A comparison of release kinetics and glutamate receptor properties in shaping rod–cone differences in EPSC kinetics in the salamander retina

Synaptic transmission from cones is faster than transmission from rods. Using paired simultaneous recordings from photoreceptors and second-order neurones in the salamander retina, we studied the contributions of rod–cone differences in glutamate receptor properties and synaptic release rates to shaping postsynaptic responses. Depolarizing steps evoked sustained calcium currents in rods and cones that in turn produced transient excitatory postsynaptic currents (EPSCs) in horizontal and OFF bipolar cells. Cone-driven EPSCs rose and decayed faster than rod-driven EPSCs, even when comparing inputs from a rod and cone onto the same postsynaptic neurone. Thus, rod–cone differences in EPSCs reflect properties of individual rod and cone synapses. Experiments with selective AMPA and KA agonists and antagonists showed that rods and cones both contact pharmacologically similar AMPA receptors. Spontaneous miniature EPSCs (mEPSCs) exhibited unimodal distributions of amplitude and half-amplitude time width and there were no rod–cone differences in mEPSC properties. To examine how release kinetics shape the EPSC, we convolved mEPSC waveforms with empirically determined release rate functions for rods and cones. The predicted EPSC waveform closely matched the actual EPSC evoked by cones, supporting a quantal release model at the photoreceptor synapse. Convolution with the rod release function also produced a good match in rod-driven cells, although the actual EPSC was often somewhat slower than the predicted EPSC, a discrepancy partly explained by rod–rod coupling. Rod–cone differences in the rates of exocytosis are thus a major factor in producing faster cone-driven responses in second-order retinal neurones.     

Cannabinoids depress excitatory neurotransmission between the subthalamic nucleus and the globus pallidus

The globus pallidus receives its major glutamatergic input from the subthalamic nucleus and subthalamic nucleus neurons synthesize CB1 cannabinoid receptors. The hypothesis of the present work was that CB1 receptors are localized in terminals of subthalamo-pallidal glutamatergic axons and that their activation leads to presynaptic modulation of neurotransmission between these axons and globus pallidus neurons. Patch-clamp studies were carried out on oblique-sagittal mouse brain slices. The subthalamic nucleus was stimulated electrically and the resulting excitatory postsynaptic currents (EPSCs) were recorded in globus pallidus neurons. The mixed CB1/CB2 receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate (WIN55212-2; 3 x 10(-7) M) had no effect on EPSCs. WIN55212-2 (10(-5) M) decreased the amplitude of EPSCs by 44+/-8%. The inhibition by WIN55212-2 (10(-5) M) was prevented by the CB1 antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazolecarboxamide (10(-6) M). WIN55212-2 (10(-5) M) did not change the amplitude of spontaneous EPSCs (sEPSCs) recorded in globus pallidus neurons but lowered their frequency. Moreover, WIN55212-2 (10(-5) M) had no effect on currents elicited by direct activation of postsynaptic receptors on globus pallidus neurons by glutamate (10(-3) M) ejected from a pipette. In a final series of experiments, the firing of subthalamic nucleus neurons was recorded; WIN55212-2 (10(-5) M) did not change the firing of these neurons. The results show that activation of CB1 receptors inhibits glutamatergic neurotransmission between the subthalamic nucleus and the globus pallidus. Lack of effect of cannabinoids on the amplitude of sEPSCs and on currents evoked by direct stimulation of postsynaptic glutamate receptors indicates that the mechanism is presynaptic inhibition of glutamate release from axon terminals. Cannabinoids seem to act preferentially presynaptically: in contrast to their action on axon terminals, they have no effect on somadendritic receptors regulating firing rate. Cannabinoids elicit catalepsy in vivo. The observed inhibition of glutamatergic neurotransmission in the globus pallidus would favor catalepsy.     

