were also included in the study. All of the rats were trained to find a submerged platform in a Morris water maze. Rats with the fetal brain transplants were more impaired in some measures of maze learning than were rats with hippocampal lesions only. The results indicate that transplants of fetal brain tissue are not always associated with recovery of behavioral function after brain damage and may even increase a lesion-induced behavioral impairment in tasks that require complex cognitive functioning.

Fetal Ammon's horn transplants improve acquisition of a radial arm maze and a low-rate operant schedule in trimethyltin-treated rats.

The results of previous studies indicated that block grafts of fetal hippocampal tissue made into cavities produced by aspiration lesions of the hippocampus in rats given the neurotoxin trimethyltin (TMT) significantly worsened the TMT-induced deficit in water maze acquisition. The purpose of the present study was to test the hypothesis that a procedure for transplantation that produced less destruction to the host brain and resulted in transplants with less mass might produce recovery in a spatial learning task in TMT-exposed rats. Acquisition of an externally cued (spatial) version of the radial arm maze (RAM), an internally cued version of the RAM, and of a differential reinforcement of low rate (DRL) operant schedule was assessed in normal rats, rats given TMT, and rats given TMT and stereotaxic implants of either fetal Ammon's horn or entorhinal cortex. The rats receiving Ammon's horn transplants made significantly fewer reentries into the baited arms in both maze configurations and fewer reentries into the nonbaited arms in the spatial RAM than rats in the TMT-only and TMT/entorhinal cortex transplant groups. The rats receiving transplants of Ammon's horn made significantly fewer responses and received more reinforcements during training on the DRL-20 schedule than rats receiving just TMT or rats receiving TMT and transplants of fetal entorhinal cortex. These results support the proposal that transplantation procedures that cause less damage to the host brain and result in transplants that do not occupy a large extent of the ventricular space increase the probability of behavioral recovery.

Selected contribution: PKC activation inhibits Ca(2+) signaling in tracheal epithelial cells kept in simulated microgravity.

Microgravity has been shown to alter protein kinase C (PKC) activity; therefore, we investigated whether microgravity influences mechanically stimulated Ca(2+) signaling and ATP-induced Ca(2+) oscillations, both of which are modulated by PKC. Rabbit tracheal epithelial outgrowth cultures or suspended epithelial sheets were rotated in bioreactors to simulate microgravity. Mechanical stimulation of a single cell increased the cytosolic Ca(2+) concentration in 35-55 cells of both outgrowth cultures and epithelial sheets kept at unit gravity (G) or in simulated microgravity (smicroG). In outgrowth cultures, 12-O-tetradecanoylphorbol-13-acetate (TPA; 80 nM), a PKC activator, restricted Ca(2+) "waves" to about 10 cells in unit G and to significantly fewer cells in smicroG. TPA only slightly reduced the spread of Ca(2+) waves in epithelial sheets kept in smicroG but did not inhibit Ca(2+) waves of sheets kept in unit G. In both cell preparations from both conditions, TPA inhibited ATP-induced Ca(2+) oscillations; however, the effect was more pronounced in cells kept in smicroG. These results suggest that PKC activation is more robust in cells subjected to smicroG.

Effects of electrical stimulation of the pontine A5 cell group on blood pressure and heart rate in the rabbit.

The effects of electrical stimulation of the A5 noradrenergic cell group of the ventrolateral pons was assessed in rabbits. Stimulation administered through either concentric bipolar or monopolar electrodes produced current-intensity related increases in mean arterial pressure (MAP). Decreases in heart rate (HR) accompanied the increases in MAP, but were essentially eliminated by bilateral vagotomy or destruction of the nucleus and tractus solitarii (NTS), thereby indicating that the HR decelerations were secondary to activation of baroreceptor reflexes. Neither vagotomy nor midcollicular section of the brainstem altered the MAP response to A5 stimulation. Bilateral destruction of the NTS slightly enhanced the response. Several rabbits received microinjections of 6-hydroxydopamine (6-OHDA) into the A5 region 2 weeks before the experiment. Other rabbits received vehicle injections and served as control subjects for the non-specific effects of the 6-OHDA injections. 6-OHDA injections, but not vehicle injections, prevented the vasopressor effects of A5 stimulation. However, stimulation of the A1 noradrenergic nucleus of the ventrolateral medulla produced decreases in MAP in rabbits given prior microinjections of 6-OHDA into A5. These observations are interpreted to indicate that the 6-OHDA injections produced neurotoxic effects which were relatively restricted to the A5 region. Furthermore, the data from all of these experiments are interpreted as indicating that the vasopressor effects observed as a consequence of electrical stimulation of A5 are due to excitation of the noradrenaline-containing neuron cell bodies of this region and that this effect is mediated via pathways arising from this region and terminating in the intermediolateral cell column of the spinal cord.

