Multiple paternity and sperm storage in captive Hermann's tortoises, Testudo hermanni boettgeri determined from amniotic fluid adhering to the eggshell.

We identified multiple paternity in 52.9% of the clutches of Hermann's tortoise Testudo hermanni boettgeri using polymorphic microsatellite markers. In addition we demonstrated sperm storage across seasons. DNA was extracted from the amniotic fluid adhering to the eggshell's inner surface, a procedure suitable for easy, non-invasive DNA sampling in conservation and breeding programs. To improve the informative value of monomorphic single tandem repeat (STR) markers we additionally analyzed single nucleotide polymorphism (SNP) variability.

Attention and normalization circuits in macaque V1.

Attention affects neuronal processing and improves behavioural performance. In extrastriate visual cortex these effects have been explained by normalization models, which assume that attention influences the circuit that mediates surround suppression. While normalization models have been able to explain attentional effects, their validity has rarely been tested against alternative models. Here we investigate how attention and surround/mask stimuli affect neuronal firing rates and orientation tuning in macaque V1. Surround/mask stimuli provide an estimate to what extent V1 neurons are affected by normalization, which was compared against effects of spatial top down attention. For some attention/surround effect comparisons, the strength of attentional modulation was correlated with the strength of surround modulation, suggesting that attention and surround/mask stimulation (i.e. normalization) might use a common mechanism. To explore this in detail, we fitted multiplicative and additive models of attention to our data. In one class of models, attention contributed to normalization mechanisms, whereas in a different class of models it did not. Model selection based on Akaike's and on Bayesian information criteria demonstrated that in most cells the effects of attention were best described by models where attention did not contribute to normalization mechanisms. This demonstrates that attentional influences on neuronal responses in primary visual cortex often bypass normalization mechanisms.

Connections of the superior colliculus to shoulder muscles of the rat: a dual tracing study.

Previous investigations indicate that the superior colliculus (SC) is involved in the initiation and execution of forelimb movements. In the present study we investigated the tectofugal, in particular the tecto-reticulo-spinal projections to the shoulder and arm muscles in the rat. We simultaneously retrogradely labeled the premotor neurons in the brainstem by injection of the pseudorabies virus PrV Bartha 614 into the m. rhomboideus minor and m. acromiodeltoideus, and anterogradely visualized the tectofugal projections by intracollicular injection of the tracer FITC dextrane. Our results demonstrate that the connection of the SC to the skeletal muscles of the forelimb is at least trisynaptic. This was confirmed by long survival times after virus injections into the muscles (98-101 h) after which numerous neurons in the deep layers of the SC were labeled. Transsynaptically retrogradely labeled brainstem neurons connected disynaptically to the injected muscles with adjacent tectal terminals were predominantly located in the gigantocellular nuclear complex of the reticular formation. In addition, putative relay neurons were found in the caudal part of the pontine reticular nucleus. Both tectal projections to the nucleus gigantocellularis and the pontine reticular nucleus were bilateral but ipsilaterally biased. We suggest this projection to be involved in more global functions in motivated behavior like general arousal allowing fast voluntary motor activity.

Visual response properties of neurons in cortical areas MT and MST projecting to the dorsolateral pontine nucleus or the nucleus of the optic tract in macaque monkeys.

Neurons in cortical medial temporal area (MT) and medial superior temporal area (MST) projecting to the dorsolateral pontine nucleus (DLPN) and/or to the nucleus of the optic tract and dorsal terminal nucleus (NOT-DTN) were identified by antidromic electrical stimulation in five macaque monkeys. Neurons projecting to either target were located in close proximity to each other, and in all subregions of MT and MST sampled. Only a small percentage of the antidromically identified projection neurons (4.4%) sent branches to both the NOT-DTN and the DLPN. Antidromic latencies of neurons projecting to the NOT-DTN (0.9-6 ms, median 2.1 ms) and to the DLPN (0.8-5 ms, median 2.0 ms) did not differ significantly. Visual response properties of the neurons antidromically activated from either site did not differ significantly from those of cells that were not so activated. On the population level only neurons activated from the NOT-DTN had a clear preference for ipsiversive stimulus movement, whereas the neurons activated from the DLPN and neurons not antidromically activated from either target had no common directional preference. These results are discussed in terms of specification of cortico-subcortical connections and with regard to pathways underlying slow eye movements in different visuomotor behaviours.