Cannabinoid CB1 receptor signaling dichotomously modulates inhibitory and excitatory synaptic transmission in rat inner retina

In the inner retina, ganglion cells (RGCs) integrate and process excitatory signal from bipolar cells (BCs) and inhibitory signal from amacrine cells (ACs). Using multiple labeling immunohistochemistry, we first revealed the expression of the cannabinoid CB1 receptor (CB1R) at the terminals of ACs and BCs in rat retina. By patch-clamp techniques, we then showed how the activation of this receptor dichotomously regulated miniature inhibitory postsynaptic currents (mIPSCs), mediated by GABA_A receptors and glycine receptors, and miniature excitatory postsynaptic currents (mEPSCs), mediated by AMPA receptors, of RGCs in rat retinal slices. WIN55212-2 (WIN), a CB1R agonist, reduced the mIPSC frequency due to an inhibition of L-type Ca²⁺ channels no matter whether AMPA receptors were blocked. In contrast, WIN reduced the mEPSC frequency by suppressing T-type Ca²⁺ channels only when inhibitory inputs to RGCs were present, which could be in part due to less T-type Ca²⁺ channels of cone BCs, presynaptic to RGCs, being in an inactivation state under such condition. This unique feature of CB1R-mediated retrograde regulation provides a novel mechanism for modulating excitatory synaptic transmission in the inner retina. Moreover, depolarization of RGCs suppressed mIPSCs of these cells, an effect that was eliminated by the CB1R antagonist SR141716, suggesting that endocannabinoid is indeed released from RGCs.     

Cannabinoids inhibit excitatory neurotransmission in the substantia nigra pars reticulata

The substantia nigra pars reticulata belongs to the brain regions with the highest density of CB(1) cannabinoid receptors. Since the level of CB(1) receptor messenger RNA is very low in the pars reticulata, most of the receptors are probably localized on terminals of afferent axons. The hypothesis was tested that terminals of glutamatergic afferents of substantia nigra pars reticulata neurons possess CB(1) cannnabinoid receptors, the activation of which presynaptically modulates neurotransmission.Rat midbrain slices were superfused and the electrophysiological properties of substantia nigra pars reticulata neurons were studied with the patch-clamp technique. Focal electrical stimulation in the presence of bicuculline evoked excitatory postsynaptic currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate glutamate receptors. The excitatory postsynaptic currents were reduced by the metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD; 10(-4)M). The mixed CB(1)/CB(2) cannabinoid receptor agonists R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2, 3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone (WIN55212-2; 10(-8)-10(-5)M) and (-)-cis-3-[2-hydroxy-4-(1, 1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940; 10(-6)M) also produced inhibition. The maximal inhibition by WIN55212-2 was 54+/-6%. The CB(1) cannabinoid antagonist N-piperidino-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide (SR141716A; 10(-6)M) prevented the effect of WIN55212-2, but had no effect when superfused alone. WIN55212-2 (10(-6)M) increased the amplitude ratio of two excitatory postsynaptic currents evoked with an interstimulus interval of 100ms. Currents evoked by short ejection of glutamate on to the surface of the slices were not changed by WIN55212-2.The results show that activation of CB(1) cannabinoid receptors inhibits glutamatergic synaptic transmission between afferent axons and neurons in the substantia nigra pars reticulata. The lack of effect of the cannabinoids on glutamate-evoked currents and the increase of the paired-pulse ratio indicate that the mechanism of action is presynaptic inhibition of transmitter release.     