Cerebellar nuclear lesions in rats: subsequent avoidance behavior and ascending anatomical connections.

Bilateral lesions of the rat cerebellar dentate and lateral interposed nuclei produced transient deficits in movement and posture, and facilitated acquisition of two-way active avoidance. Bilateral lesions of the fastigial and medial interposed nuclei of the rat cerebellum also produced transient deficits in movement and posture, but impaired acquisition of the avoidance task. Analysis of degeneration patterns after unilateral lesions to either the lateral or medial nuclear region indicated that the lateral area has a denser rostral projection than the medial area, while the medial nuclear region has a heavier caudal projection. It is suggested that these differences in anatomic connections may be related to the observed differences in lesion effect on two-way active avoidance.

Cholinergic and non-cholinergic septo-hippocampal projections: a double-label horseradish peroxidase-acetylcholinesterase study in the rabbit.

The existence of a massive cholinergic projection from cells in the medial septal nucleus (MS) and nucleus of the diagonal band (DB) to the hippocampal formation has been recognized for some time. However, the actual percentages of cholinergic and non-cholinergic neurons in the MS and DB which project to the hippocampus have not been reported. A procedure which combines horseradish peroxidase (HRP) and acetylcholinesterase (AChE) histochemistry in the same tissue was used to determine these percentages in the rabbit. Less than 50% of the neurons in the MS and DB which were labeled with reaction product following an HRP injection into the dorsal hippocampus also stained for AChE. Moreover, 70% of all neurons containing HRP reaction product were located in the DB, but neurons in the DB could not be differentiated from those in the MS on the basis of size or morphology. These data are taken to indicate that much of the MS-DB hippocampal projection is not cholinergic. Substance P is suggested as another possible transmitter within this anatomical system.

Embryonic hippocampal grafts ameliorate the deficit in DRL acquisition produced by hippocampectomy.

Transplants of fetal neural tissue survive and develop in lesion cavities produced in adult rats. The present experiment tested the effect of grafting fetal hippocampal or brainstem tissue on the ability of rats with hippocampal lesions to perform on a differential reinforcement of low response rate (DRL) operant schedule. The DRL interval was 20s. Eighty-six percent of the hippocampal grafts and 69% of the brainstem grafts developed to maturity. Inspection of sections from rats in which the mature transplant had been injected with Fast blue, indicated that these grafts formed connections with the host brain. Consistent with previous reports, rats with hippocampal lesions were impaired in performance of the DRL task. Rats given fetal grafts of hippocampal tissue into the hippocampal lesion site on the day of lesion production were significantly better in performance of the DRL requirement than were lesion-only rats or rats receiving grafts of fetal brainstem tissue. The results of this study confirm that grafts of fetal brain tissue can both develop in a lesion site in an adult brain and ameliorate lesion-induced behavioral deficits.

Angiotensin II binding sites in the hamster brain: localization and subtype distribution.