Retinal projections to the accessory optic system in pigmented and albino ferrets (Mustela putorius furo).

We investigated if a reduced specificity of the retinal projection to the accessory optic system might be responsible for the loss of direction selectivity in the nucleus of the optic tract and dorsal terminal nucleus (NOT-DTN) and, in consequence of this, the optokinetic deficits in albino ferrets. Under electrophysiological control we performed dual tracer injections into the NOT-DTN and the medial terminal nucleus (MTN). Retrogradely labelled ganglion cells were found in the visual streak, the dorsal, and the ventral retina both after injections into the NOTDTN and the MTN indicating that both nuclei receive input from the same retinal regions. The distribution and spacing of labelled ganglion cells did not differ between pigmented and albino ferrets. However, retinal ganglion cells projecting simultaneously to both the NOT-DTN and the MTN occurred only in albino ferrets. These results suggest that a reduced specificity of the projection pattern of direction specific ganglion cells may contribute to the loss of direction selectivity in the NOT-DTN in albino ferrets.

Sensitivity to relative disparity in early visual cortex of pigmented and albino ferrets.

To investigate binocular interactions as the neuronal substrate for disparity sensitivity in the ferret (Mustela putorius furo), we measured the effects of relative horizontal disparities on responses of neurons in areas 17 and 18 of the visual cortex. Stimulation by moving bars and sinusoidal gratings showed that about half of our sample in pigmented ferrets was sensitive to relative horizontal disparity. This also included many neurons, which were classified as only monocularly activated when testing either eye alone. However, the tuning width was about two or three times coarser (median tuning width 4 degrees of visual angle) than that in the cat. In albino ferrets, only 8% of the neurons in the early visual cortex displayed some sort of disparity-dependent binocular interactions, but none could be clearly identified as relative disparity-coding neuron.

Receptor protein tyrosine phosphatases are expressed by cycling retinal progenitor cells and involved in neuronal development of mouse retina.

Receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including cell proliferation, migration and differentiation. Here we investigated potential roles of RPTPs in the developing mouse retina. Using a degenerate oligonucleotide-based reverse transcription polymerase chain reaction approach, we identified 11 different RPTPs in the retina at embryonic stage 13 (E13). Subsequently, the expression patterns of RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma in the retina from embryonic stages to adult were analyzed in detail using quantitative real-time-PCR, in situ hybridization, immunohistochemistry and Western blotting. At E13, all six RPTPs are expressed in actively cycling retinal progenitor cells and postmitotic newborn retinal neurons. With ongoing maturation, RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma display a different spatiotemporal regulation of mRNAs and proteins in the pre- and postnatal retina. Finally, in adulthood these six RPTPs localize to distinct cellular compartments of multiple retinal neurons. Additional studies in RPTPgamma(-/-) and RPTPbeta/zeta(-/-) (also known as PTPRZ1, RPTPbeta or RPTPzeta) mice at postnatal stage P1 reveal no apparent differences in retinal laminar organization or in the expression pattern of specific retinal cell-type markers when compared with wild type. However, in RPTPbeta/zeta(-/-) retinas, immunoreactivity of vimentin, a marker of Müller glial cells, is selectively reduced and the morphology of vimentin-immunoreactive radial processes of Müller cells is considerably disturbed. Our results suggest distinct roles of RPTPs in cell proliferation and establishing phenotypes of different retinal cells during retinogenesis as well as later in the maintenance of mature retina.

Identification of a tyrosinase (TYR) exon 4 deletion in albino ferrets (Mustela putorius furo).

Albinism is due to a lack of pigmentation in hair, skin and eye, and has been shown to occur in several animal species. Mutations of the tyrosinase (TYR) gene account for albinism in domestic cats, rabbits, cattle, mice and rats. In this study, we demonstrate that a TYR mutation accounts for albinism in the ferret (Mustela putorius furo). The coding sequence of the five exons of TYR was determined in genomic DNA from wild-type pigmented 'sable' coloured and albino ferrets. It was not possible to amplify TYR exon 4 in albino ferrets originating from different breeds. The deletion of exon 4 in albino ferrets was confirmed by Southern blot hybridization of genomic DNA from albino and pigmented ferrets. This is the first report of a deletion of a TYR exon in a non-human mammal.