Cannabinoids inhibit striatonigral GABAergic neurotransmission in the mouse

The substantia nigra pars reticulata (SNR) belongs to the brain regions with the highest density of CB(1) cannabinoid receptors. Anatomical studies indicate that the great majority of CB(1) receptors in the SNR are localized on terminals of GABAergic axons arriving from the caudate-putamen (striatonigral axons). The aim of the present experiments was to clarify the role of CB(1) receptors on terminals of striatonigral axons.Oblique sagittal slices, including the caudate-putamen and the substantia nigra, were prepared from brains of young mice. Electrical stimulation in the caudate-putamen elicited GABAergic inhibitory postsynaptic currents (IPSCs) in the SNR, which were studied by patch-clamp techniques. The long latency of IPSCs (14+/-1 ms) suggests that striatonigral axons were indeed activated within the caudate-putamen. The synthetic CB(1)/CB(2) cannabinoid receptor agonist WIN55212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate; 10(-5) M) decreased the amplitude of IPSCs by 93+/-1%. CP55940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; 10(-5) M), another CB(1)/CB(2) receptor agonist, also reduced IPSC amplitude, by 76+/-4%. The CB(1) cannabinoid receptor antagonist SR141716A (N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide; 10(-6) M) prevented the inhibition produced by WIN55212-2 (10(-5) M). Depolarization of SNR neurons led to suppression of IPSCs; this suppression was prevented by SR141716A (10(-6) M). Three observations indicate that the agonists inhibited neurotransmission presynaptically. (1) CP55940 (10(-5) M) enhanced the ratio of amplitudes of two IPSCs which were elicited by two electrical stimuli 100 ms apart (paired pulses). (2) WIN55212-2 (10(-5) M) did not change the amplitude of miniature IPSCs recorded in the presence of tetrodotoxin. (3) WIN55212-2 (10(-5) M) also had no effect on currents elicited in SNR neurons by ejection of the GABA(A) receptor agonist muscimol from a pipet.In summary, we have established a method which allows selective examination of GABAergic neurotransmission between striatonigral axons and SNR neurons. Using this method, the function of CB(1) cannabinoid receptors on terminals of striatonigral axons was unequivocally clarified. Activation of these receptors causes strong presynaptic inhibition of GABAergic neurotransmission between striatonigral axons and SNR neurons. This effect may be one explanation of the catalepsy observed in animals after cannabinoid administration. Endocannabinoids released from SNR neurons can modulate striatonigral neurotransmission by inhibiting GABA release from terminals of striatonigral axons.     

Endogenous adenosine reduces glutamatergic output from rods through activation of A2-like adenosine receptors

Adenosine is released from retina in darkness; photoreceptors possess A2 adenosine receptors, and A2 agonists inhibit L-type Ca2+ currents (ICa) in rods. We therefore investigated whether A2 agonists inhibit rod inputs into second-order neurons and whether selective antagonists to A1, A2A, or A3 receptors prevent Ca2+ influx through rod ICa. [Ca2+]i changes in rods were assessed with fura-2. ICa in rods and light responses of rods and second-order neurons were recorded using perforated patch-clamp techniques in the aquatic tiger salamander retinal slice preparation. Consistent with earlier results using the A2 agonist N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine (DPMA), the A2A agonist CGS-21680 significantly inhibited ICa and depolarization-evoked [Ca2+]i increases in rods. The A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and A2A antagonist, ZM-241385, but not the A3 antagonist, VUF-5574, inhibited effects of adenosine on Ca2+ influx in rods. DPCPX and ZM-241385 also inhibited effects of CGS-21680, suggesting they both act at A2A receptors. Both A2 agonists, CGS-21680 and DPMA, reduced light-evoked currents in second-order neurons but not light-evoked voltage responses of rods, suggesting that activation of A2 receptors inhibits transmitter release from rods. The inhibitory effects of CGS-21680 on both depolarization-evoked Ca2+ influx and light-evoked currents in second-order neurons were antagonized by ZM-241385. By itself, ZM-241385 enhanced the light-evoked currents in second-order neurons, suggesting that endogenous levels of adenosine inhibit transmitter release from rods. The effects of these drugs suggest that endogenous adenosine activates an A2-like adenosine receptor on rods leading to inhibition of ICa, which in turn inhibits l-glutamate release from rod photoreceptors.     