This study was designed to characterize the distribution of angiotensin II (AII) binding sites in the hamster brain. Brain sections were incubated with [125I][sar1,ile8]-angiotensin II in the absence and presence of angiotensin II receptor subtype selective compounds, losartan (AT1 subtype) and PD123177 (AT2 subtype). Binding was quantified by densitometric analysis of autoradiograms and localized by comparison with adjacent thionein stained sections. The distribution of AII binding sites was similar to that found in the rat, with some exceptions. [125I][sar1,ile8]-angiotensin II binding was not evident in the subthalamic nucleus and thalamic regions, inferior olive, suprachiasmatic nucleus, and piriform cortex of the hamster, regions of prominent binding in the rat brain. However, intense binding was observed in the interpeduncular nucleus and the medial habenula of the hamster, nuclei void of binding in the rat brain. Competition with receptor subtype selective compounds revealed a similar AII receptor subtype profile in brain regions where binding is evident in both species. One notable exception is the medial geniculate nucleus, predominately AT1 binding sites in the hamster but AT2 in the rat. Generally, the AII binding site distribution in the hamster brain parallels that of the other species studied, particularly in brain regions associated with cardiovascular and dipsogenic functions. Functional correlates for AII binding sites have not been elucidated in the majority of brain regions and species mismatches might provide clues in this regard.

The motor innervation of the single-bellied digastric muscle in the rabbit: a retrograde horseradish peroxidase study.

The digastric muscle of the rabbit consists of a single anterior belly which inserts onto the lower jaw. Horseradish peroxidase was injected into the muscle and into subcutaneous regions overlying the lower jaw to determine the sites of origin of the motor innervation to both the digastric muscle and the platysma muscles. After digastric muscle injection, labelled cells were found in the ipsilateral retrotrigeminal nucleus as well as in the intermediate subnucleus of the main facial nucleus on both sides. Subcutaneous injections produced labelling which was found bilaterally in the intermediate subnucleus and in the ventromedial portion of the medial subnucleus. These results are interpreted in relation to the common embryological origin of these two muscles and their innervation.

The effects of stimulation of the A5 region on blood pressure and heart rate in rabbits.

The effects of stimulation of the A5 cell group of the caudal ventrolateral pons electrically or with L-glutamate on heart rate and blood pressure were determined in rabbits. Electrical stimulation caused blood pressure increases and reflex bradycardia. L-glutamate caused decreases in blood pressure and heart rate which were blocked by the L-glutamate antagonist aminophosphoheptanoic acid. Transection of the brainstem at the level of the midbrain did not alter the effects of either electrical or chemical stimulation. Lesions of the nucleus and tractus solitarius (NTS) attenuated the effects of L-glutamate, but did not change the effectiveness of electrical stimulation. Injections of 6-hydroxydopamine three to four weeks before the experiments blocked the effects of electrical stimulation but only reduced the effects of L-glutamate injection. The A5 group may have two functional subdivisions. Some A5 cells may produce blood pressure depressor and bradycardic effects by means of projections to the NTS and the spinal cord. Other A5 cells may produce blood pressure pressor effects by means of projections to the spinal cord.

Anatomical and behavioral sequelae of fetal brain transplants in rats with trimethyltin-induced neurodegeneration.

The effect of transplants of either fetal hippocampal or dorsal ventricular ridge (DVR) tissue into the brains of adult male rats exposed to TMT was determined for two behavioral tasks. Administration of TMT produced deficits in acquisition and performance of an operant differential reinforcement of low response rates (DRL) schedule and learning in the Morris water maze. The fetal transplants developed well within the TMT-damaged brains of the adult rats and numerous axons could be shown to cross the host-transplant interface. The transplants significantly reduced the DRL deficit produced by exposure to TMT. However, the TMT-induced deficit in water maze acquisition was made significantly worse by the hippocampal transplants. The improvement in DRL performance is attributed to the effect on the host brain of an unidentified trophic substance produced by the transplants. However, this positive effect may not protect the brain sufficiently to produce recovery in tasks demanding more complex neural computations than are required to withhold lever-press responses. The transplant-induced deficit observed in some aspects of water maze acquisition and performance may be attributable to either a tumor-like deleterious effect of the mass of the transplant or to abnormal neuronal activity transmitted from the transplant to the host brain. The results of the present study, and those from other similar studies, suggest that transplants of fetal tissue may be useful in producing changes in the brain of an animal exposed to an environmental neurotoxin, but that research should be focused upon development of transplant methodology that will minimize adverse effects of the grafts.