Deficits of visual motion perception and optokinetic nystagmus after posterior suprasylvian lesions in the ferret (Mustela putorius furo).

We recently described an area in the ferret posterior suprasylvian (PSS) cortex characterized by a high proportion of direction selective neurons. To answer the question whether area PSS subserves functions similar to cat posteromediolateral suprasylvian area (PMLS) and monkey medial temporal area (MT) we investigated the contribution of area PSS to visual motion perception and optokinetic nystagmus. Ferrets were tested on global motion detection before and after bilateral lesions involving area PSS and control lesions of other extrastriate visual areas. Following PSS lesions motion coherence thresholds were significantly increased both in pigmented and albino ferrets, whereas control lesions sparing PSS did not affect visual motion perception. Optokinetic nystagmus was strongly reduced to absent after PSS lesions. These results indicate that area PSS is crucial for global motion processing in the ferret and in that sense may be functionally equivalent to PMLS in the cat and area MT in the monkey.

Motion perception deficits in albino ferrets (Mustela putorius furo).

Albino ferrets contrary to their pigmented conspecifics show no optokinetic nystagmus. Therefore, in this study motion perception was compared between pigmented and albino ferrets (Mustela putorius furo) trained to discriminate between coherently moving random dot patterns and dynamic noise stimuli in a two-alternative forced choice task. Fully coherently versus incoherently moving patterns could be distinguished by ferrets of both phenotypes. Motion coherence thresholds, however, were significantly higher in albinos. These results indicate that albino ferrets are not motion blind as could be expected from their total lack of optokinetic reactions. However, they are severely impaired in global motion perception.

A motion-sensitive area in ferret extrastriate visual cortex: an analysis in pigmented and albino animals.

In search of the neuronal substrate for motion analysis in the ferret (Mustela putorius furo), we extracellularly recorded from extrastriate visual cortex in five pigmented and two albino ferrets under general anaesthesia and paralysis. Visual stimulation consisted of large area random dot patterns moving either on a circular path in the frontoparallel plane or expanding and contracting radially. Strongly direction-selective neurons were recorded in a circumscribed area in and just posterior to the suprasylvian sulcus, thus named by us the posterior suprasylvian area (area PSS). Altogether, we recorded 210 (90%) and 95 (72%) PSS neurons in pigmented and albino ferrets, respectively, that were direction selective. In these neurons responses during random dot pattern stimulation in the preferred direction were at least twice as strong than stimulation in the non-preferred direction. Response strength in preferred direction and tuning sharpness of PSS neurons in albinos were significantly reduced when compared to pigmented animals (median values: 34.1 versus 14.8 spikes/s and 142 versus 165 degrees for pigmented and albino ferrets, respectively). Inter-spike-intervals during visual stimulation were significantly shorter in pigmented (median 9 ms) than in albino PSS neurons (median 14 ms). Our data indicate that area PSS may play a crucial role in motion perception in the ferret.

GABA-immunoreactive starburst amacrine cells in pigmented and albino rats.

In this study we tested whether the critical anatomical substrate for retinal direction selectivity is altered in albino mammals. We used dual immunostaining for GABA and choline acetyltransferase and quantitatively analyzed the number of double-labelled starburst amacrine cells in wild-type and albino rats. In albino rats, the percentage of ON-amacrine cells with high GABA content was significantly lower than in pigmented animals. OFF-amacrines did not significantly differ between the two rat strains. Thus, the decreased GABA content in ON-amacrine cells could reflect an altered neuronal substrate for retinal direction selectivity. These results are discussed in relation to the optokinetic deficits described in albino mammals.

Contribution of inhibitory mechanisms to direction selectivity and response normalization in macaque middle temporal area.