Light-induced resistance changes in retinal rods and cones of the tiger Salamander

1. The electrical properties of retinal rods and cones of the larval tiger salamander were investigated with intracellular electrodes, and the cells identified by means of dye injections.2. Both types of photoreceptors are hyperpolarized by illumination. Following stimulation with brief flashes of dim light, rod responses show a slower time course than cone responses; with bright flashes, rod responses can be recognized because of their long recovery time.3. Values of input resistance were derived from the voltage displacement induced by constant current pulses in darkness or at the peak of the photoresponse. The input resistance following illumination was also calculated from the effect of steady polarizing currents on the amplitude of the photoresponse.4. In darkness, the input resistance of the rod cells is time- and voltage-dependent, but the voltage-current relations of most cells have a linear region which includes the physiological limits of membrane potential. At the peak of the photoresponse, the input resistance (slope of the linear region of the v-i relations) is decreased.5. Cone cells show approximately linear v-i relations. As reported by previous authors, illumination increases the input resistance.6. These results support the current view that the cone photoresponse is the consequence of a reduction in the permeability of channels which in darkness shunt the membrane. In rods, however, it appears that the main effect of illumination is to increase the permeability of the membrane to ions for which the equilibrium potential is more negative than the membrane potential in darkness.     

Functional GABA(B) receptors are expressed at the cone photoreceptor terminals in bullfrog retina

GABA(B) receptors at the cone terminals in bullfrog retina were characterized by immunocytochemical and whole-cell patch clamp techniques in retinal slice preparations. Somata, axons and synaptic terminals (pedicles) of cones were both GABA(B) receptor (GABA(B)R) 1 and GABA(B)R2 immunoreactive. Physiologically, barium/calcium currents of cones to voltage steps were significantly reduced in size when GABA was puffed to cone terminals in the presence of picrotoxin that is supposed to block both GABA(A) and GABA(C) receptors. Similar reduction in barium currents was obtained with puff application of baclofen to cone terminals. These results suggest the presence of functional GABA(B) receptors at the bullfrog cone terminals. Suppression of barium currents of cones by baclofen was dose-dependent. Moreover, barium currents of cones were potentiated by background illumination, as compared with those recorded in the dark. 6,7-Dinitroquinoxaline-2,3-dione, an antagonist of non-NMDA receptors that hyperpolarizes horizontal cells and reduces GABA release from these cells, and saclofen, a GABA(B) receptor antagonist, both potentiated barium currents of cones in the dark, thereby mimicking the effects of background illumination. It is suggested that changes in calcium influx into the cone synaptic terminals due to activation of GABA(B) receptors may provide a negative feedback mechanism for regulating signal transmission between cones and second-order neurons in the retina by modifying the amount of glutamate released from the cones.     

Retinal fine structure in the European eel Anguilla anguilla. VIII. Photoreceptors of the sexually mature silver eel stage

The morphology of the photoreceptors of the sexually mature silver eel has been investigated by electron microscopy and these findings compared with observations made on the photoreceptors of the glass, yellow and sexually immature silver eel stages of the lengthy life cycle of the European eel (Anguilla anguilla). As in previous stages the photoreceptors of the sexually mature silver eel are adequately described as either rods or single cones. The rod: cone ratio at this stage is about 200: 1 which is the highest ratio of any of the stages investigated. Rods and perhaps also cones are capable of photomechanical movements. In the light-adapted stage studied, rod inner segments are the same width in the ellipsoid region as the outer segments but the rod myoid region is much thinner. Rod outer segments in the mature silver eel are slightly longer than that observed in the immature silver eel but remain much the same diameter as in previous stages. Cone outer segments taper distally and are not as wide as the inner segments. Both rods and cones display organelles in the inner segment region indicative of active protein production as it is well established that the inner segment is the synthetic center of these cells. In most cones degenerative changes were noted in the mitochondria of the ellipsoid and in the presence empty vacuoles within their cytoplasm. Cone nuclei are large and vesicular and in the light-adapted state are always located sclerad to the external limiting membrane. Rod nuclei are smaller and more electron dense and located within the outer nuclear layer. Both rods and cones still show both invaginated (ribbon) and superficial synaptic sites. In the sexually mature silver eel, therefore, the rod: cone ratio becomes higher still; the rod outer segments become somewhat longer in length and cone cell degenerative changes are even more widespread.     

Photoreceptor fine structure in light- and dark-adaptation in the butterfly fish (Pantodon buchholzi)

The morphology of the retinal photoreceptors of the butterfly fish Pantodon buchholzi has been studied by electron microscopy in both light- and dark-adaptation. The photoreceptors in this species are readily divisible into rods and cones based on morphological criteria. No double or twin cones are present. The rod photoreceptors show marked retinomotor movements. In light-adaptation they are extremely elongate cells while in the dark-adapted state they are much shorter. Cones seem to respond but minimally to the circadian cycle. Rod outer segments are composed of membranous discs of uniform diameter displaying several incisures. The inner segment has a small distal ellipsoid and a thin myoid region which is lost in dark-adaptation. The nuclei of rods are condensed and always located vitread to the external limiting membrane. The rod synaptic spherule displays 2 or 3 invaginated sites. The single cones display a tapering outer segment. The wider inner segment contains a large electron-dense ellipsoid with small glycogen deposits located peripherally. The cone nuclei are large and vesicular and usually located sclerad to the external limiting membrane. The synaptic pedicle of cones is larger and more electron-lucent and contains more synaptic sites than do the rods. No mosaic pattern of arrangement of the photoreceptors is apparent. Except for the obvious lengthening or shortening of the rods, the morphology of the photoreceptors changes but little during the circadian cycle.     

Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress

Glycinergic interplexiform cells provide a feedback signal from the inner retina to the outer retina. To determine if cones receive such a signal, glycine was applied on cultured porcine cone photoreceptors recorded with the patch clamp technique. A minor population of cone photoreceptors was found to generate large currents in response to puff application of glycine. These currents reversed close to the calculated equilibrium potential for chloride ions. These glycine-elicited currents were sensitive to strychnine but not to picrotoxin consistent with the expression of α–β-heteromeric glycine receptors. Glycine receptors were also activated by taurine and β-alanine. The glycine receptor antibody mAb4a labelled a minority of the cone photoreceptors identified by an antibody specific for cone arrestin. Finally, expression of the β subunit of the glycine receptor was demonstrated by single cell RT-PCR in a similar proportion (∼13%) of cone photoreceptors freshly isolated by lectin-panning. The identity of cone photoreceptors was assessed by their specific expression of the cone arrestin mRNA. The population of cone photoreceptors expressing the glycine receptor was not correlated to a specific colour-sensitive subtype as demonstrated by single cell RT-PCR experiments using primers for S opsin, cone arrestin and glycine receptor β subunit. This glycine receptor expression in a minority of cones defines a new cone population suggesting an unexpected role for glycine in the visual information processing in the outer retina.     

The visual cells of the chicken as revealed by phase contrast microscopy

Phase microscopic investigations of Kolmer-fixed, depigmented sections of the adult chicken retina have provided photomicrographic evidence of the existence of three different photoreceptors: single rods, single cones, and double cones. The rod extends the entire thickness of the visual cell layer and is characterized by a uniformly thick outer segment and a hyperboloid-containing inner segment which is devoid of an oil droplet. The single cone is the shortest element; it contains a red oil droplet. The double cone consists of two unequal members, a tall, slender chief cone and a broad accessory cone. The chief component contains a large yellow oil droplet, whereas the accessory cone houses a small, oval, yellowish-green droplet and a characteristically large, oval paraboloid. The rod hyperboloid and the accessory cone paraboloid contain glycogen. No colorless droplets have been observed. Owing to the close association between oil droplet color and cone type, three colored layers of oil droplets are formed within the thickness of the retina: a proximal row of red droplets (the short, single cones), an intermediate layer of yellowish-green droplets (the accessory cones), and a distal row of yellow droplets (the tall chief cones).     

Ectopic synaptogenesis during retinal degeneration in the royal college of surgeons rat