Inhibitory mechanisms contribute to directional tuning in primary visual cortex, and it has been suggested that, in the primate brain, the middle temporal area (MT) inherits most of its directional information from primary visual cortex (V1). To test the validity of this hierarchical scheme, we investigated whether directional tuning in MT was present upon blockade of local gamma-aminobutyratergic (GABAergic) inhibitory mechanisms. Direction selectivity during the initial 50 ms after response onset was abolished in many MT cells when the local inhibitory network was inactivated whereas direction selectivity in later response periods was largely unaffected. Thus, direction selectivity during early response periods is often generated autonomously within MT whereas direction selectivity during later response periods is either inherited from other visual areas or locally mediated by mechanisms other than gamma-aminobutyric acid type A receptor (GABA(A)) inhibition. GABAergic inhibition may also mediate contrast normalization. Our data suggest that GABA(A) inhibition implements a local direction-selective static nonlinearity, rather than a full normalization in MT. These findings put constraints on strict hierarchical models according to which MT performs more complex computations based on local motion measurements provided by earlier areas, arguing for more distributed and independent information processing.

GABA content in the retina of pigmented and albino rats.

Reduction of the melanin precursor DOPA associated with albinism leads to spatiotemporal disturbances in retinal neurogenesis and thus seems to be responsible for numerous neuronal alterations found in albino retinae. To investigate whether these cellular alterations are reflected in retinal neurotransmitter concentrations we compared the levels of GABA and glutamate in the retina of adult pigmented Long Evans and albino Wistar rats using reversed phase-liquid chromatography (RP-HPLC). When normalized to retinal weight, GABA levels showed a statistically insignificant trend to be lower and glutamate values to be higher in albinos than in pigmented animals. The ratio of glutamate to GABA was significantly higher in albino than in pigmented retinae. As numerous studies have shown that the balance between GABA and glutamate plays a crucial role for establishing direction selectivity, these results are discussed in relation to direction selectivity and defects in the optokinetic system of albinos.

Differences in calcium signalling in rat peripheral sensory neurons.

Calcium influx and the resulting increase in intracellular calcium concentration [Ca2+]i can induce enhanced sensitivity to temperature increases in nociceptive neurons. Using the patch-clamp technique and simultaneous calcium microfluorimetry we show that experimental elevation of [Ca2+]i using the calcium ionophore ionomycin resulted in a significant potentiation of heat-activated currents. This was not the case when rises in [Ca2+]i were elicited by depolarization of the cell membrane by current injection via the patch pipette. Our data provide first, however, indirect evidence that in sensory neurons calcium ions may be guided into different intracellular microdomains depending on the type of ion channel or pore through which they enter the cell. We conclude that the compartmentalization of sensory neurons for calcium ions may be decisive on further signalling cascades accounting, for example, for neuronal plasticity.

Development of the optokinetic response in macaques: a comparison with cat and man.

In macaque monkeys, an optokinetic response (OKR) can be elicited monocularly both in temporonasal and, albeit weaker, in nasotemporal direction very early after birth. The further maturation of equal strengths of OKR in both directions depends on stimulus velocity: at low-stimulus velocities (10-20 degrees /s) symmetry is reached at 3-4 weeks of age, at higher-stimulus velocities (40-80 degrees /s) it is reached only at 4-5 months of age. Retinal slip neurons in the NOT-DTN are direction selective for ipsiversive stimulus movement shortly after birth. Most of these neurons receive input from both eyes; many are dominated by the contralateral eye. Electrophysiological and neuroanatomical evidence suggests that the cortical input to the NOT-DTN starts to become functional by postnatal day 14, at the latest. Based on these behavioral and physiological data, as well as on comparison with data from kittens and human infants, we hypothesize that the very early monocularly elicited bidirectional optokinetic response is due to the direct retinal input from both eyes to the NOT-DTN. As the cortical projection matures, it gains more and more influence upon the response properties of retinal slip neurons in the NOT-DTN, and the retinal influence gradually decreases.

Fast Ca2+-induced potentiation of heat-activated ionic currents requires cAMP/PKA signaling and functional AKAP anchoring.

Calcium influx and the resulting increase in intracellular calcium concentration ([Ca(2+)](i)) can induce enhanced sensitivity to temperature increases in nociceptive neurons. This sensitization accounts for heat hyperalgesia that is regularly observed following the activation of excitatory inward currents by pain-producing mediators. Here we show that rat sensory neurons express calcium-dependent adenylyl cyclases (AC) using RT-PCR and nonradioactive in situ hybridization. Ionomycin-induced rises in [Ca(2+)](i)-activated calcium-dependent AC and caused translocation of catalytic protein kinase A subunit. Elevation of [Ca(2+)](i) finally resulted in a significant potentiation of heat-activated currents and a drop in heat threshold. This was not prevented in the presence of suramin that nonspecifically uncouples G protein-dependent receptors. The sensitization was, however, inhibited when the specific PKA antagonist PKI(14-22) was added to the pipette solution or when PKA coupling to A kinase anchoring protein (AKAP) was disrupted with InCELLect StHt-31 uncoupling peptide. The results show that heat sensitization in nociceptive neurons can be induced by increases in [Ca(2+)](i) and requires PKA that is functionally coupled to the heat transducer, mostly likely vanilloid receptor VR-1. This calcium-dependent pathway can account for the sensitizing properties of many excitatory mediators that activate cationic membrane currents.