Rod photoreceptor-specific mutations cause ectopic synapses to form between cone photoreceptor terminals and rod bipolar cell dendrites in degenerating retinas of rhodopsin transgenic (P347L) pigs and retinal degeneration mice. Since the mutations occur in rod photoreceptor-specific genes in these two models, it is not known if ectopic synaptogenesis occurs specifically due to some rod photoreceptor cell-autonomous properties of a mutation or as a general consequence of photoreceptor degeneration. In the Royal College of Surgeons (RCS) rat, a mutation in the receptor tyrosine kinase gene, Mertk, causes failure of the retinal pigment epithelial (RPE) cells to phagocytose shed photoreceptor outer segments; subsequently, both rod and cone photoreceptors die. The non-phagocytic phenotype of the RCS rat is RPE cell-autonomous and the photoreceptors degenerate secondarily. Here we show that in 35-day-old RCS rats, where a majority of rod and cone photoreceptors remained, rod bipolar cell dendrites had abnormal (flat-contact type) synaptic contacts with rod and cone terminals.Demonstration of ectopic synapses in the RCS rat suggested that ectopic synaptogenesis could occur as a result of photoreceptor degeneration, even when the rods and cones were developmentally normal. This further supported the hypothesis that ectopic synaptogenesis may be a common step in the disease progression of different forms of retinal degeneration that include photoreceptor death as a feature, such as retinitis pigmentosa.     

Retinal fine structure in the European eel Anguilla anguilla. VI. Photoreceptors of the sexually immature silver eel stage

This report deals with the fine structure of the photoreceptors of the sexually immature silver eel and compares these findings with previous observations on the glass and yellow eel stages of the life cycle of the European eel. The photoreceptors of the sexually immature glass eel are readily categorized as either rods or cones. No multiple cones are observed. The rod:cone ratio is about 100:1, which is greater than that of either the glass or yellow eel stages. Rods and perhaps cones also are capable of retinomotor responses. In the light-adapted condition, rod inner segments are the same width as the outer segments but narrow abruptly below the ellipsoid to form the myoid. Rod outer segments in the immature glass eel are longer but much the same diameter as that noted for the glass and yellow eel. Cone inner segments are much wider than the outer segments which taper distally. Both rods and cones display organelles in the inner segment region indicative of protein production. Most of the cones observed at this stage showed some degenerative signs mostly as empty vacuoles within their cytoplasm. Cone nuclei are large and vesicular and in the light-adapted state are invariably located sclerad to the external limiting membrane while rod nuclei are small and dense and located vitread to this membrane. Both rods and cones display both invaginated and superficial synaptic sites. The most obvious changes noted in the immature silver eel photoreceptor population are a marked rise in the rod:cone ratio and a lengthening of rod outer segments.     

Functional expression of cell surface cannabinoid CB(1) receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons

At present, little is known about the mechanisms by which cannabinoids exert their effects on the central nervous system. In this study, fluorescence imaging and electrophysiological techniques were used to investigate the functional relationship between cell surface cannabinoid type 1 (CB(1)) receptors and GABAergic synaptic transmission in cultured hippocampal neurons. CB(1) receptors were labelled on living neurons using a polyclonal antibody directed against the N-terminal 77 amino acid residues of the rat cloned CB(1) receptor. Highly punctate CB(1) receptor labelling was observed on fine axons and at axonal growth cones, with little somatic labelling. The majority of these sites were associated with synaptic terminals, identified either with immunohistochemical markers or by using the styryl dye FM1-43 to label synaptic vesicles that had undergone active turnover. Dual labelling of neurons for CB(1) receptors with either the inhibitory neurotransmitter GABA or its synthesising enzyme glutamate decarboxylase, demonstrated a strong correspondence. The immunocytochemical data was supported by functional studies using whole-cell patch-clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs). The cannabinoid agonist WIN55,212-2 (100nM) markedly inhibited (by 77+/-6.3%) the frequency of pharmacologically-isolated GABAergic mIPSCs. The effects of WIN55,212-2 were blocked in the presence of the selective CB(1) receptor antagonist SR141716A (100nM).In conclusion, the present data show that cell surface CB(1) receptors are expressed at presynaptic GABAergic terminals, where their activation inhibits GABA release. Their presence on growth cones could indicate a role in the targeting of inhibitory connections during development.     

Photoreceptor fine structure in the archerfish (Toxotes jaculatrix)

The fine structure and arrangement of the photoreceptor cells of the archerfish (Toxotes jaculatrix) have been studied by electron microscopy. Rods, twin cones, and single cones are present. In the light-adapted state, the rods are very tall cells reaching almost to the base of the retinal epithelial cells. The outer segment is composed of membraneous discs of uniform diameter displaying a single incisure. The rod inner segment displays a distal small ellipsoid and an extremely thin myoid region. The nuclei of rods are electron-dense, and the synaptic spherule displays two or three invaginated sites. The single cone is similar to the individual members of a twin cone and displays a tapering outer segment and accompanying accessory outer segment. The wider cone inner segment contains a large, centrally loated ellipsoid and a peripheral region rich in endoplasmic reticulum, polysomes, and microfilaments. Twin cones display subsurface cisternae along their entire contiguous surfaces. The cone nuclei are large and vesicular and located vitread to the external limiting membrane. The synaptic pedicle of cones is lager and moe electron-lucent and contains more invaginated “ribbon” synaptic sites (ten to 12) than do rods. In addition, small “coated” invagination and larger synaptic vesicle-filled processes are also seen within cone pedicles. In the light-adapted state the cone photoreceptors are arranged in a repeating square mosaic pattern with one single cone surrounded by four twin cones.     

Retinal photoreceptor fine structure in the velvet cichlid (Astronotus ocellatus).

Summary The structure and arrangement of the retinal photoreceptors of the velvet cichlid fish (Astronotus ocellatus) have been studied by light and electron microscopy. Rods, single cones and double (twin) cones are present. In the light-adapted state, rods are very tall cells that reach deep into the retinal epithelial (RPE) layer. The long outer segment is composed of discs of uniform diameter displaying one or two incisures. The rod inner segment shows a distal ellipsoid of mitochondria, and then narrows dramatically in the myoid region. Rod nuclei are electron dense and located deep in the outer nuclear layer. Rod synaptic spherules are small and show two to three invaginated synaptic sites as well as superficial synapses. Single cones are similar to the individual members of a double cone and all display a short tapering outer segment, a large ellipsoid of mitochondria and a myoid rich in rough endoplasmic reticulum, polysomes, Golgi zones and autophagic vacuoles. Double cones have extensive subsurface cisternae along their entire contiguous surfaces. Cone nuclei are large and vesicular and located close to or through the external limiting membrane. The synaptic pedicles of cones are larger, more electron lucent, and display more invaginated (ribbon) synapses as well as conventional (superficial) synaptic sites than do the rod spherules. Rod photoreceptors certainly undergo retinomotor movements and it is probable that cones do as well. In the light-adapted state the cone photoreceptors are arranged in a repeating square mosaic pattern with one single cone surrounded by four double (twin) cones.     

Cannabinoid receptor modulation of synapses received by cerebellar Purkinje cells

The high density of cannabinoid receptors in the cerebellum and the degradation of motor coordination produced by cannabinoid intoxication suggest that synaptic transmission in the cerebellum may be strongly regulated by cannabinoid receptors. Therefore the effects of exogenous cannabinoids on synapses received by Purkinje cells were investigated in rat cerebellar slices. Parallel fiber-evoked (PF) excitatory postsynaptic currents (EPSCs) were strongly inhibited by bath application of the cannabinoid receptor agonist WIN 55212-2 (5 microM, 12% of baseline EPSC amplitude). This effect was completely blocked by the cannabinoid CB1 receptor antagonist SR 141716. It is unlikely that this was the result of alterations in axonal excitability because fiber volley velocity and kinetics were unchanged and a cannabinoid-induced decrease in fiber volley amplitude was very minor (93% of baseline). WIN 55212-2 had no effect on the amplitude or frequency of spontaneously occurring miniature EPSCs (mEPSCs), suggesting that the effect of CB1 receptor activation on PF EPSCs was presynaptically expressed, but giving no evidence for modulation of release processes after Ca(2+) influx. EPSCs evoked by climbing fiber (CF) stimulation were less powerfully attenuated by WIN 55212-2 (5 microM, 74% of baseline). Large, action potential-dependent, spontaneously occurring inhibitory postsynaptic currents (sIPSCs) were either severely reduced in amplitude (<25% of baseline) or eliminated. Miniature IPSCs (mIPSCs) were reduced in frequency (52% of baseline) but not in amplitude, demonstrating suppression of presynaptic vesicle release processes after Ca(2+) influx and suggesting an absence of postsynaptic modulation. The decrease in mIPSC frequency was not large enough to account for the decrease in sIPSC amplitude, suggesting that presynaptic voltage-gated channel modulation was also involved. Thus, while CB1 receptor activation reduced neurotransmitter release at all major classes of Purkinje cell synapses, this was not accomplished by a single molecular mechanism. At excitatory synapses, cannabinoid suppression of neurotransmitter release was mediated by modulation of voltage-gated channels in the presynaptic axon terminal. At inhibitory synapses, in addition to modulation of presynaptic voltage-gated channels, suppression of the downstream vesicle release machinery also played a large role.     