Directional asymmetry of neurons in cortical areas MT and MST projecting to the NOT-DTN in macaques.

The cortical projection to the subcortical pathway underlying the optokinetic reflex was studied using antidromic electrical stimulation in the midbrain structures nucleus of the optic tract and dorsal terminal nucleus of the accessory optic system (NOT-DTN) while simultaneously recording from cortical neurons in the superior temporal sulcus (STS) of macaque monkeys. Projection neurons were found in all subregions of the middle temporal area (MT) as well as in the medial superior temporal area (MST). Antidromic latencies ranged from 0.9 to 6 ms with a median of 1.8 ms. There was a strong bias in the population of cortical neurons projecting to the NOT-DTN for ipsiversive stimulus movement (towards the recording side), whereas in the population of cortical neurons not projecting to the NOT-DTN a more or less equal distribution of stimulus directions was evident. Our data indicate that there is no special area in the posterior STS coding for ipsiversive horizontal stimulus movement. Instead, a specific selection of cortical neurons from areas MT and MST forms the projection to the NOT-DTN and as a subpopulation has the same directional bias as their subcortical target neurons. These findings are discussed in relation to the functional grouping of cortical output as an organizational principle for specific motor responses.

Cortical input to the nucleus of the optic tract and dorsal terminal nucleus (NOT-DTN) in macaques: a retrograde tracing study.

Using retrograde tracing methods, we investigated the cortical projection to the nucleus of the optic tract and dorsal terminal nucleus of the accessory optic system (NOT-DTN) in macaque monkeys. Tracer injections at electrophysiologically identified recording sites in the NOT-DTN resulted in retrogradely labelled neurons in layer V of various cortical areas. The strongest projection always arose from the middle temporal area (MT) and the adjoining cortex anterior to MT in the superior temporal sulcus. A less dense projection came from the middle superior temporal area (MST). In addition, retrogradely labelled cells were consistently found in areas V1 and V2 at moderate to high density. Furthermore, sparse to moderate labelling occurred in prestriate area V3. These findings were compared with the label resulting from control injections into the superior colliculus in two additional cases. Our results indicate that the cortical input to the NOT-DTN as the sensorimotor interface for the pathway subserving stabilizing eye movements during the optokinetic reflex and smooth pursuit mainly arises from the motion-sensitive areas MT and MST in the superior temporal sulcus, as well as from areas V1 and V2. Clearly the projection to the NOT-DTN does not arise from a single cortical area.

Retinal ganglion cells projecting to the nucleus of the optic tract and the dorsal terminal nucleus of the accessory optic system in macaque monkeys.

Using classical neuroanatomical retrograde tracing methods we investigated the retinal ganglion cells projecting to the nucleus of the optic tract and dorsal terminal nucleus of the accessory optic system (NOT-DTN) in macaque monkeys. Our main aim was to quantify the strength of the projection from the ipsilateral retina to the NOT-DTN. We therefore examined the number, distribution, and soma size of retinal ganglion cells involved in this projection. Electrophysiologically controlled small injections into the NOT-DTN revealed a clearly bilateral retinal projection originating mainly from the central retina but also involving peripheral retinal regions. Labelled cells were found nasally in the contralateral retina and temporally in the ipsilateral retina with some overlap in the fovea. The projection from the ipsilateral retina was 36-43% of that from the contralateral retina. On average, only 1-6% of the local population of ganglion cells projected to the NOT-DTN. Small soma size and large dendritic fields imply that in monkey rarely encountered, 'specialized' ganglion cells provide the direct retinal input to the accessory optic system (AOS). These results are discussed with respect to the symmetry of monocular horizontal optokinetic nystagmus (OKN) in primates.