The dynamic range and domain-specific signals of intracellular calcium in photoreceptors

Vertebrate photoreceptors consist of strictly delimited subcellular domains: the outer segment, ellipsoid, cell body and synaptic terminal, each hosting crucial cellular functions, including phototransduction, oxidative metabolism, gene expression and transmitter release. We used optical imaging to explore the spatiotemporal dynamics of Ca(2+) signaling in non-outer segment regions of rods and cones. Sustained depolarization, designed to emulate photoreceptor activation in the darkness, evoked a standing Ca(2+) gradient in tiger salamander photoreceptors with spatially-averaged intracellular Ca(2+) concentration within synaptic terminals of approximately 2 microM and lower (approximately 750 nM) intracellular calcium concentration in the ellipsoid. Measurements from axotomized cell bodies and isolated ellipsoids showed that Ca(2+) enters the two compartments via both local L-type Ca(2+) channels and diffusion. The results from optical imaging studies were supported by immunostaining analysis. L-type voltage-operated Ca(2+) channels and plasma membrane Ca(2+) ATPases were highly expressed in synaptic terminals with progressively lower expression levels in the cell body and ellipsoid. These results show photoreceptor Ca(2+) homeostasis is controlled in a region-specific manner by direct Ca(2+) entry and diffusion as well as Ca(2+) extrusion. Moreover, quantitative measurement of intracellular calcium concentration levels in different photoreceptor compartments indicates that the dynamic range of Ca(2+) signaling in photoreceptors is approximately 40-fold, from approximately 50 nM in the light to approximately 2 microM in darkness.     

Photoreceptor fine structure in the goldeye (Hiodon alosoides)(Teleost)

Summary The retinal photoreceptors of the goldeye (Hiodon alosoides) are arranged in large bundles of 40–50 cells optically isolated from other bundles by the retinal epithelial cells. Within each bundle are found both rods and cones in roughly equal numbers. Rod photoreceptors show marked retinomotor responses to project beyond the photoreceptor bundle in light-adaptation and to lie entirely within the bundle in dark-adaptation. In all stages of the light cycle cone outer segments remain at the apex of the photoreceptor bundle. In light-adaptation, rod inner segments display an apical ellipsoid separated from a basal ellipsoid by the greatly elongated myoid. In dark-adaptation the rod inner segment is much the same diameter throughout its length. In both rods and cones, profiles of rough and smooth endoplasmic reticulum and Golgi zones are present in a supranuclear location. The nuclei of rods display little heterochromatin and are located vitreal to the external limiting membrane in light-adaptation, whereas in dark-adaptation more heterochromatin is noted and the nuclei lie scleral to the external limiting membrane. Cone nuclei display the same changes in chromatin pattern as rods but they show changes in nuclear location opposite that of the rods. Throughout its length, the rod photoreceptor cytoplasm is more electron dense than that of the cone. The synaptic spherule, of rods displays 2–3 invaginated synaptic, sites while the cone pedicle is larger and presents 8–10 invaginated synaptic sites. Both rods and cones also appear to have superficial synaptic sites. Membrane specializations are found along the length of the inner segments where rods and cones are contiguous. These may act as sites of intercellular communication and the whole photoreceptor bundle may therefore be considered, as a macroreceptor.